Biofertilizers: Study and Impact [1 ed.] 1119724678, 9781119724674

Citation preview

Biofertilizers

Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106 Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

Biofertilizers Study and Impact

Edited by

Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi

This edition first published 2021 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2021 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. Wiley Global Headquarters 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no rep­ resentations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchant-­ ability or fitness for a particular purpose. No warranty may be created or extended by sales representa­ tives, written sales materials, or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further informa­ tion does not mean that the publisher and authors endorse the information or services the organiza­ tion, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Library of Congress Cataloging-in-Publication Data ISBN 9781119724674 Cover image: Wikimedia Commons Cover design by Russell Richardson Set in size of 11pt and Minion Pro by Manila Typesetting Company, Makati, Philippines Printed in the USA 10 9 8 7 6 5 4 3 2 1

Contents Preface xxi 1 Biofertilizer Utilization in Forestry Wendy Ying Ying Liu and Ranjetta Poobathy 1.1 Introduction 1.2 Mechanisms of Actions of Biofertilizers 1.2.1 Facilitation of N Acquisition 1.2.1.1 Mutualistic N2 Fixation 1.2.1.2 Non-Symbiotic N2 Fixation 1.2.2 Facilitation of P Acquisition 1.2.2.1 Phosphate Solubilizing Microorganisms 1.2.2.2 Mycorrhizas 1.2.3 Potassium Solubilization 1.2.4 Production of Siderophores 1.2.5 Modulation of Phytohormones 1.2.6 Phytoprotection 1.3 Factors Influencing the Outcome of Forestry-Related Biofertilizer Applications 1.4 Applications of Biofertilizers in Forestry 1.5 Conclusion and Future Prospects References 2 Impact of Biofertilizers on Horticultural Crops Clement Kiing Fook Wong and Chui-Yao Teh 2.1 Introduction 2.2 Microbial Strains Used in Biofertilizers 2.3 Impact of Biofertilizer Application on Horticultural Crops 2.3.1 Increased Yield and Quality of Crops 2.3.1.1 Vegetable Crops 2.3.1.2 Fruit Crops 2.3.1.3 Ornamental Plants

1 2 3 3 4 5 5 6 7 8 9 10 12 13 16 18 20 39 40 41 41 41 44 46 48 v

vi  Contents 2.3.2 Enhanced Nutritional Content of Produce 2.3.2.1 Mineral-Biofortified Crops 2.3.2.2 Enhanced Secondary Metabolites 2.3.2.3 Improved Vitamin Content 2.3.3 Improved Tolerance Against Biotic Stress 2.3.3.1 Fungal and Bacterial Pathogens 2.3.3.2 Viral Pathogens 2.3.3.3 Insect Pests 2.3.3.4 Nematodes 2.3.3.5 Weeds 2.3.4 Improved Tolerance Against Abiotic Stress 2.3.4.1 Drought 2.3.4.2 Salinity 2.3.4.3 Heavy Metal 2.3.4.4 Cold Stress 2.3.4.5 Heat Stress 2.3.5 Improved Vegetative Propagation Efficiency 2.3.5.1 Propagation by Cuttings 2.3.5.2 Grafting 2.4 Future Perspectives and Challenges Ahead 2.5 Conclusion References

49 49 50 51 52 52 56 58 61 64 65 66 68 70 71 73 73 73 74 75 79 79

105 3 N2 Fixation in Biofertilizers Rekha Sharma, Sapna Nehra and Dinesh Kumar 3.1 Introduction 106 3.2 Biofertilizers 108 3.2.1 Origin 108 3.3 Biofertilizer: Transporter Constituents 108 3.4 Mechanism of Actions of Biofertilizers 109 3.5 Biochemistry of Manufacture of Biofertilizer 109 3.6 Benefits of Biofertilizer Over Biochemical Fertilizers 110 3.7 Variances Among Organic and Biofertilizer 111 3.8 Types of Biofertilizers 111 3.9 Microorganisms Utilized to Make Biofertilizer 111 3.10 Microorganism in Nitrogen Fixation 113 3.10.1 Biofertilizers: Symbiotic N-Fixers 113 3.10.2 Biofertilizers: Free Living N-Fixers 114 3.10.3 Biofertilizers: Associative Symbiotic N-Fixers 114

Contents  vii 3.11 Phosphorus Solubilizing Microbes 3.12 Conclusion and Future Prospect Acknowledgments Abbreviations References 4 Organic Farming by Biofertilizers Anuradha and Jagvir Singh 4.1 Introduction 4.2 Biofertilizers 4.2.1 Benefits of Biofertilizers 4.2.2 Method of Biofertilizer Application 4.2.2.1 Seed Treatment 4.2.2.2 Seedling Treatment 4.2.2.3 Setts and Tuta Treatment 4.2.2.4 Soil Treatment 4.2.3 Precautions During Application of Biofertilizers 4.3 Classification of Biofertilizers 4.3.1 Nitrogen Fixer Bacteria 4.3.1.1 Commercial Applications 4.3.2 Cyanobacteria as Biofertilizers 4.3.2.1 Commercial Applications 4.3.2.2 Factors Affecting Cyanobacteria Biofertilizer 4.3.3 Mycorrhiza as Biofertilizers 4.3.3.1 Ectotrophic Mycorrhiza 4.3.3.2 Endotrophic Mycorrhiza 4.3.3.3 Changes in Mineral Compounds 4.3.3.4 Manure Value and Its Importance 4.3.4 Azolla as Biofertilizer 4.3.5 Vermicompost 4.3.5.1 Method of Vermicompost 4.4 Organic Farming 4.4.1 Objectives of Organic Farming 4.4.2 Benefits of Organic Farming 4.4.3 Benefit for Environment 4.4.4 Methods of Organic Farming 4.4.5 Techniques for Organic Farming 4.4.5.1 Crop Diversity 4.4.5.2 Soil Management

115 115 116 116 117 121 122 123 126 126 126 127 127 127 127 128 128 129 130 130 131 131 132 132 133 133 134 135 135 136 136 136 137 137 137 138 138

viii  Contents 4.4.5.3 Weed Management 138 4.5 Traditional Agriculture vs. Organic and Inorganic Farming 139 4.5.1 Problems Created by Traditional Farming 139 4.6 Reasons for Doing Organic Farming 140 4.6.1 To Save Soil Health 140 4.6.2 To Preserve Nutrients 141 4.6.3 To Reduce the Cost of Agriculture 141 4.6.4 To Prevent Hazardous Elements in Animal Products 141 4.6.5 To Protect the Environment 141 4.6.6 Natural and Good Taste 142 4.7 Advantage of Organic Farming 142 4.7.1 Good Nutrition 142 4.7.2 Good Health 142 4.7.3 Freedom From Poison 142 4.7.4 Less Money 143 4.7.5 Great Taste 143 4.7.6 Environmental Safety 143 4.8 Disadvantages of Organic Farming 143 4.8.1 Lack of Information 143 4.8.2 Lack of Outline 143 4.8.3 Making More Money in the Beginning 144 4.9 Conclusion 144 Acknowledgement 144 References 144 5 Phosphorus Solubilizing Microorganisms 151 Rafig Gurbanov, Berkay Kalkanci, Hazel Karadag and Gizem Samgane 5.1 Phosphorus Pollution 152 5.2 Phosphate Solubilization 153 5.3 Microbial Mechanisms of Phosphate Solubilization 155 5.3.1 Organic Phosphate Solubilization 156 5.3.2 Inorganic Phosphate Solubilization 156 5.4 Phosphate-Solubilizing Bacteria 158 5.5 Phosphate-Solubilizing Fungi 160 5.5.1 Phosphate-Solubilizing Fungi as Plant Growth Promoters 162 5.5.2 The Methods of using Phosphate-Solubilizing Fungi in Agriculture 164

Contents  ix 5.6 Bacteria-Fungi Consortium for Phosphate Solubilization 5.7 Conclusions References 6 Exophytical and Endophytical Interactions of Plants and Microbial Activities A. Mbotho, D. Selikane, J.S. Sefadi and M.J. Mochane 6.1 Introduction 6.2 Beneficial Interactions 6.2.1 Arbuscular Mycorrhizal Fungi 6.2.2 Plant Growth-Promoting Microorganisms 6.2.3 Rhizobia 6.2.4 Endophytes 6.3 Pathogenic (Harmful) Interactions 6.3.1 Oomycetes 6.3.2 Fungi 6.3.3 Bacteria 6.3.4 Viruses 6.4 Conclusion References 7 Biofertilizer Formulations Sana Saif, Zeeshan Abid, Muhammad Faheem Ashiq, Muhammad Altaf and Raja Shahid Ashraf List of Abbreviations 7.1 Introduction 7.1.1 Evolution of Biofertilizers 7.1.2 Biofertilizers: A Sustainable Approach 7.2 Biofertilizer Formulations 7.2.1 Selection of Strain 7.2.1.1 Microbial Strains 7.3 Types of Formulations 7.3.1 Carrier-Based/Powder Formulations 7.3.1.1 Selection of Carrier Material 7.3.1.2 Sterilization of Carrier 7.3.2 Granular Formulations 7.3.3 Liquid Formulations 7.3.3.1 Inoculant Preparation 7.3.3.2 Common Additives 7.3.4 Cell Immobilization

165 167 167 183 184 185 186 189 193 194 194 195 198 199 200 203 204 211 212 212 212 213 215 215 215 227 230 230 235 236 236 237 238 239

x  Contents 7.3.4.1 Polymer Entrapped Formulations 7.3.4.2 Advantages and Constrains 7.3.5 Fluid Bed-Dried Formulation 7.3.6 Mycorrhizal Formulations 7.4 Stickers 7.5 Additives 7.6 Packaging 7.7 Conclusion References

239 243 243 244 246 246 246 247 247

8 Scoping the Use of Transgenic Microorganisms as Potential Biofertilizers for Sustainable Agriculture and Environmental Safety 257 Vasavi Rama Karri and Nirmala Nalluri 8.1 Introduction 258 8.2 Role of Nitrogen in Plant Growth and Development 260 8.2.1 Microorganisms Involved in Nitrogen Fixation 260 8.3 Importance of Phosphorus 261 8.3.1 Microbes Involved in Phosphate Solubilization 262 8.3.2 Reducing the pH of Soil 262 8.3.3 Mineralization 263 8.3.4 Chelation 263 8.3.5 Promotion of Plant Growth by PSMs 263 8.3.6 Approach of Using PSMs as Biofertilizer and the Future Perspective 264 8.4 Significance of Potassium (K) 265 8.4.1 Microorganisms Involved in Potassium Hydrolyzation 265 8.4.2 Effect of KSB on Plant Growth and Yield 266 8.4.3 Abilities and Objections of K Solubilizing Bacteria 266 8.5 Biofertilizers Used in Agriculture 267 8.5.1 Mycorrhiza 268 8.5.2 Plant Growth-Promoting Rhizobacteria (PGPR) 268 8.6 Role of Biotechnology in Agricultural Sector 268 8.6.1 Development of Potent Microbial Strains Through Genetic Engineering Approach to Produce Efficient Biofertilizers 269 8.6.2 Genetically Altered Transgenic Azotobacter vinelandii as an Effective Diazotrophs Biofertilizer 270 8.6.3 Phytostimuators and Biofertilizers 271 8.6.4 Azospirillum 272

Contents  xi 8.6.5 Generation of Genetically Modified Transgenic Azospirillum Strains With Enhanced Levels of Phytoharmone Secretion 8.6.6 Development of Rhizobium Strains With Increased Competitiveness by Genetic Modification 8.6.7 Effect of GM Rhizobial strains on Arbuscular Mycorrhizal (AM) Fungi 8.6.8 Release of Genetically Manipulated Rhizobium for Field Trails 8.7 Conclusion Acknowledgements References 9 Biofertilizer Utilization in Agricultural Sector Osikemekha Anthony Anani, Charles Oluwaseun Adetunji, Osayomwanbo Osarenotor and Inamuddin 9.1 Introduction 9.2 Application of Biofertilizer as Bioaugmentation Agent for Bioremediation of Heavily Polluted Soil 9.3 Advantages of Biofertilizer in Comparison With Synthetic Fertilizer 9.4 Specific Examples of a Biofertilizer for Crop Improvement in Agricultural Sector 9.5 Management of Biotic and Abiotic Stress 9.6 Combinatory Effect of Biofertilizer With Other Substance and Their Effect on Crops 9.7 Conclusion and Recommendation to Knowledge References

274 275 278 279 280 281 281 293 294 295 296 298 301 303 305 306

10 Azospirillum: A Salient Source for Sustainable Agriculture 309 Rimjim Gogoi, Sukanya Baruah and Jiban Saikia 10.1 Introduction 309 10.1.1 The Genus Azospirillum 311 10.1.2 Properties of Azospirillum spp. 312 10.1.2.1 Chemotaxis 312 10.1.2.2 Aerotaxis 313 10.1.2.3 Formation of Cysts and Aggregates or Flocs 313 10.1.2.4 Survivability in Rhizosphere and Bulk Soil 314 10.1.2.5 Competition With Other Soil Microorganisms 316

xii  Contents 10.1.2.6 Association With Plant Roots 10.2 Azospirillum and Induction of Stimulatory Effects for Promoting Plant Growth 10.3 Applications in Various Fields 10.4 Current Status 10.5 Challenges in Large-Scale Commercial Applications of Azospirillum Inoculants 10.6 Programs Employed for Enhanced Applications of Azospirillum Inoculants 10.7 Conclusion and Future Prospects References 11 Actinomycetes: Implications and Prospects in Sustainable Agriculture V. Shanthi 11.1 Introduction 11.2 Role in Maintaining Soil Fertility 11.2.1 Nitrogen Fixation 11.2.2 Phosphate Solubilization 11.2.3 Potassium Solubilization 11.3 Role in Maintaining Soil Ecology 11.4 Role as Biocontrol Agents 11.4.1 Production of Antibiotics 11.4.2 Production of Siderophores 11.4.3 Production of Hydrogen Cyanide 11.4.4 Production of Lytic Enzymes 11.5 Role as Plant Stress Busters 11.5.1 Resistance From Heavy Metal Toxicity 11.5.2 Resistance Against Drought/Water Deficit 11.5.3 Resistance Toward Salinity 11.6 Conclusion 11.7 Future Perspectives References 12 Influence of Growth Pattern of Cyanobacterial Species on Biofertilizer Production Jasti Tejaswi, Kaligotla Venkata Subrahmanya Anirudh, Lalitha Rishika Majeti, Viswanatha Chaitanya Kolluru and Rajesh K. Srivastava 12.1 Introduction 12.2 Habit and Habitat of Cyanobacteria

316 318 320 324 327 328 329 330 335 336 338 338 340 342 342 345 346 348 349 349 351 352 354 355 355 356 357 371

371 373

Contents  xiii 12.3 Morphology and Mode of Reproduction 12.4 Role of a Fertilizer in Plant Growth 12.4.1 Synthetic Fertilizers 12.4.2 Organic Fertilizers 12.4.3 Biofertilizer 12.5 Cyanobacteria as Biofertilizer 12.6 Production of Cyanobacteria 12.7 Methods for In Vitro Culture of Cyanobacteria 12.7.1 Macro- and Microelements 12.7.2 Temperature 12.7.3 Light and Cell Density 12.7.4 Media 12.8 Methods for Gene Transfer into Cyanobacteria 12.8.1 DNA-Mediated Transformation 12.8.2 Electroporation 12.8.3 Conjugation 12.8.4 Biolistic Method 12.9 Conclusion and Future Prospects 12.10 Abbreviations References

373 375 376 377 377 379 381 382 382 383 383 383 384 385 385 386 386 386 387 388

13 Biofertilizers Application in Agriculture: A Viable Option to Chemical Fertilizers 393 Rajesh K. Srivastava 13.1 Introduction 394 13.2 Chemical Fertilizer 397 13.2.1 Customized Fertilizers 400 13.2.2 Fortified Fertilizer 400 13.3 Biofertilizers 400 13.3.1 Biocompost 403 13.3.2 Trichocard 404 13.3.3 Trichocard Production 405 13.3.4 Azotobacter 405 13.3.5 Phosphorus 406 13.3.6 Vermicompost 406 13.4 Conclusion 408 13.5 Abbreviations 408 References 408

xiv  Contents 14 Quality Control of Biofertilizers Swati Agarwal, Sonu Kumari and Suphiya Khan 14.1 Introduction 14.2 Biofertilizer Requirement and Supply 14.3 Process of Biofertilizer Quality Control 14.4 Requirement of Quality Control 14.5 Standards for Biofertilizers Quality Control 14.6 Methods for Quality Testing 14.6.1 Microbiological Methods 14.6.2 Serological Methods 14.6.3 Molecular Methods 14.7 Conclusion Acknowledgement References

413 413 414 416 417 419 421 422 422 423 423 423 424

15 Biofertilizers: Characteristic Features and Applications 429 Tanushree Chakraborty and Nasim Akhtar 15.1 Introduction 430 15.2 Types of Biofertilizers 430 15.3 Characteristic Features and Applications of Biofertilizers 431 15.3.1 Cyanobacteria Biofertilizer 431 15.3.2 Actinomycetes 435 15.3.3 Rhizobium leguminosarum bv. Trifolii 436 15.3.4 Arbuscular Mycorrhizal Fungi (AMF) 436 15.3.5 Bacillus thuringiensis 437 15.3.6 Microalgae 438 15.4 Phosphate Solubilizing Bacteria (PSB) and Fungus (PSF) 438 15.4.1 Azotobacter 439 15.4.2 Azospirillum 440 15.4.3 Paenibacillus 440 15.4.4 Phyllosphere Associated Methylobacterium 441 15.4.5 MO Plus Biofertilizer 441 15.5 Effect of Biofertilizer on Various Plants (Experimental Design) 442 15.5.1 Azotobacter spp. (AZT) and Azospirillum spp. (AZP) on Eucalyptus grandis 442 15.5.2 Bradyrhizobium Strains and Streptomyces griseoflavus on Some Leguminous, Cereal, and Vegetable Crops 443 15.5.3 Rhizobium and Rhizobacteria on Trifolium repens 444

Contents  xv 15.5.4 Arbuscular Mycorrhizal and Phosphate Solubilizing Fungi on Coffee Plants 445 15.5.5 Glutamicibacter halophytocola KLBMP 5180 on Tomato Seedlings 446 15.6 Screening of Microbes for Biofertilizer 447 15.6.1 Screening for Phosphate Solubilization 447 15.6.2 Screening for Potassium Solubilizing 447 15.6.3 Screening for Nitrogen-Fixing 448 15.6.4 Screening for Zinc Solubilization 448 15.6.5 Screening for Ammonia Production 448 15.6.6 Screening for Hydrogen Cyanide (HCN) Production 448 15.6.7 Screening for Siderophores 448 15.6.8 Screening for Auxin Production 449 15.6.9 Screening for Gibberellic Acid Production 449 15.6.10 Screening for Production of Chitinase 449 15.7 Limitations of Biofertilizers 449 15.8 Success of Biofertilizer 450 15.9 Debottlenecking 453 15.10 Optimization of Biofertilizer 456 15.10.1 Optimization of Phosphate Solubilization 456 15.11 Concomitant of Biofertilizer 458 15.12 New Approach 458 15.13 Conclusion and Future Prospects 459 References 460 16 Fabrication Approaches for Biofertilizers Andrew N. Amenaghawon, Chinedu L. Anyalewechi and Heri Septya Kusuma 16.1 Introduction 16.2 Biofertilizers 16.3 Types of Biofertilizers 16.3.1 Nitrogen-Fixing Biofertilizers 16.3.1.1 Rhizobium 16.3.1.2 Azospirillum 16.3.1.3 Azotobacter 16.3.2 Phosphorus-Solubilizing Biofertilizers 16.3.3 Phosphate-Mobilizing Biofertilizer (Mycorrhizae) 16.3.4 Potassium Biofertilizer 16.3.5 Growth-Promoting Biofertilizers 16.3.6 Blue-Green Algae (Cyanobacteria)

491 492 492 493 493 494 494 495 495 496 497 497 498

xvi  Contents 16.4 Preparation Approaches for Biofertilizers 16.4.1 Inoculant Formulation 16.4.2 Carriers for Biofertilizer Preparation 16.4.2.1 Sterilized Carriers 16.4.3 Carrier Form 16.5 Methods of Biofertilizer Formulation 16.5.1 Solid-Based Carrier Bioformulation 16.5.1.1 Peat Formulations 16.5.2 Liquid Inoculants Formulation 16.5.3 Polymer-Based Formulation 16.5.3.1 Alginate Formulations 16.5.4 Fluidized Bed Dried Formulation 16.5.5 Particles From Gas Saturated Solutions (PGSS) Method 16.5.6 Bionanoformulations 16.6 Application Modes for Biofertilizers 16.6.1 Seed Treatment 16.6.2 Seedling Root Dipping 16.6.3 Soil Application 16.7 Factors Affecting the Preparation of Biofertilizers 16.8 Beneficial Effects of Biofertilizers 16.9 Challenges and Limitations of Biofertilizers 16.10 Future Prospects 16.11 Conclusion References 17 Biofertilizers From Waste Rafaela Basso Sartori, Ihana Aguiar Severo, Álisson Santos de Oliveira, Paola Lasta, Leila Queiroz Zepka and Eduardo Jacob-Lopes 17.1 Introduction 17.2 Waste Sources 17.3 Technologies for Waste Treatment 17.3.1 Conventional Technologies 17.3.2 Emerging Technologies 17.3.2.1 Nutrients Recovery From Wastes by Microalgae 17.3.2.2 Overall Process Operations 17.4 Main Applications of Microalgae Biofertilizers 17.4.1 Fertility and Soil Quality 17.4.1.1 Nitrogen Fixation

499 499 500 500 501 501 501 502 503 504 504 504 505 505 506 506 506 507 507 508 509 509 510 511 517

518 519 521 521 522 523 526 528 528 528

Contents  xvii 17.4.1.2 Carbon Sequestration 529 17.4.1.3 Soil Organic Matter, Improvement, and Recovery 530 17.4.2 Promotion of Plant Growth, Disease, and Pest Control 531 17.4.2.1 Plant Colonization and Hormone Production 531 17.4.2.2 Disease and Pest Control 532 17.5 Conclusion and Recommendations 532 References 533 18 Biofertilizers Industry Profiles in Market Kashish Gupta 18.1 Biofertilizers and Biofertilizer Technology 18.1.1 Benefits of Different Biofertilizers 18.2 Limitations in Usage of Biofertilizers 18.3 Biofertilizer Market Segments 18.4 Biofertilizers Market Drivers in India 18.5 Present Scenario of Biofertilizer Market 18.6 Key Players of Biofertilizers in Indian Market 18.7 Problems in Promotion of Biofertilizer 18.8 Popular Marketed Biofertilizers in Indian Market 18.9 Recent Trends in Biofertilizer: Liquid Biofertilizer 18.9.1 Specialties of Liquid Biofertilizer 18.10 Conclusion and Future Scope References

541

19 Case Study on Biofertilizer Utilization in African Continents Osikemekha Anthony Anani and Charles Oluwaseun Adetunji 19.1 Introduction 19.2 Specific Examples of Biofertilizer for Crop Improvement, Environmental Bioremediation, and Their Advantages and Challenges in Africa 19.3 Conclusion and Future Recommendations References

561

20 Biofertilizers: Prospects and Challenges for Future Tanushree Chakraborty and Nasim Akhtar 20.1 Introduction 20.2 Definition 20.2.1 Helper Bacteria 20.2.2 The Point of Difference

575

541 542 543 544 546 547 549 550 553 554 554 555 556

562 563 570 570

576 579 579 580

xviii  Contents 20.3 20.4 20.5 20.6 20.7

Advances in Biofertilizer Preparation of Biofertilizer The Carrier Materials Production System of Biofertilizer Mechanism of Growth-Promoting Activity of Biofertilizers 20.8 Advantages and Limitations 20.9 Future Aspects 20.10 Conclusion References

580 581 581 582 583 584 584 585 586

21 Biofertilizers: Past, Present, and Future Mukta Sharma and Manoj Sharma 21.1 Introduction 21.2 Biofertilizer: A Brief History 21.3 Biofertilizer Classification 21.4 Different Paradigms of Biofertilizers 21.4.1 Impregnation of Fertilizers and Fertilizer Use Efficiency 21.4.2 Inoculants of Mixtures of Microorganisms 21.4.3 Different Formulations of Inoculants 21.4.4 Inoculant Carrier 21.4.5 Biofertilizer Carriers and Liquid Formulations 21.4.6 Controlled Release Techniques: Encapsulation, Lyophilization, and Drying 21.5 Biofertilizers: Current Status 21.6 Biofertilizers: Future Paradigm 21.7 Conclusion References

591

22 Algal Biofertilizer Muhammad Mudassir Iqbal, Gulzar Muhammad, Muhammad Shahbaz Aslam, Muhammad Ajaz Hussain, Zahid Shafiq and Haseeba Razzaq 22.1 Introduction 22.2 Algae and Algal Biofertilizers 22.2.1 Algae is a Polyphyletic Functional Group 22.2.2 Multifaceted Role of Algal Biofertilizer in Sustainable Cultivation 22.2.3 Biostimulants From Algae 22.3 Techniques of Application of Algal Biofertilizer

607

592 593 594 596 596 597 597 598 599 600 601 601 602 603

608 609 609 610 612 613

Contents  xix 22.3.1 Algal Extracts as Biofertilizer 22.3.2 Addition of Algal Strains and Algal Biofertilizer to Soil 22.4 Cultivation of Algae and Production of Algal Biofertilizer 22.5 Conclusion References

613 619 625 630 630

Index 637

Preface Great attention has been paid to reduce the use of conventional chemical fertilizers harming living beings through food chain supplements from the soil environment. Therefore, it is necessary to develop alternative sustainable fertilizers to enhance soil sustainability and agriculture productivity. Biofertilizers are the substance that contains microorganisms (bacteria, algae, and fungi) living or latent cells that can enrich the soil quality with nitrogen, phosphorous, potassium, organic matter, etc. They area costeffective, biodegradable, and renewable source of plant nutrients/ supplements to improve the soil-health properties. Biofertilizers emerge as an attractive alternative to chemical fertilizers and as a promising costeffective technology for eco-friendly agriculture and a sustainable environment that holds microorganisms which enhance the soil nutrients’ solubility leading a raise in its fertility and stimulate crop growth and healthy food safety. This book provides in-depth knowledge about history and fundamentals to advances biofertilizers, including latest reviews, challenges, and future perspectives. It covers fabrication approaches and various types of biofertilizers and their applications in agriculture, environment, forestry, and industrial sectors. Also, organic farming, quality control, quality assurance, food safety, and case studies of biofertilizers are briefly discussed. Biofertilizers’ physical properties, affecting factors, impact, and industry profiles in the market are well addressed. This book is an essential guide for farmers, agrochemists, environmental engineers, scientists, students, and faculty who would like to understand the science behind the sustainable fertilizers, soil chemistry, and agroecology, etc. Chapter 1 focuses on the various action mechanisms observed in microorganisms, those that drive effective biofertilizer functions for forestryrelated utilization. Besides, the chapter discusses the factors influencing the success of forestry-related biofertilizer applications as well as the current use and prospects of biofertilizers in the forestry sector.

xxi

xxii  Preface Chapter 2 highlights the impact of applying biofertilizers on horticultural crops including the possible mechanisms, leading to improved crop growth and stress tolerance. Possible challenges of biofertilizer application and recommended solutions to these problems are also discussed to ensure the efficient use of biofertilizers in the horticulture industry. Chapter 3 discusses various microorganisms which as act as biofertilizers and also the nitrogen-fixing bacteria including different symbiotic and asymbiotic nitrogen-fixing microbes and other substitutes for easy making of biofertilizers. The major focus is given to innovative methods, for example, growing of microorganisms, accumulation of microorganisms, and conveniences for distribution, applying, and framing of microorganisms for moving from greenhouse and laboratory to field test. Chapter 4 highlights the usefulness of organic manure in biofarming. Various types of agrochemicals have spoiled our life, environment, and ecosystem. This chapter provides a detailed discussion about how organic manure can save the life of our earth and how it is better than agrochemicals. Chapter 5 reviews the scientific literature on environmental phosphorus pollution and mechanisms of phosphate solubilization through intact bacteria and fungi or their enzymes. Moreover, inoculation methodologies, factors affecting the inoculum efficiency, and applications of single or multiple species as promising biofertilizer components for sustainable and ecological farming practices are summarized. Chapter 6 reviews plant-microbe associations occurring both exo- and endophytically on different plant species. The beneficial and pathogenic outcomes of these interactions are discussed, highlighting the microorganisms and the plants involved. Furthermore, the importance of research of these interrelationships is considered concerning use in agriculture for the development of agricultural agents. Chapter 7 discusses the different formulation technologies of biofertilizers used to mitigate the harmful effects of chemical fertilizers. The complete formulation process is discussed, highlighting the significance of each step, i.e., types of selected microbes, choice of suitable carrier, and addition of sticking materials while unifying the biofertilizer formulation. Chapter 8 describes the scope of exploiting efficient transgenic microorganisms produced by genetic engineering strategy as potential biofertilizers to enhance the yield of crops through the sustainable farming approach. Furthermore, environment-friendly benefits of utilizing various types of microorganisms alternative to chemical fertilizers in improving soil fertility of agricultural lands are also emphasized.

Preface  xxiii Chapter 9 intends to provide detailed information on the application of biofertilizer as a sustainable biotechnological tool that could lead to an increase in agricultural production. Detailed information is provided on the modes of action of these biofertilizers while specific examples of cases where biofertilizer has been utilized for improving an increase in agricultural production are also discussed. Chapter 10 discusses the various characteristic properties, utility, and challenges of free-living nitrogen-fixing bacteria of the genus Azospirillum as a commercial inoculant, aimed to enhance the nitrogen availability in the soil for sustainable agriculture. Mechanistic routes aiding in nitrogen fixation by the bacteria are comprehensively elaborated. Chapter 11 discusses the beneficial role of actinomycetes in the field of sustainable agriculture. Its unique ability to mitigate soil ecosystem and to promote plant growth by producing important agro-active substances is highlighted. Also, the role of actinomycetes to serve as a potential and efficient source of biofertilizer is discussed. Chapter 12 discusses the influence of growth conditions and other parameters including morphological patterns of cyanobacterial species, found in biofertilizer quality and nutrients richness. Chapter 13 provides the positive influences of biofertilizers’ application on agricultural sector via improving the productivity and yields of the crop. Further, it discusses the role of biofertilizers’ production and promotion, with a viable option to chemical fertilizers that have minimized the productivity of the crop with negative impacts on soil, water bodies, or environment components. Chapter 14 provides detail informationabout the necessity of quality control of biofertilizers. Quality supervision is crucial and should be achieved constantly to manage the microbial products in support of the clients. The rules applied for calculating the quality are restricted to the concentration and viability of the microbes. Chapter 15 focuses on the significance of biofertilizers with their microbial composition having an edge over chemical fertilizers. The emphasis is on recent advances in preparation, mechanism, growth promotion, carrier materials, and production system of biofertilizers. The chapter also discusses the prospects of biofertilizers along with its advantages and limitations. Chapter 16 provides an in-depth understanding of biofertilizers and the various approaches available for their preparation. The chapter ends with some prospects and recommendations needed for further improvements in the development, preparation, and application of biofertilizers to achieve green, cleaner, and sustainable food production.

xxiv  Preface Chapter 17 discusses the use of biofertilizers based on waste recycling as a potential substitute for chemical fertilizers. Initially, an overview of biofertilizers is presented. Furthermore, the main sources of organic waste are discussed, as well as the appropriate treatment processes. Finally, emerging technologies and the main applications of biofertilizers are presented. Chapter 18 clearly describes the know-how of biofertilizer technology, its segments, and a brief description of the types of biofertilizer available in the market. The highlight of the chapter is the recent trend in biofertilizer technology: liquid biofertilizer, its merits over conventional fertilizers, and future potentials. Chapter 19 discusses the current situation on the application of biofertilizer in Africa and their mechanism of action. Different types of biofertilizer that have been introduced are also highlighted. Moreover, specific examples are cited where biofertilizer has led to an increase in agricultural production. Chapter 20 focuses on various types of biofertilizers, their properties, significance, preparation, production, uses, and outcome. It also focuses on experimental designing on screening and selection of microbes and their optimization as biofertilizers. The chapter also deals with the success of biofertilizers, their limitations, and new approaches to overcome constraints. Chapter 21 discusses the use of microorganisms in the form of biological fertilizers. It also discusses the characteristics and features of different formulations in which the biofertilizers are used extensively. The chapter highlights the development and recent advances in biological fertilizer and its performance. Chapter 22 covers the biodiversity of algae and biochemical constituents of algal biofertilizers with the effects on plant growth and yield. The state-of-the-art techniques for the mass cultivation of algae are also part of the discussion. This chapter also focuses on novel strategies for the mass production of algal biofertilizers.

1 Biofertilizer Utilization in Forestry Wendy Ying Ying Liu* and Ranjetta Poobathy School of Biological Sciences, Quest International University, Perak, Malaysia

Abstract

The forest biomes provide crucial ecosystem services which include acting as carbon sinks, providing habitats for biodiversity, preventing soil erosion, mitigating climate change, and producing important resources such as timber, fuel, and bioproducts. However, due to various human activities, the productivity of forests has greatly reduced over time. In order to manage nutrient deficiencies and phytopathogens, chemical products are utilized in forest nurseries, plantation forests, and natural stands. However, this often leads to nutrient losses via leaching, gaseous losses, and other detrimental effects. Exploitation of biofertilizers in the bid to reduce reliance on chemicals could promote the growth and development of tree species and ultimately increase forest productivity in a more sustainable manner. Most biofertilizer utilizations are focused in the agriculture and horticulture sectors with less emphasis in forestry, with the exception of mycorrhizae. It is imperative to recognize the various mechanisms of action of biofertilizers (e.g., facilitation of nutrient uptake, phytohormone modulation, and phytoprotection) to fully exploit the potential of biofertilizer in promoting the ecosystem services of forest biomes. Hence, this chapter explores the mechanisms of action of effective microorganisms in biofertilizers, factors influencing the effectiveness of biofertilizer application, and applications of biofertilizers in the forestry sector. Keywords:  Biofertilizer, forestry, plant growth promoting microorganisms, biological nitrogen fixation, mycorrhizae, phytohormones, biocontrol, sustainability

*Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (1–38) © 2021 Scrivener Publishing LLC

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2  Biofertilizers

1.1 Introduction The forest biomes are globally important as they cover 30% of Earth’s terrestrial surface with more than three trillion trees at an estimated size of four billion hectares [1–3]. The forest ecosystem services include, but are not limited to, acting as carbon sinks, providing biodiversity, protecting soil quality, and producing a wide array of resources such as wood, timber, biomass, and coal [4, 5]. The forest ecosystems are widely distributed worldwide, in boreal, temperate, and tropical regions [6]. For some forest ecosystems, their distributions are highly correlated to land use and soil characteristics, whereby nutrient-poor soils are allocated for forests while high fertility soils are utilized for agriculture crops and grasslands [6]. The productivity of forests has been on a decline due to accelerated growth of human and livestock populations, forest fires and undiscerning exploitation of forest products [7]. These human activities have led to continuous soil erosion causing the forest soils to experience deficiencies of essential nutrients [8]. Chemical fertilizer usage in forestry, from forest nurseries to plantations and natural stands, ranges from zero to very minimal in comparison to its usage in the agriculture sector [9]. In the field, fertilizers are typically applied only once, or at maximum, a few times over a rotation that takes 25 to 30 years [10–12]. The application of chemical fertilizer, when required, allows management of nutrient deficiencies in marginal soils, increased productivity of planted forests and/or maintenance of sustainable soil nutrients after successive rotations [10, 11]. However, the use of chemical fertilizers has its drawbacks. Firstly, excessive applications in the field could lead to nutrient losses via leaching, gaseous losses, and reduced beneficial forest microbiomes [13]. Also, it is arduous to fertilize forest soils due to the remoteness, low accessibility, and low fertility of many forests [6]. Hence, it is more viable to exploit the potential of biofertilizers to reduce the reliance on chemical fertilizers to promote the growth and development of tree species and ultimately promote sustainable forest productivity. Biofertilizers are products containing beneficial microorganisms, mainly bacteria and fungi, which can inhabit the rhizosphere and/or plant interiors and subsequently promote plant growth via application onto seeds, plants, and/or soil [14, 15]. Such microorganisms, also known as plant growth promoting microorganisms (PGPMs), can directly increase plant growth by facilitating nutrient acquisition in plants and modulating phytohormones while indirectly promote plant growth by decreasing inhibitory effects of phytopathogens while making the rhizosphere more favorable for

Biofertilizer Utilization in Forestry  3 plants and other beneficial microorganisms [16–18]. The utilization of biofertilizer in agriculture and horticulture is more extensive and widespread while the utilization of biofertilizers in forestry is still under investigation or restricted to forest nurseries, with most studies focusing only on mycorrhizae [19, 20]. It is imperative to explore other beneficial microorganisms and their mechanisms of actions to fully utilize the potential of biofertilizer in promoting growth of tree species, be it in forest nurseries, plantations, and/or natural stands, in order to fully harness the ecosystem services of forest biomes.

1.2 Mechanisms of Actions of Biofertilizers Biofertilizers could improve plant health and growth, soil nutrient, and fertility status in forest biomes [21]. Nonetheless, the efficacy of the biofertilizers could be greatly affected by various external factors including soil characteristics, tree species, and native microbiome composition [22]. Hence, it is crucial to understand the mechanisms of actions of PGPMs [inclusive of the commonly used term, plant growth promoting rhizobacteria (PGPR)] utilized in biofertilizer so as to employ them under appropriate circumstances. In reality, many PGPMs are likely to employ more than a single mechanism, either simultaneously or at different times under different conditions [14, 17]. The mechanisms of action of PGPMs comprise of aiding nutrient uptake [e.g., nitrogen (N), phosphorus (P), potassium (K), iron (Fe), zinc (Zn), and sulfur (S), modulation of phytohormones (e.g., abscisic acid, cytokinin, ethylene, indole acetic acid (IAA), and gibberellin (GA)] and biocontrol ability to confer phytoprotection [17, 23–25].

1.2.1 Facilitation of N Acquisition Nitrogen is vital for plant growth and development as it is a major component of nucleic acids, membrane lipid, amino acids, proteins, chlorophylls, and enzymes [18, 26]. However, N is a key limiting nutrient in forest biomes, especially in forest ecosystems that are developed on marginal soils [27–29]. Despite N abundance in the atmosphere as N2, most plants are incapable of utilizing it and are highly dependent on soil N bioavailability, in organic and inorganic N forms, to sustain their growth. However, N2 can be converted into inorganic forms, such as ammonium and nitrate, by the aid of N2-fixing microorganisms via a highly energy intensive biological N2 fixation (BNF) process catalyzed by nitrogenase [23, 30]. Nitrogen-fixing

4  Biofertilizers microorganisms in forest biomes can be categorized into two categories: (i) symbiotic N2 fixers (e.g., rhizobia in association with leguminous plants and Frankia spp. in association with actinorhizal plants) and (ii) nonsymbiotic N2 fixers (in free-living, associative or endophytic forms) [17, 23]. Nevertheless, symbiotic N2 fixation contributes to majority of the amount of fixed N required by plants in comparison to non-symbiotic N2 fixation [17]. Most literature have referred to symbiotic N2 fixation as symbioses with the development of root nodules while others have also included associative N2 fixation as symbiotic as it fits the definition of symbiosis which is beneficial association between two different organisms [31]. Nonetheless, the former definition will be used in this review.

1.2.1.1 Mutualistic N2 Fixation Studies have shown that the legume-rhizobia interaction is a main contributor to the amount of fixed N2 in many forest biomes [32]. Many, albeit not all, tree legume species are able to fix atmospheric N2 in association with rhizobia, a group of gram-negative bacteria that are capable of forming N2 nodules on mainly leguminous roots, under N-deficient conditions [33]. Rhizobia can penetrate leguminous root tissues either via hair infection, crack entry or epidermal entry [34]. Most legumes employ the root hair infection strategy where flavonoids secreted by the plants will signal for rhizobia to secrete signal molecules (Nod factors) that binds to root hair cell receptors and then results in the curling of root hair, bacterial entry into the cells of the root hair, division of cortical cells, followed by the formation and ramification of an infection thread in developing nodule primordia [35, 36]. Subsequently, the bacteria will differentiate morphologically to form bacteroids and are eventually removed from the infection thread to form symbiosomes via the synthesis of nitrogenase [35–37]. Ammonium produced in the nodules will be transported to plant cells where it will be assimilated into amino acids for the plant’s use via the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway [38]. While rhizobia provide N source for plant growth, the legumes provide protection and photosynthates for the rhizobia as a carbon source [39]. Another common N2-fixation symbiosis in forest biomes is the interaction between actinorhizal plants and the actinomycete Frankia via nodule formation [40, 41]. Many actinorhizal trees and shrubs can form mutualisms with mycorrhizae and tripartite symbiosis (actinorhizal plant-Frankiamycorrhiza), thus providing them an edge to grow in soils with poor nutrient availability [42, 43]. The strategy employed by Frankia in infecting their host plants will depend on the host and Frankia species involved. Nonetheless,

Biofertilizer Utilization in Forestry  5 they are similar to those employed by rhizobia, which are hair infection, crack entry, or epidermal entry. For actinorhizal plants that belong in the order Fagales, their intracellular infection progress through via root hairs while for those in the Rosales, the intracellular entry of Frankia is through the roots [43]. Nodules produced by actinorhizal plants have indeterminate growth and are able to fix a substantial amount of N2 that is equivalent to those produced by legumes [43, 44].

1.2.1.2 Non-Symbiotic N2 Fixation Many free-living heterotrophic diazotrophs in the rhizosphere, such as Azotobacter and Klebsiella, are also capable of fixing atmospheric N2, albeit without direct interaction with any other organisms [45–47]. Hence, these bacteria have to source for their own energy to carry out the highly energy-­intensive BNF process. They can usually oxidize organic molecules released through decomposition or the aid of other organisms while some have chemolithotrophic abilities in utilizing inorganic compounds instead in order to obtain their energy sources [48]. It has been reported that the free-living BNF rates are strongly correlated to soil organic matter contents, with higher rates in woody residues and surface organic layers compared to mineral soil [48, 49]. Also, as oxygen inhibits nitrogenase activities, these bacteria will have to be able to act as anaerobes or microaerophiles during the N2 fixation [50, 51]. Thus, with these restrictions, free-living N2 fixation, in comparison with symbiotic N2 fixation, will not be able to contribute greatly to BNF in most of the ecosystems [48, 49]. However, it was also reported that free-living N2 fixers fix substantial amounts of N in rain forests [52, 53]. In addition, studies have shown that leguminous trees in mature tropical forests do not fix as much N due to N deposition [54]. Meanwhile, other bacteria, such as Azospirillum and Herbaspirillum, can form endophytic and/or associate mutualisms with various varieties of plants [17, 55]. This manner of N2 fixation is similar to symbiotic N2 fixation without the formation of specialized structures whereby these diazotrophs receive reduced carbon and other nutrients from the plants while they provide fixed nitrogen for the plants to use [56, 57].

1.2.2 Facilitation of P Acquisition Despite N being generally recognized as the most limiting nutrient in many forest ecosystems, P is also another major limiting nutrient [58, 59]. Phosphorus is vital for plants as a major component of energy source (ATP) to perform various metabolic processes such as signal transduction,

6  Biofertilizers macromolecule synthesis, respiration, and photosynthesis [30, 60]. It has been widely reported that high N deposition that is not matched by equivalent increase in the P inputs could cause nutritional imbalances which eventually reduce forest growth and productivity [61–63]. Demand for P has been shown to increase as growth promotion takes place following the additions of N as plant P concentrations and N:P ratios decrease [64, 65]. Also, there is evidence suggesting that P limitation has detrimentally affected the rate of N2 fixation in tropical forests that mainly consist of non-leguminous tree species [66, 67]. Phosphorus can be found abundantly in most soil (as organic and inorganic forms) but the amount of accessible P for plants to utilize is low, as >90% of soil P are insoluble and precipitated (e.g., phosphotriesters and aluminium phosphate) [30, 68]. Plants are only able to access soluions [30, ble P sources such as monobasic (H2PO4) and dibasic HPO2− 4 140]. Although chemical fertilizer provides sufficient soluble inorganic P, most of them are immobilized quickly after its application, thus rendering them unavailable to plants [17]. Hence, a sustainable alternative would be to employ microorganisms to aid in P solubilization and mineralization. These microorganisms can be divided in two major groups, which are phosphate solubilizing microorganisms (PSMs) and mycorrhizal fungi.

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1.2.2.1 Phosphate Solubilizing Microorganisms PSMs have been shown to effectively increase the accessibility of available P to plants via solubilization and mineralization of complex P compounds [69, 70]. There are a few strategies that can be employed by PSMs to solubilize insoluble P forms, of which the secretion of organic acids (OA) with low molecular weight, such as acetic, citric, and gluconic acids, is recognized as a main way P is solubilized [17, 71, 72]. The OA secreted will either chelate the mineral ions or lower the cell’s pH level in order to acidify these microorganisms and their environments, resulting in the P-ion being released from P-mineral via the swap of H+ for Ca2+ instead [73]. The efficacy of the solubilization process is highly reliant on on the type, concentration, and quality of OAs released into the soil, with the quality of OA being more crucial [74]. The ability of the PSMs to secrete various OAs simultaneously may enhance the P solubilization process further [71]. Some studies have also recommended that the transformation of insoluble P to soluble forms of P could take place without the production of OA [75, 76]. This was reported by Altomare et al. [77] whereby Trichoderma harzianum strain T-22 was able to solubilize P, potentially due to chelation and reduction processes, along with providing biocontrol to the plant

Biofertilizer Utilization in Forestry  7 without the acidification process. Besides OA production, inorganic acids secreted by chemoautotrophs (e.g., nitric and sulfuric acids) have been shown to solubilize P by converting tricalcium phosphate into mono- and dibasic phosphates [78, 79]. Phosphate-solubilizing microorganisms can mineralize organic phosphorus via the secretion of phosphatases and phytases that catalyze the hydrolysis of phosphoric esters [72]. Acid phosphates, commonly found in fungi, have been proposed to be the main mechanism in the organic P mineralization [80]. Meanwhile, phytase releases P from organic materials that are stored as phytate [72]. Although plants generally are not capable in acquiring P directly from phytate, PSMs in the rhizosphere could alleviate the effects due to the plant’s inability to do so [81]. A single PSM strain could carry out both phosphate solubilization and mineralization [17]. Examples of bacterial genera capable of solubilizing and mineralizing P include Azotobacter, Bacillus, Bradyrhizobium, Burkholderia, Enterobacter, Erwinia, Mezorhizobium, Rhizobium, Microbacterium, Paenibacillus, Pseudomonas, and many others [82]. Meanwhile, fungal PSMs include those from the genera Aspergillus, Cladosporium, Penicillium, Rhizoctonia, Rhizopus, Sclerotium, Trichoderma, and others [82, 83].

1.2.2.2 Mycorrhizas Mycorrhizal fungi can establish symbiotic relationships, either obligately or facultatively, with many plants, where these mycobionts are reliant on their host plants for photosynthates and energy while they contribute various benefits to their hosts [84]. This group of microorganisms is one of the most commonly studied PGPMs used in biofertilizer for forestry purposes [85]. The fungi can increase surface areas for better access to procure nutrients, in particular insoluble phosphorus sources by extending their mycelia from root surfaces into the soil [86]. Besides that mycorrhizal fungi are also able to improve quality and aeration of soil, reduce susceptibility of the plants to phytopathogens, and promote tolerance of host plants to environmental stresses [84, 87, 88]. Mycorrhiza can colonize their host plant’s root tissues either via intracellular or extracellular mechanisms. Hence, they can be divided into two main categories, which are endomycorrhiza and ectomycorrhiza, respectively. Endomycorrhizas can be further categorized as arbuscular, arbutoid, ericoid, monotropoid, and orchid mycorrhizaes [88, 89]. The most common mycorrhizal associations are arbuscular mycorrhizal fungi (AMF) which are obligate mycobionts (generally, Glomeromycetes) that can form mutualism with more than 80% of vascular plants [90]. Vesicles

8  Biofertilizers and arbuscules are specialized structures of AMF that assist in mobilizing and making P available to their host plants and also obtaining mineral and water sources from further areas in the soil [3, 85]. However, as AMF are obligate in nature and cannot be grown as pure cultures in isolation from their hosts, these make AMF biofertilizer production in large scale a very difficult task [91]. Although ectomycorrhizal fungi (ECMF) are extensively distributed in many ecosystems, they are only affiliated with only 3% of the vascular plant families, particularly woody trees (e.g., birch, beech, myrtle, pine, and willow) [85]. Nonetheless, as they are effective in symbiotic association with many important woody trees, they are crucial in forestry and ecosystem management. Species of ECMF (>20,000 species) are mainly members of the phyla Ascomycota and occasionally Basidiomycota [92, 93]. ECMF are unable to penetrate the cell walls of their host plants but they form an intercellular interface, the Hartig network, comprising extremely branched hyphae to form a lattice between epidermal and cortical root cells to allow metabolic exchange between both fungi and the root [85]. The thick hyphal sheath, termed as the mantle, formed by ECMF are connected to extraradical mycelia that extend into the soil to allow minerals and water to be mobilized, absorbed and translocated to the hosts, often aiding their survival during adverse conditions [94, 95]. As of late 1950s, ECMF were investigated and utilized as biofertilizer to promote plant growth as they are able to assist in plant acquisition of P, N, water, and other minerals in forestry [96]. The application of these fungi is more common in nurseries whereby tree seedlings are inoculated in the nursery before planting in field in order to ensure healthy fungi system. Successful ECMF association is highly dependent on the suitable selection of plant-host species [85]. Mycorrhizal helper bacteria (MHB), when used with ECMF, have been shown to assist in the establishment and effectiveness of the mycorrhizal symbioses [97] by stimulating mycelial growth, increase the interaction and surface areas for root-mycorrhizae colonization via phytohormone and flavonoid production, and mitigate the detrimental effect of environmental stress on mycelia [85, 98, 99].

1.2.3 Potassium Solubilization Potassium is an integral macronutrient for enzyme activation, osmotic balance, phloem transport, photosynthesis, and protein synthesis in plants [100, 101]. The concentration of soluble K is generally minimal in soil as most K occur in the forms of silicate minerals and insoluble rocks [102]. The soil is the main source of K for plants and its accessibility in soil is

Biofertilizer Utilization in Forestry  9 reliant on the dynamics and content of K in the biomes [100]. Besides that, K limitation may detrimentally affect the soil microbial community resulting in inefficient nutrient cycling in the forest biomes [103, 104]. It has been suggested that the K dynamics and distribution in plant tissues, soils, and water in the forest biomes could be strongly affected by biotic factors [105]. The availability of K could also be further affected by stressors such as timber harvesting, forest fire, and intensification of land usage [106]. Many forests of which tree species have been continuously harvested for fuel, fertilizers, and others may have seen a decrease in substantial amounts of K available in the ecosystem, which may have decelerated the K availability for the growth and development of various tree species [107, 108]. Also, the addition of other major limiting nutrients (N and P) through atmospheric deposition, without increasing the amount of K, may be detrimental to the ecology of the forest biomes [105]. Limitations of K in the soil could be addressed by using potassiumsolubilizing microorganisms (KSMs) which produce OAs (e.g., formic, malic, oxalic, and tartaric acids) to release K from various insoluble minerals which include illite, micas, and orthoclases [100]. The OAs can convert K into soluble forms via direct solubilization of rock K or excretion of chelated silicon. These acids increase the solubilization of K compounds via provision of protons and formation of complexes with metal ions, such as Ca2+, present in the soil [109]. Examples of genera of KSMs include Arthrobacter, Acidithiobacillus, Bacillus, Burkholderia, Paenibacillus, and others [110, 111].

1.2.4 Production of Siderophores Iron is an important trace element for plant growth as it acts as a major component of redox enzymes that aids in oxygen transport, cellular proliferation, chlorophyll synthesis, and nucleic acid synthesis [112]. Despite being the fourth most copious element on earth, Fe cannot be readily assimilated by plants and microorganisms as it occurs predominantly as Fe3+ in nature and tends to form highly insoluble oxides and hydroxides under aerobic conditions, particularly in calcareous soils [113]. Though iron is not a limiting factor in many forest biomes, it has reported to be a limiting factor in some mangrove forests [114–116]. Under Fe-limiting conditions, microorganisms can acquire Fe via the secretion of siderophores, which are low-molecular mass iron chelators (~400–1,500 Dalton) with high affinity for Fe3+ ions [17, 117]. Siderophores will bind to Fe3+ ions and form complexes that are then returned to the cytosol where the reduction of Fe3+ takes place, followed by the secretion of

10  Biofertilizers Fe2+ into the cell through a gating machinery, making Fe accessible to the microorganism [118]. Siderophores are divided into three major families, which are catecholates (e.g., enterobactin), carboxylates (e.g., rhizobactin), and hydroxamates (e.g., ferrioxamine B) based on their functional groups [119]. Thus far, there are 270 types of siderophores that have been structurally characterized out of more than 500 known siderophores [120]. Generally, plants may employ two strategies to sequester iron: (i) lowering of pH levels in the rhizosphere, trailed by reduction of Fe3+ ions by ­ferric-chelate reductase, allowing the root cells to successively uptake of Fe2+; and/or (ii) secretion of phytosiderophores for iron solubilization, binding, and subsequent transport into root cells [121]. Yet, these strategies are unable to provide sufficient Fe for the plants under Fe-deficient conditions, especially in calcareous and alkaline soils [121]. Hence, a better alternative would be to assimilate Fe from microorganisms. Plants can assimilate Fe from these bacterial or fungal siderophores by two strategies which are (i) transfer of their Fe-siderophore complexes to plant roots for Fe reduction to take place and (ii) direct Fe chelation followed by ligand exchange reaction with phytosiderophores to release Fe2+ [23, 122]. Although the main function of the siderophores is to scavenge for Fe, they are also able to form complexes with other metals (e.g., aluminium, cadmium, copper, and nickel) present in the rhizosphere allowing them to be utilized by the microorganisms [123]. This, in turn, could help to ameliorate the abiotic stresses caused onto plants by toxic concentrations of heavy metals in the soil. Besides improving plant growth directly, side­ rophore production could also indirectly increase plant growth by inhibiting the growth of phytopathogens by binding to most Fe3+ in the root area and reducing Fe availability for them to proliferate [124]. This was proposed as an effective biocontrol mechanism as the siderophores produced by PGPMs have higher affinity for Fe3+ compared to fungal pathogens, thus out-competing them for available Fe [17].

1.2.5 Modulation of Phytohormones Phytohormones are organic substances that, at low concentrations (lesser than 1 mM), have the ability to boost, impede or modify the developmental activities of plants, particularly in their response to the environment [30, 125]. When exposed to environmental stresses (e.g., low pH, drought, salinity, and high temperature), plants usually try to regulate the levels of endogenous plant hormones so as to reduce the detrimental effects posed by these stresses [17, 126]. Certain PGPMs are capable of in vitro phytohormone production and could modulate the levels of phytohormones to

Biofertilizer Utilization in Forestry  11 reach equilibrium, thus aiding in regulating plants’ responses to environmental stresses [17, 127]. Common phytohormones that are produced by PGPMs comprise abscisic acid, cytokinin, IAA, and GA [128, 129]. IAA has various functions such as to promote cell division and differentiation, enhance germination rates and percentages of seeds, initiate and increase the expansion of roots, regulate plants’ responses to stress, produce metabolites, and negatively affect photosynthesis process [17, 30, 130]. It has been reported that many PGPMs are capable of releasing IAA as secondary metabolites in a slow and continuous manner, subsequently changing the auxin pool and affecting the plants’ physiological functions [131, 132]. The biosynthesis of IAA by PGPMs could involve L-tryptophan–dependent or independent pathways. Tryptophan, an amino acid that occurs at plant root regions, is the main precursor to synthesize IAA [133]. In addition, tryptophan can indirectly increase IAA production by inhibiting the formation of anthranilate, a precursor that inhibits IAA biosynthesis, via negative feedback regulation on the amount of anthranilate synthase [23, 134]. The indole-3-pyruvic acid pathway is the most frequent IAA biosysnthesis pathway utilized by PGPMs, followed by the indole-3-acetamide pathway [30, 131]. GAs are diterpenoid phytohormones that are crucial in various processes including stem elongation, leaf expansion, seed germination, floral induction, breaking of dormancy in seeds and tubers, and augmentation in size and number of fruits [128, 135]. Generally, GAs are produced endogenously by plants to regulate plant growth and development, with GA1, GA3, and GA4 being the three most common types of GAs that promote plant growth and shoot elongation [136]. There are three regulatory processes that control the synthesis of GA which are biosynthesis, reversible conjugation, and catabolism [130, 137]. Ethylene can affect plant growth by stimulating root initiation, hindering root elongation, accelerating the ripening of fruits, reducing wilting, improving seed germination, and triggering the production of other phytohormones [30]. When exposed to stress conditions, plants will secrete ethylene at higher concentrations to overcome the stresses [138, 139]. An essential precursor required for ethylene production is 1-aminocyclopropane1-carboxylic acid (ACC) [17, 139]. The production of ACC can be stimulated by the presence of both endogenous and bacterial IAA [17]. However, high concentrations of ethylene can cause defoliation and alter cellular processes that will eventually decrease plant growth [23, 30, 138, 140]. The production of ACC deaminase by PGPMs can reduce the negative impacts of high concentrations of ethylene on plant growth. The beneficial microorganisms, via ACC deaminase, are able to hydrolyze ACC into

12  Biofertilizers ɑ-ketobutyrate and NH3 which are then utilized as carbon and nitrogen sources, thus reducing the ethylene levels [139].

1.2.6 Phytoprotection Despite being used to a much lesser extent in forestry compared to agriculture, pesticides are mostly applied in forest nurseries and planted forests as they are the most cost-effective manner to manage insect pests, diseases, and weeds [141]. However, continual application of pesticides can bring about environmental damage to forest biomes (e.g., contamination of ground water, reduction in soil fertility, and disruption in ecosystem dynamics) [30]. Hence, the use of PGPMs as biocontrol agents in forests could be a more sustainable approach to control pests. The biocontrol activities include antibiosis nutrient competition, hydrogen cyanide (HCN) and lytic enzyme production, and induced systemic resistance (ISR) [17, 30]. One of the most effective biocontrol mechanisms by PGPMs is antibiosis to suppress the proliferation of phytopathogens (generally fungi). Some of the identified antimicrobial compounds responsible for phytopathogen inhibition include 2,4-diacetylphloroglucinol (DAPG), ecomycins, pyrrolnitrin, pyoluteorin, oomycin A, amphisin, phenazine, and others [142]. However, overdependence on these antimicrobial compounds could lead to the development of resistance in phytopathogens [30]. Thus, selection of strains that are capable of producing more than a single antimicrobial compound as well as synthesizing HCN is preferred to address this [17]. The synthesis of HCN, a volatile secondary metabolite with low molecular weight, can confer selective advantages on the producer, despite the lack of a role in their growth and primary metabolism. Production of HCN could inhibit the synthesis of cytochrome-c oxidase and some metalloenzymes in the pathogens, consequently preventing electron transport and disrupting energy supply to the cells [131, 143]. However, the utilization of HCN on its own does not render significant biocontrol activity, but instead, it works synergistically with antimicrobial compounds produced in antibiosis [17]. It was also reported that HCN could aid in nutrient acquisition by chelating metal ions and boosting nutrient availability to the plants [144]. PGPMs could also exert their biocontrol ability by producing lytic enzymes, such as β-glucanase, chitinase, dehydrogenase, phosphatises, and proteases that are capable of directly inhibiting fungal pathogens by affecting the structural integrity of their cell walls [145]. β-glucanase and chitinase are able to degrade fungal cell walls that are normally made up of β-1,4-N-acetyl-glucoseamine and chitin. Besides lytic enzyme production, PGPMs could also suppress phytopathogen growth via competition as they

Biofertilizer Utilization in Forestry  13 could colonize surfaces of the plants quickly and utilize many accessible nutrients, rendering them unavailable to the pathogens for their growth [17]. Also, as mentioned in the previous section, siderophore production by PGPMs could restrict the proliferation of these pathogens in the rhizosphere as well due to the lack of iron. ISR in plants could be triggered by PGPMs with biocontrol ability by enhancing their ability to defend against specific detrimental environmental stimuli, triggering their innate defense mechanisms against subsequent biotic stresses [18, 30]. This resistance involves jasmonate and ethylene signalling in plants whereby both hormones can positively influence the host plant’s defense responses to pathogens [17, 23]. Besides these hormones, many bacterial components have also been shown to cause ISR, including cyclic lipopeptides, flagella, lipopolysacharrides, homoserine lactones, and others [146]. Plants with ISR are reported to respond quicker and more aggressively to pathogen attacks [17]. As ISR does not target pathogens in a specific manner, it is highly effective in mitigating various pathogenic diseases [17, 147].

1.3 Factors Influencing the Outcome of Forestry-Related Biofertilizer Applications The rhizosphere, extending a few millimetres from the soil surface, is a substrate that facilitates interactions between bacteria, fungi, and other microfauna [148–150]. These interactions affect the chemistry and physiology of the surrounding soil, hence presenting major influences on plant health and nutrition, especially when the total root length and density of a plant is taken into consideration [148, 151]. Microbes in the rhizosphere have been observed to interact within and between species as well as with the host plant, for instance, in the case of co-phosphorus solubilizing bacteria (PSB) with vesicular arbuscular mycorrhizae (AM) or N2 fixers, for instance, Azotobacter and Azospirillum [152, 153]. Rhizospheric fungi are known to confer benefits on various plant species through a number of mechanisms, upon which three functional fungal groups are apparent: biofertilizers, biocontrol agents, and bioremediators [148]. Plant growth can also be boosted via culture with fungi displaying ACC deaminase activity [154, 155], which results in longer roots as well as reduction of symptoms following pathogen-induced stress [155, 156]. Generally, the success of any biofertilizer application in forestry depends on the microbial strain and inoculum production, including formulations,

14  Biofertilizers and field experimentation strategies, as observed in the application of Azospirillum [157]. It was found that plants inoculated with PGPR strains sourced from the plants’ native rhizosphere fare better in terms of germination and yield [158, 159]. Important considerations include the workability and versatility of the microbial strains at different temperatures: in the case of a cold climate-targeted PSB, it should be physiologically active to solubilize P at freezing temperatures. This trait allowed P availability to the evergreen pine forests during extreme winter conditions [153]. On the other hand, functionality at ambient temperatures also renders the psychrotolerant PSB species good candidates as P biofertilizer for all conditions [153]. The host plant species itself influences parameters associated with microbial inoculation: variations in the species, phenology, alterations in the rhizosphere as mediated by the host, mycorrhizal dependency, as well as various unknown host plant characteristics are known to influence AMF colonization as well as spore densities in the host plant [160–162]. The host species plays the biggest role in this regard, as observed in the rhizosphere of C. setosum [162]. Carvalho et al. [163] noted that AMF distribution in salt marshes relies heavily on the host rather than on environment-related stresses. The host species may also affect the depth of AMF colonization: spores were located at 40 cm in the subsoil of the rhizospheres of Taxus chinensis and Pyrus communis [162], attributed to the deep penetration of roots of the plants. A study on the Pichavaram mangrove in south east India revealed that areas displaying Rhizophora growth recorded the highest total nitrogen content, especially during the monsoon season when the terrigenous sediments deposit on the mangrove soil [164, 165]. Soil conditions are also integral in the outcome of a biofertilizertesting experiment. Soil, as an environment, could present with difficulties due to its unpredictable nature [159, 166]. Research has indicated that the effectiveness of a PGPR relies on the soil type: plants cultivated on less fertile soils benefit the most in terms of plant growth from the application of PGPR [159, 167]. Frommel et al. (1993) concluded that low soil pH and rainfall as well as high temperatures and Verticillium soil infection contributed to the poor root colonization by PGPR [159]. The clay soil in the fringes of the Pichavaram mangrove forest is rich in calcium, magnesium, nitrogen, and phosphorous, besides producing hydrogen sulfide, partly due to the presence of various species of fungi and bacteria that confer a host of benefits to the community around them, including assisting in nitrogen fixation and phosphate availability [164]. Another factor to consider is the soil moisture content, which may affect the success of plant growth post-inoculation [159, 169]. This factor,

Biofertilizer Utilization in Forestry  15 however, depends on the PGPR type as high moisture content may reduce soil oxygen levels [159]. The potency of a biofertilizer is also affected by the area in which a particular microbe is sourced from. The most important groups of PGPRs, obtained from the ectorhizospheric zone and among endosymbionts, involve phosphate-solubilizing species such as Acinetobacter, Serratia, Alcaligenes, Erwinia, Arthrobacter, Aspergillus, Burkholderia, Azospirillum, Pseudomonas, Bacillus, Enterobacter, Flavobacterium, Penicillium, and Rhizobium [80, 170–174]. AMF are found in many ecosystems, including stress-inducing environments such as acid-saline as well as saline soils [162]. Research has indicated that the AMF distribution depends on both host physiological and root morphological characteristics [162]. Mangrove forests have received much attention in terms of the discovery and effects of biofertilizers on individual tree species [164]. Mangrove areas are known for harboring populations of nitrogen-fixing Azotobacter [164, 175], of which three species, i.e., A. vinelandi, A chroococcum, and A. beijerinckii were identified as potential biofertilizers in mangrove nurseries and prawn ponds [164, 176]. The high levels of nitrogen resulting from leaf decomposition and the presence of the listed microbes are known to attract amphipods, crabs, fishes, isopods, prawns, and young oysters [177], leading to the conception of a “mangrove vegetation trap” for seafood harvesting, based on the requirements above [164, 178, 179]. Cyanobacteria are also prominently featured in mangrove areas. In the case of the Pichavaram mangrove area in India, aerial N2-fixing cyanobacteria increase in cell number during the summer and post-monsoon seasons, growing profusely on the lower and middle portions of the aerial roots [164, 180], and comprise 15% of the total phytoplankton population [164, 181]. Phormidium species are known to be tolerant to salinity and present great potential as biofertilizer as well as shrimp feed formulations [164, 180, 182, 183]. Better results may also be obtained in field experiments involving biofertilizers if combinations of microbes are used rather than single fungal or bacterial isolates. It was found that phosphate solubilization capacities in plants can be improved via the usage of a microbial combination instead of single isolates. Justifications for this phenomenon include improved growth conditions for other isolates via the metabolic products of one isolate [174, 184]; contribution of P-solubilizing functions by other isolates when one particular isolate can no longer dissolve phosphate due to changes in the culture condition [174]; and the microbial diversity involved in the process [174, 185]. A biofertilizer enriched with N-providing free-living diazotrophic bacteria and Cunninghamella elegans, a chitosan-producing fungus, was found to be effective in restricting nematode movement as

16  Biofertilizers well as reproduction in Pinus pinaster and P. pinea, and mitigating infection symptoms such as water and total chlorophyll loss, in the former [21]. Phosphate-solubilizing microbes are also known to exert biocontrol potential [153, 186, 187], a differential influence on AMF within and outside of plant roots in terms of colonization [153, 188] as well as other growth parameters for the host plant [153, 189, 190], indicating its potency on the target plant species as well as the surrounding microbial community. However, a major hurdle in any study involving biofertilizers is the successful transfer of laboratory-based experiments to the field, which has been shown as not viable in some studies [153, 191], despite the fulfilment of all the factors listed above. This phenomenon is attributed to the mismatch in terms of the interactions between the soil biota and the biofertilizer. Hence, it is important to ensure that the ecological characteristics of all components of a biofertilizer as well as their interactions with the microbiota are well-characterized [153]. Hayat et al. [153] found that the PSM isolates, especially Trichoderma paratroviride PWF-1 and Bacillus megaterium PWB-4, did not display antagonism to one another and other P-solubilizing fungi, indicating their capability to be established as bacterial or fungal-bacterial combination in order to enhance soil P availability. The challenge in the fruition of such experiments may also lie in the role of climate, which is known to influence the effectiveness of an applied PGPR and hence the outcome of a field experiment greatly [159, 192].

1.4 Applications of Biofertilizers in Forestry As mentioned in the previous section, efforts to translate laboratory and greenhouse forestry-related biofertilizer experiments to the field have been met with minimal success throughout the years. Success is limited to tree species found in specific locations throughout the globe. Lucy et al. [159] as well as Mallik and Williams [193] summarized some examples of research involving the use of PGPR in various forest tree species. A majority of the research were conducted in the growth chamber and greenhouse; only a minority were transferred to the field [159]. Field testings, however, indicated increased biomass, shoot and root growth, nutrient uptake, foliar nitrogen content, pathogenic biocontrol, and seedling emergence in various pine, beech, oaks, and spruce species inoculated or co-inoculated with Arthrobacter sp., Pseudomonas sp., Bacillus sp., Azotobacter sp., and unidentified bacterial strains or AMF [159, 193–219]. In the case of mangrove forests, known to be lacking in soluble nutrients, especially phosphorus [155, 220–222], seedlings were reported to fare better when inoculated

Biofertilizer Utilization in Forestry  17 with Azotobacter and Azospirillum [155, 223], phosphobacteria [224–227], as well as cyanobacteria [155, 228]. Greater success in the experiments was observed when the rhizospheres of the locations were inoculated with biofertilizers sourced from areas native to both the tree and microbial species [158, 159]. Climatic conditions, which may differ from laboratory and greenhouse conditions, influence the success of any biofertilizer experiments. For instance, extreme cold environments, such as tundra ecosystems, present a challenge in terms of microbial interactions, and as such are dominated by psychrophile or psychrotolerant bacteria [153, 229] that tolerate the cold environment due to abilities such as producing cold-shock proteins, enzymes, genetic changes from thermal shifts, as well as short unsaturated membranous acids [153, 230]. Based on this premise, Hayat and team [153] isolated PSB located in the Himalayan region to quantify for the isolates’ ecological characteristics as well as their abilities to convert inorganic P to available P and interact with microbiota present within the rhizosphere of Pinus spp. The team discovered that 31% and 69% PSB displayed ordinary and distinctive P-solubilizing capacities, respectively. Of the latter, 48% were categorized as “efficient P-solubilisers” (>400 mg L−1). Out of the 16 efficient PSB isolated, 11 and 4 were located in the lower subsurface and surface of the Himalayas, respectively. The highest amount of P-solubilization was observed in Ochrobactrum anthropic, which was isolated from the pine rhizospheric subsoil. Four out of eight PSB were found to be resistant to nematode grazing, while another three and one PSB resisted grazing up to 96 and 120 hours of co-culture, respectively [153]. The study stressed the importance of isolating microbes native to the area where the tree species is found in order to increase the fertilizing potential of the target microorganism. In order to increase the success of improving plant growth and development or soil remediation efforts, microorganisms targeted as biofertilizers have to be carefully selected based on their characteristics. AMF were found to be beneficial in terms of plant developmental activities as well as increasing tolerance to high pH and salinity levels [162, 231–233]. It was found that AMF inoculation provided more efficient salt stress protection for the plants when compared to any concentration of plant-available P in the soil [162, 234]. Salt-tolerant species of AMF that necessitates further investigation into their roles in plants include Glomus mosseae [162, 235] and G. caledonium [162] found predominantly in the alkaline and saline soils of the Yellow River Delta [162]. On the other hand, Trichoderma species, a common fungal species, is known to enrich soil via nutrient solubilization, especially in barren or pathogen-infected substrates, which can lead

18  Biofertilizers to higher plant yields as well as better plant resistance against drought and pathogenic stress [155, 186, 236, 237]. Trichoderma releases and solubilizes bound nutrients in the soil [155, 227]. It was reported that an increase of 70% in biomass as well as an increase in the assayed biochemical parameters was observed in Rhizophora apiculata and R. mucronata when the seedlings were inoculated with mangrove-derived Trichoderma. Again, the results strongly indicated the potential in using biofertilizers derived from areas native to the target plant [155]. Microbes are sometimes incorporated with certain compounds or substances to create biostimulants. A biostimulant is considered as such when a substance is observed to increase or influence a plant’s quality, nutritional status, or response to stress, regardless of the nutritional situation [238, 239]. Commercial biostimulant products are currently grouped into five categories: amino acids, fulvic acids, microbial inoculants, humic acids, and seaweed extracts [238–240]. The use of a biostimulant, reinforced with A. flavipes cultured in soybean bran and with various amounts of IAA, was found to be beneficial in the growth of the eucalyptus hybrid E. grandis x E. urophylla (clone IPB2) when applied in both solid and liquid forms [239], in which increased root length and mass were observed, especially in the case of the former. Other Aspergillus species were reported to present with similar effects. It was found that A. ustus promoted shoot and root growth as well as an increase lateral root and root hair numbers in Solanum tuberosum and Arabidopsis thaliana [239, 241]. Similar effects were observed in shoots and roots of Cicer arietinum via inoculation with both Aspergillus niger and Trichoderma harzianum [239, 242]. The natural compounds present in biofertilizers add value to plant natural resistance and general nutritional status [21]. Microbial inoculations may stimulate adventitious root growth, especially in species experiencing difficulties in rooting, which leads to an increase nutrient and water absorption, hostplant biomass, as well as stress tolerance levels [239, 243].

1.5 Conclusion and Future Prospects Biofertilizers can act as viable alternatives to chemicals in enhancing plant growth and are increasingly indispensable in both the agricultural and horticultural field. Besides presenting an important role in improving crop productivity, it is evident, even with the lack of data, that biofertilizers present great potential in the remediation of forests and the environment. However, at present, there is a lack of research in the forest biofertilizer field. No field data have ever been collected for deciduous trees, while little

Biofertilizer Utilization in Forestry  19 field data were recorded for coniferous species. As such, future research in this field may present a gold mine in terms of useful data that can be used to further our understanding of various biotic interactions that occur in any forest. A majority of laboratory and greenhouse experiments have not been successfully transferred to the field. The rhizosphere, complex in nature, is a major consideration in any studies involving biofertilizer and has caused issues in studies of its structure and function, especially when it comes to addressing the molecular mechanisms that drive all biotic interactions and communications within the substrate. This process, however, is currently being facilitated by the use of modern analytical tools such as functional genomics, direct soil DNA extraction and amplification followed by community fingerprinting, reporter gene technology, confocal microscopy, as well as stable isotope profiling. Recent studies are also gearing toward elucidating the influence of such interactions on the above ground communities as a function of an ecosystem-based feedback process. Labeling of inoculants with lux or gfp genes can also be conducted for easy detection and enumeration in the field. Cross-disciplinary collaboration between sciences involving microbes, soil, plants, ecology, physiology, molecular biology, chemistry, and physics addresses the complexity of the rhizosphere and the interactions between all its inhabitants. Vast swathes of areas have not been tested for the presence of potentially potent biofertilizers. As such, efforts should be made to document microbes or plant performance in unexplored or underexplored areas globally for this purpose. Traditional and ethnobotanical knowledge from the indigenous communities should be researched for this purpose. Phytochemical and pharmacological studies of medicinally important forest plant species may reveal active compounds that are synthesized as a result of the interactions between the microbial fauna found in the rhizosphere. New therapeutic agents can then be developed based on the phytochemistry, pharmacognosy, and pharmacology of the plant species. Further optimization is required for parameters related to the application of biofertilizers for maximum output, as some research have displayed incomplete eradication of plant pathogens such as nematodes, or simply a reduction in inocula sizes, after application of the biofertilizer. However, some studies have indicated the presence of isolates resistant to pathogens, i.e., nematode grazing, indicating their potential in supplying important elements under pathogenic attack and hence protect target plants from pathogens. Traits such as these can be explored to further our understanding in the working mechanisms of a biofertilizer. Positive effects on plant developmental activities can be observed with accurate matching

20  Biofertilizers of a biofertilizer with the host plant species to the surrounding environment, which is advantageous in that a natural or an artificially constructed microbe can be used for this purpose. Future research in this area can especially benefit from studies involving synergism and environmental persistence in all components of a biofertilizer in boosting plant development, especially on the creation of an efficient and effective novel biofertilizer delivery systems catering to the traits of the plant and microbial species involved. Outcomes from this research can then be used to supplement in situ reforesting efforts through the use of biofertilizers. Future biofertilizer production strategies may also capitalize on yearround management and application strategies that are large-scaled, cheap, sustainable, and, independent of climate as well as environmental factors. Further field studies involving commercial biofertilizers and delivery mechanisms are required to determine their effectiveness. Sustainable methods of producing biofertilizers can be initiated through the use of agro-industrial waste. Ultimately, factors such as effective strains, inoculum production, host compatibility, appropriate formulations, commercial potential, and technology transfer capacities are integral in the establishment of effective biofertilizers for sustainable forestry.

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2 Impact of Biofertilizers on Horticultural Crops Clement Kiing Fook Wong1* and Chui-Yao Teh2 Department of Agricultural and Food Science, Faculty of Science, Universiti Tunku Abdul Rahman, Jalan Universiti, Perak, Malaysia 2 School of Biological Sciences, Faculty of Science and Technology, Quest International University Perak, Perak, Malaysia 1

Abstract

The ever-increasing global population, climate change, as well as pest and disease outbreak remains as challenges to the horticultural crop production. There is an urgent need to intensify crop production using sustainable methods. Plants are associated with rhizospheric microbes, which have the ability to promote crop growth and stress tolerance, enhance plant nutrition, and improve vegetation propagation. Thus, the formulation and application of biofertilizers containing these beneficial microbes is a promising approach to improve horticultural crops. In this chapter, the impact of applying biofertilizers will be discussed comprehensively which will include the possible mechanisms of biofertilizers in conferring plant growth promoting and stress tolerance traits in crops. This chapter will also look at the possible challenges that will arise from biofertilizer application and recommend solutions to ensure the most efficient use of biofertilizer in the horticulture industry. Keywords:  Abiotic stress, biofertilizer, biotic stress, crop tolerance, horticulture, yield

*Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (39–104) © 2021 Scrivener Publishing LLC

39

40  Biofertilizers

2.1 Introduction The horticulture industry is one of the fastest growing sector to fulfill the increasing demand as the dietary intake from the low- and middle-income countries has begun to shift toward a higher consumption of vegetables and fruits due to increased purchasing power [1]. In addition, the estimated world population coming to 2025 will be nearly 8.5 × 109 which means substantial amount of agricultural produce is needed [2]. Such high demand often requires the exhaustive use of chemical fertilizers and pesticides to boost yield but these practices have caused potential health issues among farmers and consumers, soil pollution and infertility, eutrophication of water sources, pesticide resistance of insects and plant pathogens, as well as compromised food safety and quality [3]. An estimated of demand for nitrogenous fertilizer exceeds 130 million tons per year and the dependence on these resources does not only damage the natural environment but it is also economically infeasible since the production of synthetic N fertilizer depends heavily on the use of fossil fuels [4, 5]. To achieve the goal of sustainable horticulture farming, the use of microbial-based fertilizers or biofertilizers is an alternative that is not only user- and environmental-friendly but also ensures continuous food production under variable environmental conditions [6]. A biofertilizer is defined as a formulated product containing one or a mixture of microorganisms that can improve the nutrient content, growth, and yield by making nutrients available for plants [7]. These beneficial microbes are often developed into dry and liquid formulations to prolong microbial viability under variable conditions and to ensure their efficiency will not be compromised when exposed to biotic and abiotic stresses [8]. Commonly used beneficial microbes, including the plant growth promoting (PGP) microbes and mycorrhizae fungi, are derived from natural resources such as the nutrient-rich root rhizosphere and phyllosphere of the plant. The PGP properties of these microbes are usually characterized by their ability to fix nitrogen, to solubilize phosphate and potassium, to produce plant growth regulators and to biodegrade organic soil matter resulting in enhanced crop growth and yield. Generally, more than 60% to 90% of applied chemical fertilizer is lost through soil-leaching and only 10% to 40% is taken up by plants [5]. Therefore, the application of biofertilizers has a great potential in integrated nutrient management to ensure nutrient use efficiency and to improve nutrient availability for crop growth and yield. Besides improving crop yield, biofertilizers were found to alleviate biotic and abiotic stresses in crops. In the face of climate change, abiotic

Biofertilizers on Horticultural Crops  41 factors including flooding, increased temperature, soil salinity and drought has severely hampered crop production [9]. Similarly, biotic stress such as the invasion of plant pathogens, nematodes, insect pests, and weeds is one of the major contributors to crop loss [10]. Certain beneficial microbes also possess dual function of increasing crop tolerance toward biotic and abiotic stresses and promoting crop growth and yield under stress conditions [11]. This chapter will emphasize on the impact of biofertilizers in promoting growth and yield, enhancing tolerance to biotic and abiotic stress and improving vegetative propagation of horticultural crops. Furthermore, this chapter will cover the future challenges on the application of biofertilizer that horticulturists and researchers should be aware of and also the possible solutions to address these challenges.

2.2 Microbial Strains Used in Biofertilizers Considering the major drawbacks of using chemical fertilizers and pesticides, the use of biofertilizers is an alternative to reduce the harmful effects of these chemicals on crop production, environment, and humans. By harnessing the microbial diversity in the plant root rhizosphere region, various microbial strains were incorporated into formulations that, through their interactions with the inoculated hosts, could benefit plant intake of nutrients and enhance plant tolerance against biotic and abiotic stresses. Important microbial strains typically used in the formulation of biofertilizers are summarized in Table 2.1.

2.3 Impact of Biofertilizer Application on Horticultural Crops 2.3.1 Increased Yield and Quality of Crops One of the obvious impact of applying biofertilizer on horticultural crops is the improvement of yield count and quality of crops. The overall improved crop growth performance begins at the root rhizosphere. The complex interaction between microbial inoculants and the rhizospheric region plays an important role in shaping the plant’s overall ability to uptake nutrient (Figure 2.1). Beneficial microorganisms tend to have a symbiotic relationship with plants whereby they derived most of their carbon sources from the root exudates and in exchange, they make nutrients available for plants to uptake [5].

42  Biofertilizers Table 2.1  Microbial strains used in biofertilizers. Types of biofertilizers Nitrogen-fixing

Group Free-living (non-symbiotic)

Azotobacter, Clostridium, Cyanobacteria (Anabaena), Klebsiella, Rhodopseudomonas, Rhodospirillum, Bacillus

Symbiotic

Rhizobia (Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium), Actinomycete (Frankia)

c

Associative

Azospirillum, Herbaspirillum, Alcaligenes, Bacillus, Pseudomonas, Enterobacter, Klebsiella, Acetobacter

Phosphate solubilizers

Bacillus, Pseudomonas, Penicillum, Aspergillus, Trichoderma, Rhizobium, Burkholderia, Flabobacterium, Pantoea

Phosphate mobilizers

Arbuscular mycorrhiza (Acaulospora, Glomus, Gigaspora, Sclerocystis, Scutellaspora), ectomycorrhizal (Amanita, Boletus, Laccaria, Pisolithus)

Potassium solubilizers

Bacillus, Paenibacillus

Silicate and zinc solubilizers

Bacillus, Thiobacillus, Saccharomyces

a

b

Phosphorus

Micronutrients

Examples of common microbial genera used in biofertilizers

(Continued)

Biofertilizers on Horticultural Crops  43 Table 2.1  Microbial strains used in biofertilizers. (Continued) Types of biofertilizers

Group

Examples of common microbial genera used in biofertilizers

Growth promotion

Plant growth promotion

Alcaligenes, Azotobacter, Actinomycetes, Rhizobium, Bradyrhizobium, Bacillus, Pseudomonas, Enterobacter, Pantoea, Xanthomonas, Flavobacterium

Adapted from Singh et al. (2014). a Free living nitrogen fixers do not form symbiotic interaction with host plants for nitrogen fixation. b Symbiotic nitrogen fixers form symbiotic interaction with host plants for nitrogen fixation. Symbiosis is usually characterized by bacterial nodules formed in plant roots. c Associative nitrogen fixers form symbiotic interaction with host plants from graminaceous plants without the formation of nodules.

Improved crop growth and yield due to: • Increased N levels • Increased soluble mineral ions • Regulation of phytohormones Attract

Attract Root exudates

Root exudates Root Attr ac t exudates Attract

N2 N fixation via root nodulating bacteria NH+

NH+

N fixation via free living and associative N fixing microbes

Insoluble P, K

Soluble P, K ions

Root nodulation



N2

Mineral ion solubilization by PGP microbes and AMF

Production of phytohormones by PGP microbes and AMF

Figure 2.1  Mechanism of crop growth and yield enhancement induced by beneficial microbes present in biofertilizers.

Some microbes such as those from the Trichoderma, Pseudomonas, or Bacillus genus produced organic acids or low molecular weight siderophores to solubilize essential minerals such as phosphorus (P), iron (Fe), and zinc (Zn) for plants to uptake. The Rhizobia genus are symbiotic N fixers that colonize roots of legumes, through the formation nodules, in order to absorb nutrients from the root exudates while providing N sources for the plants [12]. Other microbes including the free-living (Azotobacter, Beijerinckia,

44  Biofertilizers Klebsiella, and Cyanobacteria) or associative N fixers (Azospirillum) are able to fix atmospheric N as plants commonly take up N in the form of nitrate or ammonia ions [13]. Plant hormones are also produced exogenously by microbes that could cause significant changes to the play physiology. Auxins (indole acetic acid, IAA), cytokinins, gibberellins, and abscisic acid are synthesized by microbes and these phytohormones regulate the growth of plants via the stimulation of cell division [3]. Improved root growth is often observed as a result of hormonal-induced enhancement [14]. As there is greater mineral nutrient availability due to microbial solubilization and N fixation, extensive root networks are beneficial for efficient nutrient uptake which translates into better growth and yield in crops [15]. The following sections emphasize on the use of biofertilizer in improving the yield and quality of vegetables, fruits, and ornamental plants.

2.3.1.1 Vegetable Crops The use of native Rhizobium isolates improved the seed yield of climbing beans (Phaseolus vulgaris) by 89% increase (4,397.75 kg/ha over noninoculated control (2,334.81 kg/ha) and 30% increase over commercial Rhizobia-based biofertilizer (3,698.79 kg/ha) [16]. Native strains are adapted to local agro-climatic conditions which allows them to have a compatible interaction with other resident microbial populations resulting in improved soil health, nutrient availability and better yields [17]. In the same study, different agroecosystems showed varied seed yield of 4,691.26 kg/ha at lower midland compared to upper midland at 2,644.05 kg/ha indicating that different soil properties and nutrient levels might affect nodulation of rhizobia [16]. For instance, P deficient soil affects the biological nitrogen fixation (BNF) activity despite high abundance of native rhizobia strains and replenishing the soil with P helps to improve the BNF activity in plant roots [18]. The seed yield of Faba and runner bean was also improved via the application of Rhizobia biofertilizer [19, 20]. The possible mechanism of Rhizobia in improving seed yield of legumes was suggested [21]. The detection of GFP-tagged Azospirillum brasilense in developing seeds demonstrated that Rhizobia strains not only colonize roots and also actively migrate and form an intercellular colonization in the developing seeds. Such colonization behavior could have impacted the number of beans in pods and the pod size [21]. Additional study is therefore needed to unravel the how bacterial seed colonization could improve seed yield in leguminous plants. In an agriculture land rich with native Bradyrhizobia, the seed yield of cowpea was surprisingly lower (1,280 kg/ha) compared to exogenous application of new strains of Bradyrhizobia (1,600 kg/ha) [22]. Feasibility study

Biofertilizers on Horticultural Crops  45 also showed an increase of net returns by $104–163/ha in inoculated plants compared to uninoculated control. Naturally, large indigenous Rhizobia population in soil affects the effectiveness of introduced strains due to competition for nutrients and space but this is not the case in this study. The ineffectiveness of Rhizobia could be due to strain-specific factor in which native strains exhibited poor colonization pattern under unfavorable soil chemical properties when compared to introduced strains [23]. The seed yield of other legumes such as soybean and mung bean was also improved after the application of peat-based formulation of Bradyrhizobium under both N-deficient and supplemented conditions [24]. Other environmental factors such as the type of soil and amount of rain were also found to impact the colonization behavior of beneficial microbes. The use of commercial Rhizotech biofertilizer (consisting of four types of AMF) increased tuber yields of sweet potato from 12.8 to 20.1 t/ha in sandy clay soil compared to sandy loam soil which only showed improvement of yield from 7.6 to 14.9 t/ha during the short rain season. The colonization of AMF was also found to be higher during short rain season due to extended fungal hyphae on the rhizosphere, thereby improving nutrient availability for plant growth [25]. In order to exert the positive effects of biofertilizers on the yield of horticultural crops, environmental factors should be taken into account in future studies and methods to rectify the poor colonization behavior in the rhizosphere region should be included as well. Besides improving yield, the excessive use of fertilizers were also reduced after the application of biofertilizers on some vegetable crops. The yield of mustard and tomato was improved up to 108% and 203%, respectively, after applying Trichoderma-based fertilizers which, in turn, reduce the use of N fertilizer by 50% [26]. In the same way, the application of a consortium of Bacillus and Pantoea shortened the initiation and maturity of curd (compact white head) by up to 20 days, produced quality cauliflowers (enriched with amino acid, micronutrient, and macronutrient) and also reduced the use of chemical fertilizers by 25% [27]. In addition to reducing the dependency on chemical fertilizers, the application of biofertilizers also enhanced the quality of yield in vegetable crops such as broccoli and tomato. By applying individual inoculants of Bacillus, Brevibacillus, and Rhizobium, the macroand micronutrient content in the broccoli was improved [28]. Tomato plants inoculated with the commercial biofertilizers Baikal EM1 containing a consortium of Lactobacillus, Phodopseudomonas, and Saccharomyces produced larger fruit biomass by 14.30 g per fruit and also and improved yield by 19%–21% through soil application and 13%–14% through foliar application [29]. Cabrini et al. [30] also discovered that lettuce inoculated with rhizospheric yeast, Torulaspora globosa produced better yield quality

46  Biofertilizers in terms of size and number of leaves. On the other hand, the inoculation of Bacillus and Pseudomonas did not improve the quality and yield of lettuce when transplanted to the field although these strains improved the biomass of seedlings [31]. The inability of these strains to survive in harsh field environment could be the reason behind of such inconsistency. In another example, unequal size of broccoli was found after the inoculation with Bacillus amyloquefaciens although yield is greatly enhanced [32]. Considering that uniformity in crops strongly influences consumer preferences, the overall quality of the product should be standardized even if the yield is increased. Gange and Gadhave [32] reported that inconsistencies found in the microbial colonization behavior in plants, as a result of incompatibility toward indigenous strains, has led to various sizes of broccoli. Future commercial PGPR products should be tailored to specific crops and soil types so as to achieve consistent yield and quality in crops.

2.3.1.2 Fruit Crops Among fruit crops, the effect of applying biofertilizers on the yield and quality of strawberries is widely studied. Crop maturation days of strawberry plants were reduced to about 2 weeks after treated with Pedobacter and Bacillus strains compared to uninoculated control [33]. Pedobacter sp. specifically improved the quality of strawberry by increasing the fruit length and shape index (sphericity) by 28% and 36%, respectively. Applying Azospirillum and commercial effective microbes (EMs) has improved the average fruit weight of strawberry by 14.3% although there was no significant increase in the fruit number [34]. The berries produced by the Azospirillum treatment showed intense red color compared to other microbial treatments. Regardless of biofertilizer treatment, the overall sweetness index was improved in inoculated plants with a significant decrease in total titratable acidity [34]. When P. fluorescens strain Pf4 was paired with AMFs from the genus of Septoglomus, Funneliformis, and Rhizophagus, the induction of strawberry flowers were increased by 35% resulting in increased number of fruit by 22% and increased fruit weight per plant by 14% to 20% [35]. Nevertheless, when the AMFs were paired with Pseudomonas, the total fruit fresh weight was reduced by up to 17% and the number of flowers decreased tremendously. Incompatibility between strains has compromised colonization ability of AMF on strawberry plants which consequently imparted negative effects on the overall physiology of the strawberry plants [35, 36]. In a similar study, the inoculation of commercial products Rhizocell C containing B. amyloliquefaciens improved the photosynthetic capacity of strawberry plants leading to greater fruit yield and biomass

Biofertilizers on Horticultural Crops  47 in two seasons of planting compared to other commercialized product including MYC 800 (contains Rhizophagus) and Mykoflor (a consortium of AMFs such as Rhizophagus, Funneliformis, and Claroideoglomus) [37]. In other words, selecting biofertilizers with compatible microbial strains is essential in order to improve the yield and quality of strawberry. The yield, quality, and shelf life of mango were also enhanced after applying biofertilizer. Half dosage of inorganic fertilizer coupled with the application of Azotobacter increased yield (57.20 kg/plant) and average fruit weight (285.15 g) compared to control (48 kg/plant and 240.40 g, respectively). The shelf life of the mango fruit was increased from 5 to 10 days [38]. This study was also in congruent with previous studies that have utilized a consortium of AMF (Glosum) and PGPR strains (Azospirillum and Azotobacter to improve the same physiological parameters [39, 40]. The postharvest quality of mango was improved when a commercial biofertilizer, Maya Magic was applied to the fruits during the bagging process. Average fruit weight and penetration resistance were significantly improved compared to control treatment though the microbial strains in the biofertilizer were not stated in the study [41]. Seasonal variation was found to reduce the efficiency of biofertilizer in improving fruit yield. Guava trees treated with Azotobacter and farmyard manure gave better fruit yield during rainy season (38.2 kg/ tree) compared to winter season (19.0 kg/tree) [42]. Seasonal variation such as the low temperature during winter might have induced changes in the population of the introduced strains, thereby causing variation in yield [43]. Improved plant growth are associated with higher quantities of photosynthates such as starch and carbohydrates which are then utilized as energy to produce flowers and fruits [44]. At the same time, the photosynthates will be translocated to the fruits, thereby improving the postharvest quality [44]. The number of papaya fruits was increased to an average of 19.72 per tree after the application of a consortium of Azotobacter, Azospirillum, and versicular-arbuscular mycorrhiza (VAM) [45]. The overall postharvest quality was also improved with higher total soluble sugar, beta-carotene, and minimal acidity. A steady increase of fruit yield in pomegranate from 18 kg/plant to 38 kg/plant within 5 years was observed after continuous application of AMF and Azotobacter. The productivity of the tree was correlated with the enhanced vegetative growth parameters such as plant height and plant canopy [46]. Some PGPR strains have disease suppression properties that enable plants to retain their productivity even under disease infection. Wang et al. [47] observed that the inoculation of a biofertilizer containing B. amyloliquefaciens improved the yield of banana from 32.91 t/ha to 41.39 t/ha under field conditions naturally infested with the fungal disease, Fusarium wilt.

48  Biofertilizers Further transcriptome analysis revealed that bananas inoculated with rhizobacteria such Pseudomonas and Bacillus showed differential expression of genes that corresponded to growth promotion and regulation of specific functions such as flowering, photosynthesis, glucose catabolism, root growth, and plant defense genes against biotic and abiotic stress. Therefore, banana plants can still produce yield even though severe disease infestation [48].

2.3.1.3 Ornamental Plants The application of AMF biofertilizer was also found to have brought significant impact to the floriculture industry. AMF inoculation usually resulted in increased number of flowers, number of flowering plants or early flowering in ornamental plants [36, 49, 50]. However, AMF was also reported to delay flowering onset and reduce number of flowers [51–53]. Such differential response of host plants toward AMF could depend on the competition for nutrients and photosynthates between AMF and flowers, the preference of AMF in colonizing host plants and also, the ability of AMF in overcoming nutrient deficiency or other forms of environmental stress [53, 54]. Hence, the use of AMF in the floriculture industry is heavily dependent on the fertilizer regime, soil fertility, and the species or cultivars that are being produced. The positive effects of biofertilizer containing PGPR, phosphate solubilizing, and nitrogen fixing bacteria were reported to improve the overall growth and flowering characteristics of several ornamental plants such as petunia, geranium, carnation, gladiolus, chrysanthemum, dahlia, and poinsettia [55–58]. Important flowering characteristics such as the onset of flowering was significantly reduced in petunia by up to 20 days when NPK fertilizers was used in combination with phosphate solubilizing and nitrogen fixing bacteria [57]. Saini et al. [59] also successfully reduced the flowering time and increase the flower head size of treasure flower (Gazania rigens) after treated with AMF or Pseudomonas. The vase-life gladiolus floral spike was increased from 11 to 16 days whereas the total number of florets doubled from 8 to 16 when Azotobacter or phosphate solubilizing bacteria were applied [56]. These flowering characteristics are essential to fulfill the industry demands through the production of long-lasting flowers. However, wilting of the basal florets hastened when Azotobacter was applied but the mechanism behind remains unclear [56]. Similarly, poinsettia treated with P. putida showed reduced anthocyanin content which could affect the pigmentation of the bracts [58]. These observations could be due to poor microbial colonization as a result of production of

Biofertilizers on Horticultural Crops  49 antimicrobial metabolites from its host as a defense response. Additional study on the colonization behavior of these beneficial microbes on ornamental plants is therefore needed. Perhaps, future research could also be directed toward the use of biofertilizer in producing ornamental plants that cater to the needs of consumers such as enhanced petal or bract colors and larger flowers. More research should also emphasize on the economic feasibility of using biofertilizer in the floriculture industry so that industrial players could adopt this eco-friendly method to reduce fertilization costs and to fulfill consumers’ demands.

2.3.2 Enhanced Nutritional Content of Produce Besides improving crop yield and quality, the nutritional content of crops could be enhanced through biofortification using biofertilizers. Other biofortification methods such as breeding or transgenic technology are time-consuming, technically demanding and may subject to various government regulatory issues before commercialization takes place [60]. Generally, microbial biofortification of crops can be achieved through various types of mechanism as summarized in Figure 2.2 [61].

2.3.2.1 Mineral-Biofortified Crops Certain microbes are able to solubilize minerals or micronutrients such as iron (Fe) and zinc (Zn) by excreting small molecules (siderophores) or organic acids. In Fe-deficient soil, the accumulation of root phenolics attracts rhizobia strains to promote nodulation in leguminous plants. Increased secondary metabolite production Increased micronutrients content (Fe, Zn)

Increased vitamin C and B content Microbes

Microbes At

Production of • Siderophores • Organic acids • Chelating agents

tra

Fe and Zn complex

ct

Root exudates – organic acids, sugars, amino acids, secondary metabolites

rac Root exudates– Att organic acids, sugars, amino acids, secondary metabolites

t

Synthesis of vitamin B

Soluble cations Readily absorbed by roots

Signaling molecules?

Readily absorbed by roots To induce the production of vitamin C and secondary metabolites

Figure 2.2  Mechanism of biofortification of crops induced by beneficial microbes present in biofertilizers.

50  Biofertilizers These strains produced siderophores under Fe-deficient condition to solubilize Fe which then promotes the binding of Fe to transporter proteins produced from host plants [62]. So far, most studies on legumes and tomato emphasized on the estimation of Fe content in the vegetative parts instead of the beans or fruit in which human consume [63–65]. In fact, the only successful iron biofortified Phaseolus vulgaris beans was developed by HarvestPlus through selection breeding and not microbial biofortification method [66]. Tomato plants treated with Trichoderma biofertilizer showed an increase in Fe content (245.73 mg/kg) in fruits compared to control treatment (195.85 mg/kg) and conventional NPK fertilization (202.94 mg/ kg) [67]. As iron deficiency is still prevalent and it is the greatest contributor to anemia disease, further research on utilizing biofertilizer in producing iron-biofortified horticultural crops is necessary. On the other hand, Zn solubilization could be achieved when bacteria or AMF secrete organic acids to lower soil pH as slightly acidic soil was found to increase Zn bioavailability to plants [68]. The production of chelating agents such ethylenediamine-tetraacetic acid (EDTA) by bacterial strains such as Agrobacterium Ca-18, Azospirillum lipoferum, and Pseudomonas sp. was also able to chelate Zn ions which increased the bioavailability of Zn to plants [69, 70]. Several reports indicated that inoculation of liquid formulation containing Pseudomonas sp. showed accumulation of 26.12% of zinc in chickpea seeds compared to control while AMF inoculation showed an increase of zinc content from 12.61 to 16.09 ppm in tomato fruits [71, 72].

2.3.2.2 Enhanced Secondary Metabolites Flavonoids, polyphenols, anthocyanin, and antioxidants are naturally produced in plants as secondary metabolites and these beneficial compounds are widely studied for their anti-inflammatory, anticancer, anti-aging, and anti-microbial properties [73]. Biofortification of crops through biofertilizer application could be a method for humans to obtain these health supplements without having to purchase costly over-the-counter supplementary pills. Strawberries, which were treated with AMF or PGPRs (such as Azotobacter, Azospirillum, Bacillus, Klebsiella, and Pseudomonas), produced fruits with overall higher flavonoids, polyphenols, anthocyanin, and antioxidants compared to control treatments [35, 74, 75]. Interestingly, Todeschini et al. [35] described that AMF inoculation improved the growth of strawberry, whereas Pseudomonas inoculation improved the yield and anthocyanin content of the fruit under sterile soil condition. Thus, it is possible for future study to choose a potential consortium of beneficial microbes to enhance growth, yield, and secondary metabolite production

Biofertilizers on Horticultural Crops  51 at the same time. In spite of that, more research have to be carried in a natural field environment to validate the observations [35]. Economically important crops such as tomatoes were also enriched with antioxidants (lycopenes), flavonoids, and polyphenols after the application of biofertilizers containing Bacillus or Trichoderma [67, 76, 77]. Similarly, other crops such as spinach and flax also exhibited increased amount of all three metabolites as mentioned after treated with Azotobacter, Bacillus, or AMF [78, 79]. It would be interesting if clinical trials could be performed to ascertain the potential of consuming these nutritionally enhanced crops on a long-term basis.

2.3.2.3 Improved Vitamin Content Vitamins are sought-after targets of biofortification in crops. The transgenic golden rice and orange-fleshed sweet potato loaded with provitamin A are good examples of biofortification via genetic engineering and conventional breeding, respectively [80, 81]. Nonetheless, the tedious government approval of transgenic crops and lengthy breeding process warrants a muchneeded research on the application of biofertilizers in the improvement of vitamin A content in crops. The vitamin B group is another topic of interest lately as deficiency is reported in many poor nations with less diversified diet [82]. In nature, plants could take up vitamins B1 (thiamine) and B12 (cobalamin) from soil but plants could only synthesize minimal amount of B1 and B9 (folate). Attempts to utilize biofertilizer in vitamin B fortification of horticultural crops are extremely limited. Co-inoculation of AMF and Pseudomonas were found to increase vitamin B9 content in strawberries [36]. Microbial-rich organic wastes such as cow dung were utilized to improve the B1 and B12 vitamins content in mung bean sprouts and spinach [83, 84]. Identification of microbes with the ability to synthesize the B vitamins from the cow dung is needed so that these potential microbes could be formulated into biofertilizers for biofortification of vitamin B in crops. Vitamin C are mostly found in fruits. The majority of research discovered that application of biofertilizers usually result in higher vitamin C content in fruits. The utilization of microbes from the genus Phyllobacterium, Paenibacillus, Pseudomonas, and Glomus have successfully increase the vitamin C content in strawberries, lettuce, and tomatoes [36, 85–87]. In particular, the vitamin C content in strawberries treated with Pyllobacterium endophyticum was 79% higher that the uninoculated control. In some cases, the vitamin C content in fruits was reduced after the inoculation of microbes. For instance, the co-inoculation of Pseudomonas and Bacillus reduced the vitamin C level of strawberries [88]. Further

52  Biofertilizers investigation underlying the mechanisms of vitamin C synthesis in plants during plant-microbe interaction is required in order to facilitate the selection of potential microbes in vitamin C fortification.

2.3.3 Improved Tolerance Against Biotic Stress Plants are sessile organisms that are susceptible to different biotic stresses ranging from plant pathogens infection, nematode infestation, weed, and insect pest invasion. Sustainable disease management is desirable compared to conventional chemical methods as it protects both users and the environment from being exposed to hazardous chemicals [89]. Utilizing biofertilizers as a potential means of improving tolerance of plants against biotic stress is gaining popularity among growers due to its positive long-term effect on soil health and crop productivity [90, 91]. Generally, crop tolerance against biotic stress is generally as a result of the interaction between beneficial microbes and host plants which is usually followed by host defense response (Figures 2.3 and 2.4). The following section reviews comprehensively on the use of biofertilizer in improving growth and yield of horticultural crops as well as in building crop tolerance against biotic stress.

2.3.3.1 Fungal and Bacterial Pathogens Generally, crop loss due to plant pathogens amount to 25% of the global crop production annually [92]. By harnessing the microbiome in soil and plants, plant pathogens can be sustainably managed using soil or Foliar application of biofertilizer Improved crop growth and yield due to: • Microbial release of PGP hormones • Enhanced root growth and nutrient uptake • Improved photosynthesis activity

Microbial induced fungal and bacterial pathogens tolerance: • JA increase, SA decrease • Microbial biofilm formation • Microbial Secondary antimicrobial metabolites production • Microbial antibiotic production • Competition for nutrient and space • Shift in microbiome to enhance diversity of beneficial microbes

Viral infection

Fungal infection Nematode invasion Bacterial infection

Soil application of biofertilizer

Microbial-induced viral pathogen tolerance: • SA increase, JA decrease • Microbial interference of viral coat protein and particles assembly. • Degradation of viral coat protein by microbial proteasome

Microbial-induced nematode invasion tolerance: • Competition for space • Root cell wall lignification • Egg masses colonization and feeding by nematophagous fungi • JA increase to suppress nematode invasion. • SA increase when nematodeinduced root gall is detected by microbes.

Figure 2.3  Mechanism of crop tolerance against plant pathogens (bacterial, fungal, and viral pathogens) and nematodes as induced by beneficial microbes present in biofertilizers.

Biofertilizers on Horticultural Crops  53

Attracts

HIPV release

Natural predators Foliar application of biofertilizers

Foliar feeding insect larvae

Microbial-induced insect pests tolerance: • JA increase, SA decrease • ROS scavenging activity decrease • Volatile (HIPV) production after microbial inoculation to attract natural enemies • Protease release to degrade insect • Microbial production of insecticidal toxins • Microbial production of proteinaseinhibitors to interfere insect digestive system.

Microbial control of weeds:: • Microbial HCN production–interfere with host metabolism. • Microbial secondary metabolite production-interfere host metabolism • Microbial ALA production - disruption of chloroplast organelles • Microbial IAA production-inhibit root and shoot growth.

Soil application of biofertilizers Root-feeding insect larvae

Figure 2.4  Mechanism of crop tolerance against insect pests and weeds as induced by beneficial microbes present in biofertilizers.

plant-­derived beneficial microbes, by a method known as biological control (biocontrol), instead of using conventional and harmful chemical methods [93]. Most of these microbes were known to have both PGP and biocontrol properties which has garnered attention from researchers to formulate them into biofertilizers. Several successful instances of applying biofertilizers to sustain the production of horticultural crops even the invasion of fungal and bacterial plant pathogens were highlighted. Pre-treating tomato plants by foliar application of B. methylotrophicus bacterial suspension inhibited the growth of fungal pathogen Botrytis cinerea by up to 60% while improving plant biomass and fruit diameter under glasshouse and field trials [94]. The common bean seeds soaked in B. subtilis suspension also improved the overall vegetative growth and increased disease control of Curtobacterium flaccumfaciens pv. flaccumfaciens from 42% to 76% [95]. The pre-treatment of seed could have induced induced systemic response (ISR) response in the bean seedlings by triggering the jasmonic acid (JA)–dependent defense response as evidenced in the increase of phenylalanine (PAL) activity, phenolics, and lignin accumulation [95, 96]. The application of B. subtilis also improved the germination of mung beans when inoculated with three different Fusarium pathogens–Fusarium verticillioides, F. oyxsporum, and Fusarium sp. [97]. Although B. subtilis were not found to produce chitinase (an important enzyme for degrading fungal chitin cell wall), this strain produced a lipopeptide biosurfactant known as surfactin [97]. Yan et al. [98] described that surfactin helps in biofilm formation that protects microbial cells from harsh environments, improves microbial swarming motility to nutrient

54  Biofertilizers rich rhizosphere, enhances root colonization efficiency, and causes perforation of the pathogens’ membrane resulting in cell electrolyte leakage and, ultimately, cell death. The use of lactic acid bacteria, Lactobacillus plantarum has also successfully reduced the disease severity of three bacterial pathogens–Pseudomonas syringae pv. actinidiae in kiwifruit, Xanthomonas arboricola pv. pruni in Prunus, and Xanthomonas fragrariae in strawberry [99]. Metabolite analysis of the culture filtrate revealed that the presence of bactericidal metabolites such as lactic acid, plantaricin, and organic acids could be involved in disease suppression [100, 101]. However, the involvement of these metabolites produced by lactic acid bacteria in biocontrol of plant pathogens have yet to be elucidated in detail. Fungal biocontrol strains from the Trichoderma genus are also well known for their biocontrol and PGP characteristics. Trichoderma asperellum was reported to improve growth and effectively control the fungal pathogen Macrophomina phaseolina in melon, eggplant, and chickpea [102] and Fusarium solani in beans, chilli, and peanuts [103]. Bacterial wilt of tomato, caused by Ralstonia solanacearum, was reduced to an average of 50% in the first and second year of planting after the inoculation of T. asperellum [104]. Maximum tomato yield of 6.93 t/ha was achieved for the first year but the yield slightly dropped to 5.82 t/ha in the second year which could mean that the biocontrol efficiency was reduced. Rather than applying the biofertilizer once, continuous and long-term application could be more beneficial to sustain the population of biocontrol agents and to maintain the biocontrol efficiency [105]. Enhanced seedling growth and tolerance of tomato seedlings against the fungus Sclerotia sclerotium was achieved by the inoculation of T. harzianum [106]. The production of harzianolide by T. harzianum was discovered to induce expression of genes involved in the salicylic acid (SA) and JA-ethylene (JA/ET) pathways in pretreated tomato seedlings. ISR in tomato seedlings was first initiated with the activation of PAL enzyme activity through JA/ET signaling pathway which is also a key regulator enzyme in the synthesis of SA. Subsequently, the antioxidant activity was triggered via the SA-signaling pathway as a response of the accumulation of reactive oxygen species (ROS) due to oxidative stress induced by the fungal pathogen [106, 107]. Applying multiple mixture of bacterial strains has been reported to exert better biocontrol efficiency compared to the use of a single strain. A mixture of two different strains of B. velezensis improved plant growth and reduced disease severity of Xanthomonas axonopodis pv. vesicatoria on tomato, Pseudomonas syringae pv. tomato on tomato, Rhizoctonia solani on pepper, and Phytium ultimum on cucumber [108]. The synergistic production of antimicrobial metabolites or enzymes by each strains in the mixture might

Biofertilizers on Horticultural Crops  55 have resulted in the synergistic effect on disease suppression. Based on previous study conducted [109], individual B. velenzensis strains produced specific antimicrobial metabolite such as haloduracin α and bacillomycin, respectively, worked synergistically in disease suppression. Istifadah et al. [110] also used a combination of Lysinibacillus, Bacillus, Azotobacter, and Pseudomonas to decrease the disease incidence of Ralstonia solanacearum wilt in chilli plants by up to 80% and plant growth promotion was also observed in this study. The application of a consortium of P. aeruginosa and T. harzianum formulated into pesta granules has also reduced disease severity of Fusarium wilt in banana by 66.67% compared to conventional benomyl fungicide (37.50%) besides promoting the vegetation growth of banana plants [89]. Changes in the soil microbiome after introduction of new microbial strains also play an essential role in disease suppression and plant growth promotion. B. amyloliquefaciens, formulated with pig manure and neem cake as carriers, increased banana yield, and reduced disease incidences of Fusarium wilt while at the same time, stabilized the overall bacterial metabolic potential in carbohydrate, carboxylic, acid and phenolics metabolism in soil [105]. Microbial degradation of these compounds in soil could lead to lower disease incidence since they could serve as allelopathic agents against plant pathogens [111]. The long-term application of B. amyloliquefaciens has also caused higher richness and diversity of soil culturable rhizobacteria in which a healthy and stable soil microbial population is necessary to maintain continuous cropping and sustainable high crop productivity [112]. In a follow up study, the use of the same strain has also improved the rhizospheric soil bacterial population and improved disease suppression for over 3 years of field trial through bacterial taxa specific suppression mechanisms, which has yet to be fully understood at the current state [91]. A consortium of B. cereus and B. subtilis were also proposed to influence the soil microbiome of pepper plants. This has caused the degradation of organic and inorganic materials in soil which, in turn, provide nutrients for both biocontrol microbes and plants to thrive [90, 113]. The Bacillus microbial mixture applied together with mushroom composts significantly enhanced the yield and postharvest quality of pepper fruit and the control efficacy against Ralstonia wilt and Phytophthora blight in naturally infested field was 76% and 81%, respectively [113]. Furthermore, the ability of Bacillus strains in solubilizing mineral nutrients into freely available potassium (K) and phosphate (P) has helped to retain the soil fertility after one growing season [113]. In a recent study, two bacterial strains B. velenzis and P. fluorescens drastically reduced the wilt disease of tomato by reducing R. solanacearum

56  Biofertilizers population while promoting crop growth [114]. Shifts in the soil microbiome were also observed whereby the enriched Actinobacteria population in soil was suggested to be involved in the suppression of Ralstonia wilt [115]. Elsayed et al. [114] also utilized confocal laser microscopy to show that these strains actively colonized the roots and within xylem vessels. Efficient root colonizers would often compete with plant pathogens resulting in unsuccessful establishment of crop diseases [116]. In addition, the genome sequencing of the B. velenzis and P. fluorescens has identified antibiotic biosynthesis genes that produced diacetyl-phlorolglucinol (DAPG) and phenazine which have been widely reported for their broad spectrum antimicrobial activity against plant pathogens [114]. To put it simply, the interaction of beneficial microbes, host plants and plant pathogens is complex and dynamic. Such interaction is often interconnected in order to achieve the biocontrol and PGP effects desired for the development of sustainable disease management.

2.3.3.2 Viral Pathogens Viruses are obligate parasites that infect most cultivated crops with at least 450 different species and most of them are RNA viruses [117]. An estimated of 40% of total crop losses are due to viral infestation [118]. Plant viruses are commonly transmitted through insect vectors and the current method is to use insecticides which could cause long-term hazards to the environment and human health [119]. The increasing costs of pesticides and consumers’ demand for pesticide-free food have resulted in the replacement of chemicals with sustainable alternative such as the use of biofertilizers [120]. The use of PGP microbes contained in biofertilizers has been successful in managing plant viruses as well as in improving the vegetative growth and yield of various horticultural crops. Tissue-cultured bananas bacterized with Pseudomonas and Bacillus under in vitro condition reduced banana bunchy top virus (BBTV) by up to 60% after transplantation [121]. Foliar spray of tissue-cultured bananas with the same bacterial strains during the hardening and acclimatization stage also reduced BBTV infection with pronounced accumulation of defense related enzymes and pathogenesis-related (PR) proteins which suggested the priming effect of these strains in activating the host defense response against viral infection [122, 123]. Under glasshouse and field conditions, a biofertilizer containing the same microbial strains reduced BBTV infection at 80% and 52%, respectively, and also, reduced the virus titer and increased the yield by 53.33% [124]. The high population of the PGP microbes in the rhizosphere region throughout the growing period

Biofertilizers on Horticultural Crops  57 could have triggered a cascade of host defense response and improved the growth of bananas by promoting nutrient uptake [124]. Other similar studies also demonstrated that banana plants inoculated with a single or a consortium of Pseudomonas, Rhizobacteria, and Bacillus also increased tolerance against BBTV, yield and postharvest quality of banana under field conditions [125–127]. Mild cucumber mosaic virus (CMV) was found to control virulent CMV infecting tomato plants but causes side effects such as mild stunting, vigor reduction and 20% yield loss [128]. Hence, a combined application of Pseudomonas, Stenotrophomonas, Azospirillum, or Anabena with mild CMV reduced the replication of virulent CMV with decreased disease severity up to 91.3% [128] while fruit yield of tomato was also improved by 48% and 40% in glasshouse and field conditions [129]. Beris et al. [130] concluded that triple soil drench, foliar spray, and seed imbibition of tomato with B. amyloliquefaciens reduced the severity of tomato spotted wilt virus (TSWV) from 50% to 80%. Moreover, the accumulation of potato virus Y (PVY) in tomato was also reduced as there was delayed detection in the apical leaves. SA-induced PR proteins were upregulated in TSWV and PVY infected tomato plants after inoculation with B. amyloliquefaciens whereas the expression of JA-induced genes were not significant [30]. Plant defense against viruses is primarily based on SA and secondarily on JA signaling. SA is crucial for systemic resistance through the activation of mitogen activated kinase cascade that leads to the upregulation of NPR1 gene which, in turn, triggers the transcription of PR genes and RNA silencing antiviral mechanism [131]. Foliar spray with phyllosphere-derived bacteria, B. amyloliquefaciens prior to virus infection, reduced the relative contents of CMV coat protein RNA over a 3-year field trial. Viral tolerance of pepper plants was also associated to the upregulation of PR genes indicating that SA defense signaling was induced [132]. Naturally occurring virus such as broad bean wilt virus and pepper mottle virus was reduced [132]. The seed transmission rates of cucumber green mottle mosaic virus (CGMMV) and pepper mild mottle virus (PMMoV) in pepper and watermelon seeds were reduced after the application of P. oleovorans [133]. The subcellular localization of movement protein (MP) of PMMoV was also abolished since the culture filtrate of P. oleovorans remodeled the aggregation of the viral protein necessary for seed-to-seed transmission [133]. The study also proposed that the use of PGP microbes could result in several antiviral mechanisms including the interference of the production and translation of subgenomic RNA (sgRNA) in encoding the viral coat protein (CP), affecting the assembly and disassembly of viral particles and the degradation of

58  Biofertilizers free CP by microbial proteasome [133]. In one study, cucumber plants pretreated with Stenotrophomonas maltophilia delayed virus replication for more than 3 days and repressed the viral protein genes (CP, MP, and Rep) expression in cucumber leaves [134]. Maksimov et al. [118] described that most PGP microbes displayed several other antiviral mechanisms including the production of extracellular RNases that disintegrate virus particles, the synthesis of bacterial barnases that possess antiviral activity, and the production of microbial surfactants that trigger the SA defense signaling pathway during viral infection. Till now, these proposed antiviral mechanisms induced by PGP microbes have yet to be fully elucidated and such information would help in developing biofertilizers that contain desirable antiviral properties in future.

2.3.3.3 Insect Pests Annually, the total economic losses in agriculture caused by insect pests reached US$17.7 billion [135]. The use of insecticides is still widely adopted because of their effectiveness. However, the detrimental effects of insecticides on users, environment, natural predators, and the rise of resistant insects have prompted the use of beneficial microbes with insecticidal properties to manage pests in a sustainable manner [136]. The application of biofertilizers containing individual or a consortium of microbial strains, which possess dual functions of promoting crop growth and enhancing crop tolerance against various insect infestation, has also begun to receive attention [137]. Cucumber seeds imbibed in bacterial suspension of P. fluorescens strain PF169 showed increased yield of cucumber by 58%, promoted early maturation of plant by reducing the flowering time and most importantly, reduced the population growth rate of aphids by delaying on the reproduction rate of female cotton aphids, Aphis gossypii [138]. Imbibition of tomato seeds in P. putida also decreased the infestation of cotton leafworm (Spodoptera litura) and increased the plant biomass as well as yield by 60% and 40%, respectively [139]. Biochemical analysis of the tomato plants revealed that antioxidant activity was enhanced and protease activity was promoted which might indicate the accumulation of proteinase inhibitors that are detrimental against the larvae of S. litura during leaf feeding [139]. Biofertilizers containing a mixture of Glomus, Rhizobacterium, and Pseudomonas significantly increased the biomass and yield of Faba beans and reduced the aphid (Aphis fabae) population by 71.3% compared to inoculation of single microbial strain (64.0%) [140]. On the contrary, the inoculation of both Glomus mosseae and G. fasciculatum did not reduced

Biofertilizers on Horticultural Crops  59 the larval survival of the root-feeding black vine weevil Otiorynchus sulcatus resulting in poor strawberry plant performance such as reduced plant, root biomass and runner production [141]. Colonization with individual strains could otherwise revert this negative effect by preventing about 88% of eggs from developing into full grown larvae. Gadhave et al. [142] explained that incompatibility and competition between different strains could have led to poor root colonization and eventually reduced the host defense response against insect infestation. In order to induce the host defense response against silverleaf whitefly (Bemisia tabaci), the colonization of B. subtilis strain BsDN of tomato plants triggered a long-term ISR via JA signaling process [143]. The resistance response is a combination of JA-dependent and JA-independent defense pathways. In JA-dependent pathways, host anti-nutritive proteins such as proteases and proteinase inhibitors were produced during insect feeding while the SA-signaling is suppressed to increase host tolerance. Proteinase inhibitors act to block insect midgut proteinases, thus imprairng protein digestion which delays the release of peptides and amino acids from dietary protein. This leads to weak and stunted growth and eventually death [144]. In the same study, mutant tomato plants, which were unable to produce JA, were used to delineate if plants could achieve similar level of tolerance against B. tabaci. Interestingly, genes involved in photosynthesis, phenylpropanoid and terpenoid biosynthetic pathways as well as a Hsp90 chaperonin were upregulated which possibly mediated pest resistance response while also down-regulated pathogenensis and hypersensitivity response in tomato plants [143]. More studies are therefore required to understand JA-independent pathway in a tripartite relationship of plant, PGP microbes and insect pests. All three different strains of Bacillus efficiently suppressed the infestation of cabbage aphid (Brevicoryne brassicae) infestation and increased the population and parasitism of braconid endoparasitoid (Diaeretiella rapae) but the bacterial inoculation did not significantly affect the growth of broccoli [145]. Pare et al. [146] suggested that bacterial volatiles might have facilitated plant cellular defences and thus primed plants against the cabbage aphids. D’Alessandro et al. [147] suggested that rhizobacteria increases herbivore-induced plant volatiles (HIPV) production from plants that could attract and trigger natural enemy responses against herbivorous insects. The application of AMF Glomus mosseae together with the predatory mite Pytoseiulus persimilis reduced the population of two-spotted spider mites (Tetranychus urticae) in common bean plants [148, 149]. It was also suggested that AMF colonized roots may change plant aboveground attributes such as biomass and the production of HIPV compounds that might have

60  Biofertilizers attracted predators and parasitoids [151]. The major HIPV compounds produced by AMF-colonized plants was later discovered to consist of β-ocimene and β-caryophyllene and terpenoids [151, 152]. Moreover, the predatory mites were also specifically attracted to AMF-colonized bean plants by sensing prey-related cues such as egg masses produced by spider mite and webbing bearing faecal pellets [150]. Soil amendment with two Bacillus strains did not reduce aphid infestations of pepper in two field seasons but there were reduced population of green peach aphids (Myzus persicae) colonizing plants compared to control treatments [153]. Though so, the fruit weight and number of pepper fruits was higher than control plants consecutively for two harvests. In other words, plants grown in the presence of Bacillus tolerated the damages caused by aphids without a reduction in yield. Perhaps, the utilization of PGP microbes can be combined with natural enemies to control aphid populations and to conserve naturally occurring biocontrol predators in field. Soil amendment with PGPR formulation containing Paenibacillus macerans and B. amyloliquefaciens did not reduce aphid (M. persicae) densities in tomato plants within two years of field study. The yield of tomato was 1.7- to 2.3-fold greater in PGPR treatments than in untreated plots for the first harvest but in the subsequent years, no significant improvement in yield was observed [154]. Tomato plants inoculated with P. fluorescens strain WCS417r increased the susceptibility toward B. tabaci making the PGPR strain unsuitable for insect pest management [155]. The ineffectiveness of some strain of PGPR could be due to the ability of P. fluorescens to reduce the JA signaling pathway needed for ISR and improved plant nutrition and quality which leads to greater infestation [156, 157]. Megali et al. [158] also reported that the use of biofertilizers containing a consortium of lactic acid bacteria, phototrophic bacteria and actinomycetes improved the yield of tomato but failed to suppress the population rate of African cotton leafworm (S. littoralis) with a possible reason that the PGPR suppressed the insecticidal glycoalkaloid molecule tomatine production and JA-dependent defense pathway. As an alternative, fungal entomopathogens were found to have PGP and insecticidal traits. A consortium of compatible Beauveria bassiana and Metarhizium brunneum enhanced the growth of sweet pepper without affecting the population of aphid endoparasitoids Aphidius colemani [159]. The population of green peach aphid, M. persicae was greatly reduced by prolonging the larvae development time, delaying the onset of reproduction and decreasing the egg production by female aphids. The entomopathogenic fungi are known to be endophytes that colonize roots and

Biofertilizers on Horticultural Crops  61 increase root hairs during germination. The fungus produces an adhesion gene (MAD2) that contributes to plant adhesion whereas the other MAD1 gene functions for the fungi to attach to insect cuticles [160]. These beneficial fungi are also able to transfer nitrogen (N) to plants once they have obtained N by digesting the insect tissue. In return, the carbon derived from plant photosynthesis is moved to the fungus for nutrition and growth [137]. To exert their insecticidal activity, these fungi attach on the insect cuticle, release proteases to degrade the cuticle for penetration, form blastospores to absorb nutrients in the insect hemocoel, and produce insecticidal toxins such as beavericin causing insect death within a few days [137].

2.3.3.4 Nematodes Plant-parasitc nematodes (PPNs) including root-knot nematodes and cyst nematodes are causing global crop loss of more than US$157 billion per year [161]. These nematodes not only damage plant roots but also, they facilitate infections from plant pathogens such as fungi, bacteria and viruses. Chemical nematicides are commonly used, but as concern for environmental problems and human health increase, biological methods using nematophagous microbes have attracted attention of researchers and growers. There are numerous successful instances of nematode management using biofertilizers containing beneficial microbes. PGP bacterial strains B.  penetrans reduced the Meloidogyne igconita infestation to about 80% under field condition followed by enhanced plant biomass and postharvest quality of sugar beet [162]. The utilization of B. velezensis alone consistently decreased the incidence of Heterodera glycines (soybean cyst nematodes) in glasshouse, microplot, and field trials, whereas a combination of the same strain with B. altitudinis and abamectin (anthelmintic pesticide) increased early plant growth in microplot trials and also, enhanced soybean yield in field trials [163]. Seed treatment and soil application of B. subtilis together with vermicompost recorded the highest carrot yield by up to 28.85 and a drastic decrease of M. incognita population of up to 69.3% [164]. The application of Paenibacillus and Bacillus strains has also reduced the number of galls and egg mass of M. incognita and improved the plant biomass as well as nutrient uptake of tomato plants [165, 166]. Crop rotation of tomato with maize and the inclusion of a nematophagous fungi, Ponchonia chlamydosporia increased yield of tomato by up to 63% in the first season but dropped slightly to 41.67% on the second season within a year [167]. The reduction of yield could be due rainy seasons and warm

62  Biofertilizers weather conditions that result in drop of flowers and constant wet leaves causing other pathogens to thrive under such condition [168]. Weather changes might also cause the sporulation of P. chlamnydosporia to decrease, thereby reducing the nematicide activity [167]. In other words, the study could be extended to a few seasons to better understand the duration of protective effect exerted by biocontrol agents. Increased root branching in banana plants as a result of AMF Glomus sp. inoculation reduced the root infection by migratory endoparasitic nematodes, Radophilus similis and Pratylenchus coffeae [169, 170]. The suppressive effect of AMF could be due to competition for space since AMF symbiosis promotes increased root branching in which a denser and extensive root system is less favorable for nematodes because their infection sites are commonly on primary roots [171]. The application of another AMF Rhizophagus irregularis also showed efficient root colonization and reduced root necrosis caused by R. similis by up to 56% which suggested that AMF competes with nematode for space during root colonization [172]. Vos et al. [173] proposed that nematode suppressing effect of Glomus sp. against juvenile R. similis in tomato plants was because of the lignification of root cell and a water soluble compound from the mycorrhizal root extract. No further identification and characterization of that compound was conducted. Coffee plants inoculated with Glomus also indicated lignification of cell wall, thereby impeding the penetration of M. exigua into plant roots [174]. Vos et al. [175] found that the priming effect of Glomus in banana plants primarily triggered the phenylpropanoid pathway which is responsible for production of lignins resulting in enhanced banana defense against M. incognita infection. Nematophagous fungi such as Pasteuria penetrans and Paecilomyces lilacinus were equally effective in causing maximum reduction in M. incognita infection of okra which leads to the increase in plant growth [176]. These fungi function to reduce root penetration by enveloping the nematode juveniles with endospores, to interfere with the development of reproductive systems in female nematodes causing failure in forming egg masses and to extend their hyphal in the eggs for colonization which ultimately leads to egg rupture [177]. A peptidase S8 superfamily protein known as Sep1, which has serine protease activity found in B. firmus, exhibited the potential of degrading the intestinal tissues of nematodes [178]. In contrast, the PGP P. fluorescens produced the antibiotic DAPG that provides moderate protection against M. incognita at 41% but not against M. arenaria, P. minor, or H. glycines [179]. The production of DAPG was reported to be not effective against migratory ectoparasites such as P. minor whereas host

Biofertilizers on Horticultural Crops  63 ISR was found to be effective in the management of sedentary (Meloidogyne sp.) and migratory endoparasites (Radophilus sp. or Pratylenchus sp.) [180]. Therefore, nematode suppressing activity could be microbial strain specific. Combining decomposed organic materials as soil amendments with biocontrol agents is also one method to enhance nematode control. Luambano et al. [167] recommended that carbon to nitrogen (C:N) in organic materials should be moderate as higher C:N ratio could release toxic phenols that reduce the colonization degree of nematophagous fungi such as P. chlamydosporia. The organic materials were also decomposed for 30 days so that immediate nutrient is provided for P. chlamydosporia to proliferate and parasitize nematodes [167]. Suppressing nematode infestation in horticultural crops is also largely dependent on host SA and JA signaling pathway induced by PGP microbes. Martínez-medina et al. [181] explained in detail the regulation of SA/JA systemic defense response induced by Trichoderma during the invasion of M. incognita in tomato plants. During the invasion stage, Trichoderma boosts SA-dependent defences in roots which enhance host resistance against nematode attack. During the feeding stage, Trichoderma interferes the suppression of JA signaling by nematode for invasion by increasing host JA levels and thus, reducing nematode development and reproduction. If nematode parasitim is established, Trichoderma then boosts the activation of SA-dependent defense which is activated in the gall which then serves as a defence against invasion by new juveniles. de Medeiros et al. [182] confirmed that defense response of tomato against M. javanica to be systemic whereby the first generation of Trichoderma-primed tomato inherited PGP and nematode resistant traits. Additionally, Trichoderma-primed tomato plants also triggered SA-dependent defense response in the early inoculation stage as evidenced by the increase of NAPDH oxidase activity (involves in ROS production due to hypersensitivity response) and flavonoid biosynthesis. As nematodes utilize ROS depletion and suppresses flavonoid production in hosts, inoculation of Trichoderma could be beneficial to crops in order to block nematode root penetration by boosting the SA-signaling pathway. In a recent study, the use of biofertilizers containing a consortium of Glomus, Trichoderma, Pochonia, Agrobacterium, Bacillus, Streptomyces, and Pichia might not be efficient in protecting plants from nematode infestation in the long run since antioxidants activity was upregulated which could rapidly deplete ROS in host and favors nematode infection [183]. Perhaps, future research should look into the effect of a single and a consortium of microbial strains in the SA/JA signaling pathways and whether these strains could provide long-term protective effect against nematodes.

64  Biofertilizers

2.3.3.5 Weeds Weeds are a major problem in the agricultural system as they are associated with reduction of about 37% of crop yield [184]. The common weed management involves herbicide treatment which have caused many health and environmental hazards while at the same time, producing herbicide resistant weed biotypes [185]. Biological control of weeds using rhizospheric microorganisms is a promising alternative for reducing chemical usage. Bacterial or fungal strains isolated from the rhizospheric region were found to be effective in not only reducing common weeds found in the horticultural setting but also retain or increase crop yield [186]. Therefore, the following sections emphasizes on some bacterial and fungal strains that have been utilized in suppressing weed growth and/or in improving or maintaining the yield of some horticultural crops. P. aeruginosa strain KC1 reduced the biomass of two weeds, spiny amaranth, and common purslanes under glasshouse condition [187]. This strain produced hydrogen cyanide (HCN) that inhibits root growth. The presence of cyanide causes the formation of metal complexes with functional groups of plant enzymes involved in several major metabolic processes such as respiration, nitrate, and carbon dioxide assimilation as well as carbohydrate metabolism. Cyanide also interacts with a specific protein plastocyanin which could inhibit the electron transport during photosynthesis [188]. P. fluorescens strain WSM3455 also produced HCN that reduced the biomass of wild radish by 53.2%. The application of this strain did not pose any significant negative effect on the grapevine plants [189]. Another P. fluorescens strain G2-11 also suppressed the growth of green foxtail weeds by more than 77.0% and barnyard grass by up to 62.0% while enhancing the growth of soybean under field conditions. Growth reduction of soybean was only reported to be 6.5% for root and 1.2% for shoot [190]. Two species of broomrape weeds were also suppressed by P. fluorescens strain G2-11 as observed by the reduction of shoot emergence from seeds by 64% and 76%, respectively. The same strain also decreases the flowering time of Faba bean by up to 11 days and the number of flowers also increased by five-fold which has proven the dual function of this particular strain [191]. Certain chemical compounds are released by microbes to inhibit weed growth. For instance, P. fluorescens strain WH6 produced a compound known as Germination Arrest Factor (GAF) that arrested germination of seeds from grassy weeds after the emergence of plumule and coleorhiza [192]. The compound was later identified chemically as 4-formylaminooxyL-vinylgylcine and it was shown to interfere with enzymes that utilize

Biofertilizers on Horticultural Crops  65 pyridoxal phosphate as a cofactor which include enzymes associated with nitrogen metabolism and ethylene biosynthesis [193]. However, some grass species planted for seed and food were reported to be sensitive to GAF compared to dicot species [192]. Instead of applying to graminaceous crops, this microbial strain could be applied to dicots to manage the emergence of grassy weeds. P. fluorescens strain BRG100 formulated into pesta granules also suppressed the activity of green foxtail weed through the production of two herbicidal compounds known as pseudophomin A and B [194, 195]. The biosynthesis pathway of the compounds and their mechanism of growth inhibition of weed remain unknown. The biosynthesis of 5-aminoleveulinic acid (ALA) produced by B. flexus strain JMM24 improved the biomass of Indian mustard and decreased the plant biomass of the yellow vetchling weed [196]. ALA, in high concentrations, was found to cause disruption and disintegration of important cellular organelles such as mitochondria, thylakoid, and chloroplast and also reducing cellular antioxidant activity leading to cell death [197]. High concentration of IAA at 64 mM produced by Bradyrhizobium japonicum strain GD3 also led to inhibition of root growth of morning glory seedlings [198]. Transmission electron microscopy (TEM) revealed that damaged root cells were due to vesiculation, cytoplasm disorganization, and cell wall degradation [198]. IAA, at toxic concentrations, could result in the inhibition of root and shoot growth, decreased elongation of internodes and leaf growth, intensified green pigmentation on leaves, stomatal closure, and increase of ROS accumulation [199]. A phytotoxic compound known as viridiol was produced by Trichoderma virens which inhibited the growth of broadleaf and grassy weeds without causing significant loss of crop vigor and yield of vegetables such as pumpkins and tomatoes [200]. However, the exact mechanism of suppression of viridiol against invasive weed has yet to be elucidated.

2.3.4 Improved Tolerance Against Abiotic Stress Due to climate change, agricultural land is facing threats arising from temperature changes, salinization of land, inconsistent rainfall, and heavymetal contaminated soil [201]. Consequently, crops suffer from these unfavorable abiotic stresses such as drought, salinity, heavy metal toxicity, cold, and heat stress that could severely plant growth and development and ultimately reduce yield to about 70% [202]. The use of microbes in the form of biofertilizer have shown promising results in helping horticultural crops to gain an upper hand against abiotic stress via various mechanisms

66  Biofertilizers Microbial-induced plant growth and yield under abiotic stress: • Improved nutrient uptake • Regulation of growth-related phytohormones (GA) • Improved photosynthetic activity • Increased plant biomass (root and shoot) • Enhanced yield under stress Heavy metal (HM) stress

+

Pb +

Cd

Zn+ +

As

Microbial-induced HM tolerance: • Detoxification of HM. • Biosorption and bioaccumulation of HM • Methylation of HM • Chelation of HM.

Heat stress

Cu2+

Microbial-induced heat tolerance: • Regulation of stress-related phytohormones (ABA)

Cold stress

Microbial-induced cold tolerance: • Thickening of xylem cell wall • Enhanced antioxidant activity • Upregulation of cold acclimation and stress-responsive genes • Accumulation of secondary metabolites +

Na

Cl-

Na+

Ni+

ClSalinity stress

Application of microbes for stress alleviation

Drought stress

Microbial-induced salinity and drought tolerance: • Enhanced antioxidant activity • Osmolytes accumulation • Regulation of stress-related phytohormones (ABA, SA, JA) • Regulation of stress-responsive genes • Production of biofilm as host barrier • Production of ACC-deaminase

Figure 2.5  Mechanism of crop tolerance against abiotic stress as induced by beneficial microbes present in biofertilizers.

(Figure 2.5). The following sections described the potential use of various PGP microbes in enhancing abiotic stress tolerance while at the same time, improving overall vegetative growth and crop yield.

2.3.4.1 Drought Drought is a water deficit environmental condition that affects agricultural productivity across the globe and it is linked to global warming. Insufficient water reduces crop growth and yield which is detrimental to the global food security, particularly in the developing world [203]. Drought is expected to hamper crop production for more than 50% of the arable farming lands by the year 2050 [204, 205]. Currently, beneficial microbes with PGP and drought tolerant traits are exploited for their ability to improve growth and yield of horticultural crops under water deficit condition. As these microbes are abundantly found in different soil conditions including the dry and arid regions, apply them as biofertilizers could a promising alternative to help crops cope with drought stress [203]. Under polyethylene glycol induced drought stress, seed germination of chickpea was improved after seed bacterization with P. putida [206]. P. putida induced drought tolerance in chickpea was characterized by improved root growth, increased number of nodules, and enhanced antioxidant activity to relieve oxidative stress [206]. The endophytic fungus Phoma glomerata and Penicillum sp. also produced phytohormones such as gibberellic acid (GA) and IAA that functioned to enhance cucumber

Biofertilizers on Horticultural Crops  67 growth in both shoot and root under drought condition while also, improve nutrient uptake of essential nutrients such as potassium, calcium, and magnesium [207]. Cucumber plants inoculated with a consortium of Bacillus and Serratia showed darker green leaves and lesser wilt symptoms since the rubisco enzymatic activity involved in photosynthesis was maintained after 13 days of withholding water [208]. The negative impact of ethylene accumulation as a result drought stress in pea and potato plants was alleviated by microbial 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase produced by Variovorax, Achromobacter, and Pseudomonas which, in turn, produced plants with better plant vigor and root growth as compared to uninoculated plants [209, 210]. Bacterization of grapevine plantlets by B. licheniformis and P. fluorescens exhibited an increase in abscisic acid (ABA) levels by 76-fold and 40-fold, respectively, compared to un-inoculated control under drought stress [211]. Higher levels of ABA during drought are commonly observed which lead to stomato closure, thereby reducing transpiration rate [212]. After 20 to 30 days of post bacterization, the production of secondary metabolite such as terpenes increased in grapevines. Terpenes are suggested to have antioxidant properties that sequestrate free radicals and protective role in cellular membrane integrity [213, 214]. Nonetheless, more study has to be conducted to understand the role of terpenes in abiotic stress alleviation using molecular tools. Drought tolerance in ornamental plants such as lavender was also improved after the application of B. thuringiensis in arid environment [215]. The potassium, K, content was significantly improved in lavender in which the K+ ion is an important osmolyte that regulate water homeostasis, stomatal opening, osmotic potential, and transpiration under drought stress [215]. The phytohormone, cytokinin (CK), also plays an important role in drought stress tolerance. During drought stress, the endogenous cytokinin levels in plants decrease to favor root growth and promote stomatal closure to limit the transpiration process [216]. The use of CK-producing microbes could replace the loss of endogenous CK in plants, thereby improving drought tolerance. Improved CK levels in lentil during drought due to inoculation of CK-producing Methylobacterium stimulated early growth of shoots and roots, increased photosynthetic rates and improved harvest by at least 4-fold [217]. In a similar study, lettuce seedlings treated with CK-producing Bacillus increased the shoot biomass by 50% under water deficit condition [218]. The ornamental Oriental Thuja plants also displayed higher levels of CK after inoculation of B. subtilis with the leaves of seedlings showing higher relative water content under drought condition [219].

68  Biofertilizers The production of microbial extracellular polymeric substances (EPS) is an essential trait that allows bacterial to form biofilm as a protective barrier against drying and fluctuations in water potential. Sadhya et al. [220] described that the application of P. putida improved the drought tolerance of sunflower plants because the microbe produced EPS that aggregates soil around the root which has improved plants’ ability to uptake water and nutrients from soil. With the advent of high-throughput sequencing, the whole genome sequence of rhizobacterial strains has made discovery of genes related to symbiotic establishment, nitrogen fixation, and drought tolerance possible [221]. These two strains (Rhizobium sp. and Rhizobium cellulosilyticum) has successfully improved seed germination of soybean under drought condition imposed by 4% PEG [221]. The sequence information can be further exploited in future to better understand the interaction between microbe and host under drought condition so that agricultural production could be extended to semi-arid and arid regions.

2.3.4.2 Salinity About 50% of a total of 5.2 billion hectare of agriculture land is affected by salinity [222]. Salinity or salt stress is caused by sodium chloride (NaCl) which is the major constituent and the Na+ and Cl− ions are toxic for plants at high concentrations. Reclamation of saline soil using conventional methods such as scraping, leaching, flushing, or amending with additives (gypsum or CaCl2) showed limited success. This calls for sustainable methods including the use of salt-tolerant microbes in enhancing the tolerance of horticultural crops against salt stress [223]. PGP microbes utilize a variety of mechanisms to ameliorate salinity stress in crop plants including the production of an array of phytohormones such as IAA, gibberellins, and cytokinins, synthesizing of ACC deaminase to reduce the deleterious effect of ethylene due to oxidative stress, production of exopolysaccharides (EPS) or biofilm as a barrier for host plants, regulation of host cellular sodium and potassium levels to achieve homeostasis in osmotic potential, regulation of stress related genes, accumulation of osmolytes (proline) to regulate cellular osmotic potential and activation of antioxidant systems to scavenge ROS during salinity stress [224, 225]. Pantoea dispersa not only improved salt stress tolerance but also yield of chickpea by improving biomass (32%–34%), pod number (31%–34.%), pods weight (30%–32.6%), and seeds weight (27%–35%) [226]. The inoculation of B. subtilis also decreased ROS concentrations while increasing proline content and improved uptake of essential mineral nutrients

Biofertilizers on Horticultural Crops  69 (N, P, K, and Mg) in chickpea under field salinity stress [227]. The production of EPS by salinity tolerant Halomonas variabilis and Planococcus rifietoensis facilitated the formation of biofilm in chickpea roots that served as a barrier to retain moisture for root growth and to aggregate soil to the roots so that plants can continuously uptake water and nutrient during salt stress [228]. Pea plants inoculated with the same bacterial strain showed reduction in Na+ uptake, reduced membrane damage, improved chlorophyll content, and enhanced K+ uptake [228]. In lettuce seedlings, both AMF strains of Glomus sp. stimulated growth of the seedlings under moderately saline conditions while inoculation with P. mendocina increased plant biomass even under severely saline conditions which means that selected microbes can differentially alleviate salinity stress [229]. Besides that, microbial-treated lettuce seedlings increased root biomass which helps plants to enhance water and nutrient uptake during salt stress [229, 230]. P. medocina also produced EPS that chelate the harmful Na+ ions and reduced the Na+ bioavailability for plant uptake [229]. After inoculating pea plants with Variovorax paradoxus, the growth of pea plants were also improved by 54% due to increased photosynthetic activity, enhanced root growth and improved uptake of Ca, Mg, and K [231]. The ability of microbes in regulating host endogenous hormone levels also play an important role in salinity tolerance. Cucumber plants treated with Bukholderia and Promicromonospora showed an increase in SA and GA but lower ABA content [232]. Higher SA level in plants was reported to suppress ABA signaling, regulate Na+ and K+ vacuolar sequestration in roots, and reduce Na+ accumulation in shoots by reducing the host activity of Na+ ion transporters [233, 234]. Lower ABA level in plants increased leaf conductance and photosynthetic rate due to stomatal opening while high GA level in cucumber seedlings is desirable since it is involved in plant growth and development [232]. On the other hand, the application of P. putida on soybean plants indicated lower endogenous ABA and SA but higher level of JA during saline condition [235]. Lower level of SA is associated with less ROS generated during salt stress. The upregulation of JA and downregulation of SA is similar to ISR against biotic stress. In other words, ISR could have been signaled in soybean plants since they were pre-treated with P. putida 7 days before NaCl was applied. Nevertheless, the exact role of JA in salinity tolerance is still unknown [236]. From these studies, the changes of SA, JA, GA, and ABA in plants during salinity could be dependent on microbial strains. Khan et al. [237] described Aspergillus fumigatus lowered the endogenous ABA but elevated both SA and JA contents in soybean plants which contradicted with previous studies. The SA and JA signaling in plants are known to be antagonistic which warrants

70  Biofertilizers further investigation to understand the signaling mechanism of both phytohormones in plant-microbe interaction during salinity stress. The co-inoculation of Azospirillum and Rhizobium also promoted root branching in common bean seedlings and increased secretion of nod gene inducing flavonoids which promoted root nodulation under saline condition [238]. In particular, Azospirillum allows a longer, more persistent exudation of flavonoids from root which functions to activate nod genes leading to nodulation in bean roots. Inoculation with PGP microbe from Azopirillum, Enterobacter, or Pseudomonas genus were found to improved plant biomass, increased K+ cellular content, enhanced root growth for nutrient absorption, and yield in various horticultural crops during salinity stress including tomato, cucumber, pepper, and okra [239–242]. Intriguingly, these studies also demonstrated the ability of PGP microbes in improving chlorophyll and photosynthesis rate of inoculated plants during salinity stress. Yan et al. [243] explained that the expression levels of the Toc (Translocon complexes) GTPases or chloroplast protein import apparatus were upregulated in tomato plants inoculated with P. putida. This might facilitate the import of nucleus-encoded proteins from the cytosol for chloroplast development, thereby indicating that photosynthesisrelated pathways were not affected during salinity stress [244].

2.3.4.3 Heavy Metal Soil contaminated with high concentrations of heavy metal is often associated with excessive use of chemical fertilizers, mining activities, sewage sludge application, and disposal of heavy-metal waste. Plants absorb and accumulate heavy metals which could ultimately pass to humans through food chain [245]. Furthermore, plants also suffer from heavy metal stress, whereby the negative effects include damage of cellular structures and metabolism [245]. Applying biofertilizers containing heavy metal-tolerant PGP microbes is an attractive approach to improve crop tolerance toward heavy metal stress. In general, microbial bioremediation of heavy metal includes the transportation of metals across the cytoplasmic membrane, biosorption and bioaccumulation on the microbial cell walls, metal entrapment in cellular structures such as vacuoles, precipitation of heavy metal, and metal detoxification via cellular oxidation-reduction [246]. Grapevine plantlets bacterized with a consortium of Micrococcus luteus and B. licheniformis improved the biomass and also protected the plants from oxidative stress due to high concentration of cadmium (Cd) by increasing the host antioxidant activity [247]. Heavy metal tolerant Bradyrhizobium japonicum increased shoot and root lengths of lettuce

Biofertilizers on Horticultural Crops  71 seedlings under nickel (Ni), copper (Cu), or lead (Pb) stress. Further FTIR spectrum of the bacterium indicated that the presence of amine and nitro functional groups on the membrane were discovered to be responsible for metal biosorption, thereby reducing the heavy metal bioavailability for plant uptake [248]. Soybean plants inoculated with Bradyrhizobium sp. also indicated improved photosynthetic pigments and increased carotenoid content to reduce photo-oxidation of chlorophyll pigments under Cd stress. Inoculated plants also showed reduced Cd content in both root and shoot. Increased magnesium and iron contents in soybean plants were also observed which suggested that this microbial strain produce siderophores to sequester essential minerals for host uptake [249]. The plant biomass of spinach was also significantly increased with decreased accumulation of Cd, Pb, and zinc (Zn) due to the ability of a consortium of Pseudomonas and Bacillus to produce the growth hormone IAA [250]. Tomato cultivated under Cd stress did not accumulate high levels of Cd in roots and shoots after applying P. aeruginosa and Burkholderia gladioli. Metal transporter gene in tomato was also not highly expressed when inoculated with both PGP bacteria. Metal chelating compound such as total thiols and non-protein bound thiols in tomato plants was also enhanced under Cd stress after bacterial inoculation [251]. However, the mechanism behind the microbial regulation of metal transporter gene and the production of metal chelators in plants has yet to be investigated. AMF such as R. intraradices colonizes pepper better than F. mosseae under Cu stress. The AMF strains prevented metal translocation to shoots through metal chelators and retained heavy metals on the cell walls of the mycelia [252]. Reduced arsenic (As) in chickpea seeds and tolerance of host against As stress were observed after host inoculation of Trichoderma [253]. Trichoderma was reported to induce methylation of heavy metal in soil and methylated metals in soil are reduced via volatilization into the air [254].

2.3.4.4 Cold Stress Many horticultural crops, including tomato, soybean, and legumes, lack the ability to tolerate cold temperatures since they are cultivated in tropical and subtropical regions [255]. Cold or chilling stress severely affects plant growth and development, limits planting of plant species in cold regions, and causes loss of crop yield [256]. In terms of plant physiology, cold stress reduces cellular metabolism, accumulates ROS, decreases cellular osmotic potential, solidifies plasma membrane, and destabilizes protein complexes for metabolism [202]. To enable crops to withstand cold stress, microbial

72  Biofertilizers inoculants or biofertilizers have been successfully applied to enhance crop’s tolerance against low temperatures. In vitro grapevine plantlets bacterized with the endophyte Burkholderia phytofirmans were able to survive at low temperatures of 4°C [257]. The bacterization of plants showed improved biomass, reduced electrolyte leakage as a result of thickening of xylem cell wall, elevated levels of secondary metabolites such as phenolics and proline, and enhanced rate of photosynthesis and deposition of starch [257, 258]. The bacterial priming effect of the grapevine plantlets also contributed to cold tolerance through the upregulation of stress-related genes and metabolites indicating that the endophyte plays an essential role in the cold acclimation of the plantlets [259]. Furthermore, B. phytofirmans was found to induce cell wall strengthening which prevented cell collapse due to the formation of intercellular ice crystals [260]. Tomato seeds bacterized with cold-tolerant Pseudomonas of Flavobacterium were successfully germinated at 15°C. Cold tolerance was also achieved in bacterized plants as observed in the reduction of membrane damage (reduced malondialdehyde levels and electrolyte leakage), activation of antioxidant activities, and accumulation of proline in the plants [261]. Similarly, chilling resistance in tomato plants bacterized with coldtolerant Pseudomonas was evident with the reduced membrane disruption, decreased ROS levels, and upregulated expression of cold acclimation genes [262]. The inoculation of AM fungi, Funneliformis mosseae and Glomus mosseae of cucumber and tomato plants, respectively, reduced mortality rate, increased plant biomass, increased secondary metabolite production (proline, phenols, flavonoids, and lignin), enhanced ROS scavenging activity and elevated the expression of stress-related genes during chilling stress [263, 264]. The root water uptake of the common bean inoculated with Glomus sp. was also not affected under 4°C which suggest that AMF fungi could protect the root cell membrane from being damaged under cold stress condition [265]. Cold tolerance was also achieved by reducing the accumulation of endogenous ethylene in stressed plants. In a study conducted, a cold tolerant P. putida promoted the growth of canola under cold stress by producing ACC deaminase which is an enzyme that reduces the production of ethylene [266]. The accumulation of ethylene during chilling stress is not desirable as it could increase the senescence rate of plants. To date, most biofertilizer studies on enhancing crop tolerance to cold stress is still at its preliminary stage. More research should be conducted to understand the role of host defense signaling and metabolic

Biofertilizers on Horticultural Crops  73 pathways in conferring cold tolerance in crops especially when they are treated with biofertilizer.

2.3.4.5 Heat Stress Global climate change is predicted to increase temperatures to about 1.5°C–5.8°C by 2100 [267]. High temperature has caused major impact on plant growth and development particularly reduction in yield and crop quality. In order to overcome heat stress in crops, there are several instances of using biofertilizers to increase plant tolerance to heat stress while sustaining or improving the yield. Several studies have discovered the potential of PGP bacteria from the Pseudomonas, Bacillus, and Orchrobactrum genus to increase heat tolerance in cereal crops such as wheat while also improving grain yield under heat stress conditions [268–270]. Surprisingly, studies on the improvement of thermo-tolerance of horticultural crops through the biofertilizer application is still lacking. Currently, only a single study that utilized B. aryabhattai to improve the tolerance of soybean plants to thermal stress of up to 38°C. From this study, the PGP bacterium was found to produce exogenous absisic acid (ABA) which also regulated the host endogenous ABA. As a result, the bacterial-treated soybean plants showed elevated concentrations of ABA which led to stomatal closure to prevent further transpiration during heat stress condition [271]. Since biofertilizer is a promising tool to circumvent heat stress in plants, research focus should therefore be directed toward horticultural crops to prevent potential economic loss in the face of climate change.

2.3.5 Improved Vegetative Propagation Efficiency 2.3.5.1 Propagation by Cuttings Vegetative propagation of horticultural crops uses cuttings as a source of material to produce clonal planting materials. This method is also an alternative to grafting since no rootstocks are needed [272]. There is also no need for seed germination as certain seeds undergo dormancy. Difficult to root species often rely on the use of synthetic auxins which act similarly to natural plant rooting hormones but can be toxic to plants at high concentrations [273]. To replace harmful chemicals, applying PGP microbes to cuttings has begun to receive attention due to their ability to produce rooting hormones such as IAA to facilitate the rooting process of cuttings.

74  Biofertilizers Black pepper cuttings dipped in B. tequilensis for 30 minutes produced cuttings with higher root numbers and plant biomass [274]. Bacillus strains also improved the kiwifruit semi-hardwoord stem cuttings by 47.5% and hardwood stem cuttings by 42.5% [272]. The differences in rooting capacity might be due to juvenility of the cuttings. Juvenile cuttings might have better cellular plasticity than matured cuttings as differentiated cells in the juvenile cuttings are able to switch and differentiate into adventitious roots [275]. Grapevine cuttings treated with Azospirillum brasilense also improve the rooting parameters in terms of the number of roots, root architecture, and biomass of vines compared to conventional method [276]. It was also found rooting using PGP microbes is also cultivar dependent whereby not all grapevine cultivars were responsive to the microbial inoculation [276, 277]. Koyama et al. [278] discovered that when A. brasilense was applied together with 3 g/L IBA, higher number of adventitious roots was observed in olive-tree cuttings but the percentage and survival of rooted cuttings were not significantly different with other treatments. The addition of IBA might have influenced the production of natural IAA by the rhizobacteria which requires further validation. Certain strains such as Pantoea sp. produced higher rooting percentages in olive cuttings regardless of inoculation methods (i.e., dipping or immersing cuttings in bacterial suspension) and three test cultivars of olive [279]. Interestingly, this strain is not the best IAA producer compared to Pseudomonas and Bacillus but it synthesizes ACC deaminase that reduces the accumulation of ethylene upon wounding (i.e., collection of cuttings) and to prevent ethylene from inhibiting root development [280]. Most of the vegetative propagation studies of horticultural crops using PGP microbes are still at its infancy stage. In fact, more in-depth experimental designs should be conducted to study the cultivar/genotype effect on microbial colonization behavior, factors that affect microbial IAA production, the reception and transportation of exogenous auxin in cuttings exposed to PGP microbes, and the auxin signaling pathways during rooting process after inoculation. These information could be proven useful to accelerate the vegetative propagation process of horticultural crops.

2.3.5.2 Grafting In modern cropping systems, grafting is commonly used to impart pathogen resistance, manipulate plant physiology, confer disease or pest resistance, and tolerate certain soil types [281]. Grafting also increases nutrient uptake and utilization efficiency in fruits, ornamental, and vegetables. The rootstocks absorb water and ions more efficiently than rooted plants

Biofertilizers on Horticultural Crops  75 derived from cuttings, and these water and ions were then transported to the aboveground scion [282]. Considering the benefits of grafting for farmers, various grafting methods were developed to improve the success rate of grafting union between rootstocks and scions which include the use of PGP microbes. Combined application of P. putida and B. simplex (i.e., the scions were immersed in bacterial suspension for an hour) resulted in 100% callusing and grafts survival rate compared to single inoculation and control treatments [283]. Bacterized graft shoots also root faster than control with better developed and thick shoots which could be due to natural plant growth regulator, IAA produced by the microbial strains. IAA promotes active cell division leading to callus formation which then differentiates to adventitious roots [283, 284]. Köse et al. [285] also submerged the scion for at least 3 hours in individual strains of Pseudomonas and Bacillus and grafting results showed that the success rate was from 80% to 93.3%. Similarly, these two strains were known to produce IAA and produce ACC deaminase which reduced the host ethylene levels in order to facilitate callus formation [286]. Cacao grafting was improved to 62% as compared to water treatment (38%) through foliar spray of B. subtilis suspension on the graft unions [287]. Currently, there are still limited research on the effect of these PGP microbes on other horticultural crops and the mechanism of initiating callus and root differentiation on the graft union have yet to be studied.

2.4 Future Perspectives and Challenges Ahead It is evident that the potential of biofertilizers does not confine to growth and yield enhancement of horticultural crops. Stress tolerance toward various biotic and abiotic stress of crops was also largely improved through microbial-mediated defense response. Biofertilizers is also successfully applied to increase horticulture planting materials through means of vegetative propagation. Despite that, there are still a myriad of factors that affect the efficiency of biofertilizers in delivering consistent results. Factors that lead to inefficient biofertilization and recommendations to overcome these issues were summarized comprehensively in Table 2.2. Among them, non-reproducible results from biofertilizer application are associated with crop genotypes and physiological age, method and frequency of application, and the viability counts or contaminants present in commercial biofertilizers. Most importantly, academia-industry collaboration on biofertilizer production and knowledge transfer to farmers have also received less attention in which emphasis are often restrain in lab-scale optimization and production of biofertilizers. Social feedbacks from farmers are

76  Biofertilizers Table 2.2  Challenges and recommendations to improve the efficiency of biofertilizers. Challenges

Points to consider

Recommendations

Manufacturing process of biofertilizer

• Single or a consortium of inoculants. • Shelf life of biofertilizer. • Types of carrier used to contain the inoculants.

• Ensure the consortia are compatible to each other [288, 289]. • Identify suitable carriers and additives to protect inoculants from harsh soil or phyllosphere conditions to increase viability [290].

Marketing and quality control of biofertilizers

• Concentration of viable cells. • Contaminants present in biofertilizers.

• Present regulations should be enforced and updated regularly so that manufacturers abide by the requirements for a quality biofertilizer before commercialization is commenced [7, 291].

Biosafety issues of inoculants

• Some inoculants may be opportunistic human pathogens.

• Use polyphasic approach to characterize inoculants to ensure inoculants are not human pathogens and to follow Environmental and Human Safety Index (EHSI) to compare inoculates with pathogens [292, 293].

Persistence of microbial inoculant in soil

• Develop methods to track the persistence of inoculants that lead to plant growth and stress tolerance.

• The molecular marker-assisted method including T-RFLP, DGGE, or TGGE could be used to qualitatively assess the persistence of inoculants and changes in the microbiome [294]. • Use of qRT-PCR to specifically target and quantify the inoculants present soil and root [295, 296]. (Continued)

Biofertilizers on Horticultural Crops  77 Table 2.2  Challenges and recommendations to improve the efficiency of biofertilizers. (Continued) Challenges

Points to consider

Recommendations

Microbial inoculant interaction with biotic and abiotic stress

• Competition between native and introduced inoculants. • Survival of inoculants in harsh conditions in soil (e.g., salinity, heavy metal toxicity, and soil pH).

• Future study on the key genes involved in the interaction process between inoculants and native microbes to understand the metabolic potential of soil microbial communities in response to inoculation which allow the design of biofertilizers to specific soil/crops [294]. • Identify strains that are tolerant to abiotic stress to increase survival rate in soil [297].

Fertilization scheme

• Viability of inoculants affected by the application of organic or chemical fertilizers.

• Optimization study of suitable dosages in combined treatments of fertilizers and inoculants to achieve positive outcomes [298, 299]. • Choosing suitable organic fertilizers except for sewage sludge which reduces soil microbial community [300].

Cultural practices in farm

• Soil disturbance such as tillage reduces microbial community (e.g., AMF).

• Conservation tillage enhances the stability of rhizopsheric microbial community which directly improve crop production [301, 302].

Application methods

• Performance of biofertilizers affected by application methods.

• Automated application of biofertilizer to ensure equal distribution [294]. • Repeat application of biofertilizer as boosters to improve microbial colonization [303, 304]. • Further optimization work needed for biofertilizer applica­ tion in different agriculture field production setting to meet farmers’ requirement. (Continued)

78  Biofertilizers Table 2.2  Challenges and recommendations to improve the efficiency of biofertilizers. (Continued) Challenges

Points to consider

Recommendations

Crop genotype, physiological factors, and plant health

• Inoculant performance differs in various crop phenotypes, health conditions of plants, physiological stages (i.e., seed, seedlings, or matured plants).

• Proper optimization study needed to study the effect of crop genotypes and physiological stages on the colonization behavior of inoculants [305–307]. • Future study to study the root exudates of crop of various genotypes, physiological stages and health conditions which could directly affect microbial colonization [308, 309].

Knowledge transfer to industrial level for optimum formulation

• Transfer of technical knowledge for industry to produce quality biofertilizers.

• Engaging researchers-industry collaboration to produce quality biofertilizers [304].

Awareness of farmers

• Farmers’ poor perception on biofertilizer (i.e., chemical fertilizers/ pesticides are easily available and more efficient than biofertilizers).

• Engaging stakeholders in educating farmers in biofertilization [310, 311]. • Monitoring farmers’ and stakeholders requirements and feedbacks on the biofertilization efficiency on crop production for continuous improvement [312–314].

also essential so that constant improvements could be made to improve the quality of the biofertilizers. To conclude, successful biofertilization on horticultural crops require a thorough consideration of numerous factors so that growers could benefit from the sustainable and consistent use of biofertilizers instead of relying heavily on chemical fertilizers.

Biofertilizers on Horticultural Crops  79

2.5 Conclusion Biofertilizers have indeed brought positive impacts to the horticulture industry by ensuring that crop productivity is sustained under unpredictable environmental conditions. Yet, inconsistencies still exist in many of the research as reviewed in this chapter. In order to bridge the gap, future investigations should be directed toward a holistic understanding of the  mechanisms and factors that affect the efficiency of biofertilizers. On the other hand, policy makers should enforce tight regulations to ensure the biofertilizers have met the necessary requirements prior to commercialization. Stakeholders, too, should be actively involved in transferring knowledge to farmers and to constantly monitor the outcomes from field trials. Combining these approaches would foster the use of biofertilizers as a sustainable choice instead of using chemical fertilizers or pesticides.

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100  Biofertilizers aquaporins in Phaseolus vulgaris under drought, cold or salinity stresses. New Phytol., 173, 808–816, 2007. 266. Cheng, Z., Park, E., Glick, B.R., 1-Aminocyclopropane-1-carboxylate (ACC) deaminase from Pseudomonas putida UW4 facilitates the growth of canola in the presence of salt. Can. J. Microbiol., 53, 912–918, 2007. 267. Chitara, M.K., Keswani, C., Bisen, K., Singh, V., Singh, S.P., Sarma, B.K., Singh, H.B., Improving crop performance under heat stress using thermotolerant agriculturally important microorganisms, in: Advances in PGPR Research, H.B., Singh (Eds.), pp. 296–305, CABI, United Kingdom, 2017. 268. Ali, S.Z., Sandhya, V., Grover, M., Linga, V.R., Bandi, V., Effect of inoculation with a thermotolerant plant growth promoting Pseudomonas putida strain AKMP7 on growth of wheat (Triticum spp.) under heat stress. J. Plant Interact., 6, 239–246, 2011. 269. Sarkar, J., Chakraborty, B., Chakraborty, U., Plant growth promoting rhizobacteria protect wheat plants against temperature stress through antioxidant signaling and reducing chloroplast and membrane injury. J. Plant Growth Regul., 37, 1396–1412, 2018. 270. Ashraf, A., Bano, A., Ali, S.A., Characterisation of plant growth-promoting rhizobacteria from rhizosphere soil of heat-stressed and unstressed wheat and their use as bio-inoculant. Plant Biol., 21, 762–769, 2019. 271. Park, Y.-G., Mun, B.-G., Kang, S-.M., Hussain, A., Shahzad, R., Seo, C.-W., Kim, A.-Y., Lee, S-.U., Oh, K.Y., Lee, D.L., Lee, J.I., Yun, B.W., Bacillus aryabhattai SRB02 tolerates oxidative and nitrosative stress and promotes the growth of soybean by modulating the production of phytohormones. PLoS ONE, 12, e0173203, 2017. 272. Erturk, Y., Ercisli, S., Haznedar, A., Effects of plant growth promoting rhizobacteria (PGPR) on rooting and root growth of kiwifruit (Actinidia deliciosa) stem cuttings. Biol. Res., 43, 91–98, 2010. 273. Mariosa, T.N., Melloni, E.G.P., Melloni, R., Ferreira, G.M.R., Souza, S.M.P., Silva, L.F.O., Rhizobacteria and development of seedlings from semi-hardwood cuttings of olive (Olea europaea L.). Rev. Cienc. Agrar., 60, 301–306, 2017. 274. Dastager, S.G., Deepa, C.K., Pandey, A., Growth enhancement of black pepper (Piper nigrum) by a newly isolated Bacillus tequilensis NII-0943. Biologia, 66, 801–806, 2011. 275. Diaz-Sala, C., Direct reprogramming of adult somatic cells toward adventitious root formation in forest tree species: The effect of the juvenile-adult transition. Front. Plant Sci., 5: 310, 2014. 276. Toffanin, A., D’Onofrio, C., Carrozza, G.P., Scalabrelli, G., Use of beneficial bacterial Azospirillum brasilense SP245 on grapevine rootstocks grafted with ‘Sangiovese’. Acta Hort., 1136, 177–184, 2016. 277. Kamble, A.K., Mukunda, G.K., Raut, N.B., Nachegowda, V., Murthy, B.N.S., Nagarajaiah, Seenappa, K., Studies on rooting of cuttings of grapes (Vitis

Biofertilizers on Horticultural Crops  101 vinfera L.) using biofertilizers with special reference to wine varieties. Int. J. Chem. Stud., 6, 1406–1498, 2018. 278. Koyama, R., Júnior, W.A.R., Zeffa, D.M., Faria, R.T., Saito, H.M., Gonçalves, L.S.A., and Roberto, S.R. Association of indolebutyric acid with Azospirillum brasilense in the rooting of herbaceous blueberry cuttings. Hort., 5, 68, 2019. 279. Montero-Calasanz, M.C., Santamria, C., Albareda, M., Daza, A., Alternative rooting induction of semi-hardwood olive cuttings by several auxin-producing bacteria for organic agriculture systems. Span. J. Agric. Res., 11, 146–154, 2013. 280. Gupta, S. and Pandey, S. Unravelling the biochemistry and genetics of ACC deaminase – An enzyme alleviating the biotic and abiotic stress in plants. Plant Gene, 18, 100175, 2019. 281. Gambetta, G.A., Rost, T.L., Matthews, M.A., Passive pathogen movement via open xylem conduits in grapevine graft unions. Am. J. Enol. Viti., 60, 241–245, 2009. 282. Nawaz, M.A., Imtiaz, M., Kong, Q., Cheng, F., Ahmed, W., Huang, Y., Bie, Z., Grafting: A technique to modify ion accumulation in horticultural crops. Front. Plant Sci., 7, 1457 2006. 283. Sabir, A., Improvement of grafting efficiency in hard grafting grape Berlandieri hybrid rootstocks by plant growth-promoting rhizobacteria (PGPR). Sci. Hort., 164, 24–29, 2013. 284. Yin, H., Yan, B., Sun, J., Jia, P., Zhang, Z., Yan, X., Chai, J., Ren, Z., Zheng, G., Liu, H., Graft-union development: a delicate process that involves cellcell communication between scion and stock for local auxin accumulation. J. Exp. Bot., 63, 4219–4232, 2012. 285. Köse, C., Gϋleryϋz, M., Şahin, F., Demirtaş, I., Effects of some plant growth promoting rhizobacteria (PGPR) on graft union of grapevine. J. Sustain. Agr., 26, 139–147. 286. Grichko, V.P., Glick, B.R., Amelioration of flooding stress by ACC deaminase-containing plant-growth promoting bacteria. Plant Physiol. Biochem., 39, 11–17, 2001. 287. Falcäo, L.L., Silva-Werneck, J.O., Vilarinho, B.R., da Silva, J.P., Pomella, A.W.V., Marcellino, L.H., Antimicrobial and plant growth-promoting properties of the cacao endophyte Bacillus subtilis ALB629. J. Appl. Microbiol., 116, 1584–1592, 2014. 288. Malusà, E., Sas-Paszt, L., Zurawicz, E., Popinska, W. The effect of a mycorrhiza-bacteria substrate and foliar fertilization on growth response and rhizosphere pH of three strawberry cultivars. Int. J. Fruit Sci., 6, 25–41, 2007. 289. Vestergård, M., Henry, F., Rangel-Castro, J.I., Michelsen, A., Prosser, J.I., Chistensen, S., Rhizosphere bacterial community composition responds to arbuscular mycorrhiza, but not to reductions in microbial activity induced by foliar cutting. FEMS Microbiol. Ecol., 64, 78–89, 2008.

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3 N2 Fixation in Biofertilizers Rekha Sharma1, Sapna Nehra2 and Dinesh Kumar3* Department of Chemistry, Banasthali Vidyapith, Banasthali, Rajasthan, India Department of Chemistry, Dr. K. N. Modi University, Newai, Rajasthan, India 3 School of Chemical Sciences, Central University of Gujarat, Gandhinagar, India 1

2

Abstract

Nowadays, soil managing methods cause a serious threat to the environment and human health by the use of inorganic chemical-based fertilizers. For growing crop production and soil productiveness, biofertilizer has been recognized as a substitute in sustainable agricultural fields. Microorganisms that are generally used as biofertilizer constituents comprise plant growth-promoting rhizobacteria (PGPRs), phosphorus and potassium solubilizer, nitrogen fixers (N-fixer), cyanobacteria, ecto- and endomycorrhizal fungi, and additional beneficial microbes. The usage of biofertilizers corresponds to water uptake and improved nutrients, plant tolerance, and plant growth to biotic and abiotic features. For protecting the atmosphere and sustainability and productivity of soil as cost-effective and ecofriendly contributions for the agriculturalists, these biofertilizers will play a vital part. The degradation of the local environment and augmented soil destruction results from synthetic fertilizer use. Microalgae, possessed nitrogen fixation capability, are measured as proficient biofertilizers to save the natural atmosphere and cost-effective benefits. The root and shoot development of soybean, cowpea, and mung bean suggestively endorsed by the use of biofertilizer under N-supplemented or N-limited conditions. In this chapter, we will comprise the vital additions of biofertilizers in nodulation, plant growth, uptake of nitrogen, potassium, and phosphorus (NKP), nitrogen fixation, and seed yield. Keywords:  Biofertilizer, nitrogen fixation, nodulation, plant growth, eco-friendly

*Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (105–120) © 2021 Scrivener Publishing LLC

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3.1 Introduction The demand for significant cash crops and leguminous crops is increasing worldwide because of their applications in domestic consumption and their export. In all regions and states, about 4.6 billion hectares of pulses are propagated yearly [1]. By the use of grain legumes, around one-third of protein is resulting from human dietary protein content [2]. Because of their inimitable aptitude to harvest considerable quantities of biological nitrogen through biological symbiotic nitrogen fixation, legumes are an important rich source of protein [3]. So, to advance harvest manufacture and sustain soil productiveness, legume–rhizobia symbiosis could be able to offer a low-cost and simple method [4]. The augmented legume yield, nitrogen acquisition, and nodulation can be done through the Rhizobium strains immunization with the soil [5]. Nitrogen fixation is reducing molecular nitrogen into ammonia by the use of rhizobia, which plays a role in biological nitrogen fixation (BNF) [6]. The necessity for chemical nitrogen fertilizers is deuces by the use of BNF, which is agronomically important in this area [7]. For the improvement and maintenance of crop yield and growth, nitrogen is important. Though, in agriculture, the enduring unnecessary usage of chemical fertilizers partakes surprising ecological effects [8], counting soil organic matter deterioration, soil fertility degradation, and reduced nutrient use efficiency and nutrient and water embracing capacities [9, 10]. For the manufacture of leguminous harvest, rhizobium inoculants are comparatively low cost [11]. For a decrease of ecological complications and sustainably managing, the usage of effective inoculants could be measured as a vital approach through reducing the usage of chemical fertilizers [12]. Many viable PGPB (plant growth-promoting bacteria) inoculants appear to endorse development through the following methods: • Biostimulants (manufacture of phytohormone), • Biofertilizers (upgrading of nutrient acquisition), or • Bioprotectants (destruction of plant ailment) [13]. By the use in solubilizing insoluble phosphates, fixing atmospheric nitrogen, in the soil endorsing nodulation capability, and manufacturing materials that endorse plant progress [14], the usage of biofertilizers advances soil productiveness, which upsurges crop through 16%–60% [15]. The rhizobia acclimate to ecological circumstances, enabling their fruitful nodulation and endurance through the host plant, so it is mainly utilized as biofertilizers [16].

N2 Fixation in Biofertilizers  107 In developing nations worldwide, soil infertility is the most significant problem among resource-poor farmers, which restraining crop yield [17]. In big portions of the world which cause the elementary values of virtuous agricultural training, the soil quality preservation could be controlled. The following factors are the consequences of soil deterioration: • speedily deteriorating manufacture levels, • reducing soil productiveness, • decrease the difficulties of land deprivation. Because of ecological and biological issues, the deceased harvests in legumes are accompanying through abridged nitrogen fixation and deteriorating soil productiveness. BNF shows an important part in the maintainable manufacture of non-legumes and, also, legumes that are the main basis of nitrogen for farmers through diminutive fertilizer, and establishes the utmost potential solutions. It attains little yield in return by the use of inorganic chemical fertilizer because of the augmented populace, which resulted in a deterioration in soil productiveness by the nonstop farming of the identical part of a land year by year. The identified amounts of potassium, phosphorus, and nitrogen are used to produce chemical fertilizers—the groundwater and air contamination resulted from the use of chemical fertilizers [18]. Using chemical fertilizers comprises the hazard of polluting the atmosphere and groundwater, and insecticides hasten acidification of soil [19]. It also assembles them to be vulnerable to undesirable ailments by deteriorating the roots of plants. Incidentally, to ensure bio-safety, various efforts have been made for manufacturing biofertilizer abundant with various nutrients. To upsurge yield manufacture and soil productiveness, biofertilizer has been recognized as an alternative to chemical fertilizer in sustainable agricultural fields. In sustainability and productivity of soil for the farmers, these potential biofertilizers play a key role and protect the atmosphere as profitable and biodegradable contributions [17]. As the rhizobia plants, there are various microbes prosperous in the soil. A significant amount of these microbes establishes a comprehensive organization and have a useful association with plants, which partakes helpful possessions on the development of plants [18]. Through the potassium and phosphate mineralization or solubilization, nitrogen fixation, biodegradation of biological substance in the soil, proclamation of development adaptable matters in plant, and manufacture of antibiotics, these biofertilizers keep the soil atmosphere abundant incomplete types of micro and macronutrients [21].

108  Biofertilizers

3.2 Biofertilizers 3.2.1 Origin In the past biofertilizers, for example, Azotobacter, Blue-green algae (BGA), Rhizobium, and Azospirillum, have remained in usage. From a peer group to a peer group of farmers, the knowledge of useful bacterial inoculum was continuously carried. The capability of biofertilizer has been proved, which originated by the production and culture of minor scale compost [17]. It resulted that when the culture hastens the decay of farming harvest and organic deposits by many methods and offers a vigorous yield of crops [22]. The agronomist Nobbe and Hilther commercially introduced the origin of biofertilizers in 1895 with the launch of “Nitragin” through the finding of Azotobacter and after BGA and a host of additional microbes. These microbes are being used as biofertilizer to date [23]. Biofertilizers are generally equipped by transporter founded inoculants comprising efficient microbes [20]. Microbes used through biofertilizer comprise K-solubilizers, for example, Bacillus mucilaginous, N-fixers, for example, Rhizobium Spp., Azotobacter chroococcum, and Cyanobacteria, plant growth-promoting Rhizobacteria (PGPR), P-solubilizers, for example, Aspergillus fumigatus, Bacillus megaterium, sulfur oxidizers (S-oxidizers), and Vesicular-Arbuscular Mycorrhiza (VAM), for example, Glomus mosseae.

3.3 Biofertilizer: Transporter Constituents To upsurge the efficiency of the biofertilizers, these are generally modified through transporter substantial, which also upsurges ration capability for water [24]. A virtuous transporter substantial should partake the subsequent features, according to Somasegaran and Springer [25]: • simple to disinfect through gamma irradiation or autoclaving, • readily accessible and inexpensive in suitable quantities, • non-toxic to both the microbes and the vegetations, to which it is practical, • simple to develop and should be free of lump forming resources, • decent water-holding capability of more than 50%, • a decent wetness absorption capacity, • decent pH buffering capacity,

N2 Fixation in Biofertilizers  109 • decent adhesion to seeds, • elevated content of biological substances. Khosro and Yousef [17] detailed that the amalgamation of microbes hooked on transporter constituents allows efficiency, extended-term storage, and simple management of the biofertilizer. During extensive stowage of time, they likewise described that the disinfection of transporter constituents is vital to preserving high numbers of inoculants, for example, comprise talcum dust, sawdust, earthworm cast, and manure. Because of the disinfection method varieties, no alteration in the chemical and physical possessions of the substantial, gamma-irradiation is the recording method of transporter disinfection. Similarly, autoclaving is the additional technique of transporter disinfection. Though, the production of toxic substances can be done through autoclaving, which can alter the possessions of some transported resources resulted in the killing of some bacterial classes [26].

3.4 Mechanism of Actions of Biofertilizers Azospirillum was proposed to release auxins, ethylene, and gibberellins among the PGPRs species. The raised stages of IAA in the roots of lodgepole pine could likewise persuade phytohormone synthesis when injected through Paenibacillus polymyxa bacteria. In attendance of a carrier material, i.e., agro-waste, the Bacillus and Rhizobium were originating to manufacture IAA at diverse temperatures and pH. The ethylene is accountable for dicot plants for their development inhibition, unlike additional phytohormones [27].

3.5 Biochemistry of Manufacture of Biofertilizer Anaerobic bio-digestion is a method in which microbes collapse in decomposable constituents in the nonattendance of oxygen. In the breaking down of complex materials into simpler substances in anaerobic digestion, manufacturing biofertilizer comprises three following biochemical stages [28]. (i) To harvest unpretentious fragments, for example, longchain fatty acids and other substances by which it degrades

110  Biofertilizers compound biological substance through cellulolytic microbes. (ii) Manufacturing simple intermediates by fermentation of products from stage one, for example, carbon dioxide, pyruvic acids, and acetic acids. (iii) Last, a mixture of gases known as biogas produced by methanogens process on the crops which can be represented by Equations 3.1 to 3.3:

CH3COOH → CH4 +   CO2

(3.1)

Acetic acid   Methane  Carbon dioxide 2CH3CH2OH + Ethanol

CO2

CH4 + 2CH3COOH (3.2)

Carbon dioxide

CO2 + 4H2 Carbon dioxide   Hydrogen

→

Methane + Acetic acid

CH4 + 2H2O Methane

(3.3)

Water

3.6 Benefits of Biofertilizer Over Biochemical Fertilizers Because of their rapid proclamation of nutrients, inorganic fertilizers have the advantage of fast action and are inexpensive. Therefore, these have developed exceedingly prevalent worldwide. Though, the results showed that they have drawbacks that could not be ignored, so there has been a considerable study done on the disadvantages of inorganic fertilizers. Because of the usage of inorganic fertilizer, the utmost difficulties related to some pollution and produced crops of our ordinary atmosphere ensued [29]. Inorganic fertilizers were useful to the soil producing contamination because of their upsurge solubility in water, by which they could be leached profoundly hooked on underground water into the soil unreachable for roots of a plant. Because of their deliberate release of nutrients, biofertilizers have extensive long-lasting properties. The inclusive soil productiveness is augmented by the use of enduring usage of biofertilizer, which corresponds to the accumulation of nutrients in the soil. Various plant diseases, for example, Rhizoctonia root rot, pythium root rot, parasitic nematode, and chill wilt, could be controlled by the use of biofertilizers [29].

N2 Fixation in Biofertilizers  111 The soil particles are organized with each other and upsurge the water retention capacity of the soil, whereas avoiding erosion, desertification, and soil eructing by the use of biofertilizer, which plays as a soil conditioner by the addition of organic matter to the soil [30].

3.7 Variances Among Organic and Biofertilizer Previously, the organic fertilizer was known as the term biofertilizer, though, there is an immense variance among organic fertilizer and bio-­ fertilizer. According to Vishal and Abhishek [31], biofertilizers like fungi, algae, bacteria, in combination or alone, which can benefit in growing crop production, are made of microbial inoculants comprising living cells of microorganisms. Organic fertilizers are attained from plant sources, for example, green manure or animal sources, for example, animal manure.

3.8 Types of Biofertilizers Biofertilizers are of various types based on microorganisms used for the particular biofertilizer are comprised in Table 3.1.

3.9 Microorganisms Utilized to Make Biofertilizer The K-solubilizer, N-fixers, P-mobilizers, and P-solubilizer, which is utilized in combination with fungi or alone, are the components of organisms used as biofertilizers. Outmost microorganisms partake adjacent association with plant roots used in biofertilizers. Rhizobacteria inhibit root surfaces or rhizosphere soil because of its symbiotic interaction with legume roots. The insoluble phosphorus made through the phosphomicroorganisms such as fungi and bacteria, this insoluble P accessible for the plants. Through the concealing of biological acids, which generate the dissolution of bound systems of phosphate and lower the soil pH, to produce soluble forms of insoluble phosphate in soil by the application of few species of fungi and many soil bacteria. Whereas Azolla, BGA, and rhizobia are bio-inoculants and crop-specific, for example, Azospirillum, Azotobacter, VAM, and PSB can be observed as wide-ranging biofertilizers. VAM are fungi that are found accompanying through the mainstream of improved accretion of plant nutrients and farming crops. The photosynthetic bacteria Rhodobacter, facultative anaerobes, Clostridium

112  Biofertilizers Table 3.1  The different types of microorganisms utilized for different types of biofertilizers. Type of biofertilizer

Groups

Examples

Nitrogen-fixing biofertilizers

Symbiotic

Frankia, Rhizobium, Azollae, Anabaena

Free-living

Bejerinkia, Azotobacter, Klebsiella, Clostridium, Nostoc, Anabaena

Associative symbiotic

Azospirillum

Ectomycorrhiza

Pisolithus Spp., Laccaria Spp., Amanita Spp., and Boletus Spp.

Arbuscular Mycorrhiza

Gigaspora Spp., Scutellospora Spp., Sclerocystis Spp., Acaulospora Spp., and Glomus Spp.

Orchid Mycorrhiza

Rhizoctonia solani

Ericoid Mycorrhiza

Pezizella ericae

Fungi

Penicillum Spp., Aspergillus awamori

Bacteria

Phosphaticum, Bacillus subtilis, Bacillus circulans, Bacillus megaterium var

PGP Rhizobacteria

Pseudomonas

Pseudomonas fluorescens

Biofertilizers for micronutrients

Bacillus Spp.

Zinc and silicate solubilizers

Phosphate mobilizing biofertilizers

Phosphate solubilizing biofertilizer

pasteurianum obligate aerobes, some methanogens, and Azotobacter (cyanobacteria) are an example of free-living N-fixing microorganisms which are causing anaerobic conditions. The Bacillus mucilaginous is the utmost used K-solubilizer whereas Bacillus circulans, Bacillus megaterium, Pseudomonas straita, and Bacillus subtilis are P-solubilizers [32]. Different biofertilizers comprised in Figure 3.1.

N2 Fixation in Biofertilizers  113 Biofertilizers

N2 Fixing Microbes

Symbiotic N2 Fixers

Rhizobium

Nutrient Solubilizing Microbes

Non-Symbiotic N2 Fixers Azotobactor and Clostridium Azospirillum Azolla Blue green algae (BGA)

Phosphorus Solubilizing Microbes (PSM) Potassium Solubilizing Microbes (KSM) Vesicular Arbuscular Mycorrhiza (VAM)

Figure 3.1  Different microorganism as biofertilizers.

3.10 Microorganism in Nitrogen Fixation 3.10.1 Biofertilizers: Symbiotic N-Fixers Various genera of family Rhizobiaceae, for example, Azorhizobium, Allorhizobium, Mesorhizobium, Bradyrhizobium, Rhizobium, and Sinorhizobium, are the most attainable symbiotic nitrogen fixers. A symbiotic bacterium, i.e., Rhizobium found in communal association in root nodules of host plants. Through the reduction of molecular N2 to NH3 derivatives, Rhizobium principally fixes the environmental N2 gas in the root nodules. These fixed NH3 derivatives further utilized to produce a variety of nitrogen-containing compounds, proteins, and vitamins by the plants. By the role of dinitrogenase with Fe and Mo as its cofactor, the process of N-fixation, which equally comprises a multifaceted nitrogenase enzyme made of iron as its cofactor known as dinitrogenase reductase [33]. The nif genes are found in free-living besides symbiotic microorganisms, responsible for the process of Nitrogen fixation [34]. Deprived of the use of nitrogen fertilizer to validate the adequate source of N2 for legumes injection of Rhizobium is an important preparation in farming [35]. In connotation through the N2-fixing Anabaena azollae (BGA), Azolla partake capability to fix the environmental N2. For the commercial nitrogen fertilizers, Azolla plays a role as an alternating source of nitrogen [36].

114  Biofertilizers Acetobacter is a necessary aerobic nitrogen-fixing bacteria, which is a significant inoculant for sugarcane and colonizes the roots of diverse sorts of sugarcane [37].

3.10.2 Biofertilizers: Free Living N-Fixers Azotobacter, which generally exists in the neutral besides the alkaline soils, is a significant free-living N-fixer of Azotobacteriaceae family. Azotobacter fixes environmental N2 deprived of any symbiotic association and do not essential a precise host in non-leguminous plants, mainly vegetables, rice, and cotton. Azotobacter is likewise able to improve the grain harvest and develop, especially in wheat crops [38, 39]. Pseudomonas and Azotobacter immunization and insemination reduced the submission of the chemical fertilizers through 25%–50% in the field [40]. Among many classes of Azotobacter, Azotobacter chroococcum is the leading occupant of the arable soils the C-substrate used in culture media partake the capability to fix around 2–18 mg/g of N2 [41]. The Azotobacter insignis, A. beijerinckii, A.  vinelandii, and A. macrocytogenes, are the additional species of Azotobacter used for the fixation of atmospheric nitrogen [42]. To conceal vitamin B complex, phytohormones, and also additional bioactive mixtures, the varied classes of Azotobacter were reported that could further promote uptake of the minerals and growth of the roots and act as biocontrol agents against root pathogens [43]. Some additional compounds, such as antifungal compounds which are utilized to hinder the development of many pathogenic microbes, produced through Azotobacter indicum [44]. BGA or cyanobacteria are principally free-living nitrogen fixers and photosynthetic. Nostoc, Anabaena, Aulosira, and Calothrix are the leading fixers for atmospheric nitrogen [45]. The excretion of many developments endorsing constituents on BGA augmenting the water holding capacity [46], which comprise various amino acids, vitamins, and phytohormones [47], averting the development of the unwanted plant, decreasing the salinity of the soil, and through manufacture of numerous organic acids increment in the availability of soil productivity [48].

3.10.3 Biofertilizers: Associative Symbiotic N-Fixers Azospirillum is mostly linked through diverse grasses is the utmost associative symbiotic nitrogen fixer [49–52]. Currently, the Azospirillum thiophilum, A. zeae, A. picis, A. rugosum, A. melinis, A. oryzae, A. largimobile, A. lipoferum, A. humicireducens, A. irakense, A. halopraeferens, A. himalayense, A. amazonense, A. formosense, A. doebereinerae, A. fermentarium,

N2 Fixation in Biofertilizers  115 A. brasilense, and A. canadense are the 17 species of Azospirillum. Among all these species, the Azospirillum brasilense and Azospirillum lipoferum are the utmost well described and considered [53]. From the aerial part of plants besides soil partaking capability to fix nitrogen, these species have been isolated. The cytokinins, gibberellins, and IAA are the growth promoters which have been produced by Azospirillum [54]. The results showed that through persuading variations in osmotic adjustments and pliability of the cell wall, Azospirillum could benefit in the existence of the plants under stressful circumstances [55, 56]. In many nations, such as Argentina, Africa, Belgium, Australia, Germany, Brazil, India, France, Mexico, Italy, Uruguay, Pakistan, and the USA, Azospirillum strains are promoted as biofertilizers [57, 58].

3.11 Phosphorus Solubilizing Microbes Phosphorus (P) performs a vital role in the development of crops and their biological growth among the vigorous macronutrient [59]. Its solvable concentration is inaccessible for the plants and tiny despite its in height total attentiveness. It occurs in organic and inorganic forms in soil. Subsequently, the submission of the chemical fertilizers regularly looks inorganic P in the soil. Instead, biological substance systems of P accounts for around 20%–80% of the soil are an imperative reservoir of immobilized P [60]. The hydrated oxides, for example, manganese, aluminum, iron, apatite, and hydroxyapatite, are the key reserves that are slightly soluble and signify the mineral systems of the P in the soil. PSM partake the capability of adapting the unsolvable system of P to a solvable system, are well known to endorse development plant parts. The chelation, exchange reactions, and acidification are the diverse methods that are intricated in this adaptation. The acid phytases and phosphatases show a significant part in the soil P solubilization process by the P-solubilizers [61, 62]. It has resulted that through the manufacture of several phytohormones, for example, IAA and even harvest siderophores, PSM promote the growth of the plants, which act as biofertilizers [63].

3.12 Conclusion and Future Prospect In the area of PGP microorganisms as biopesticides and biofertilizers, it has attained a considerable wide-reaching development. The associates of the diverse bacteria reimburse the development of the soil and the plant’s health. In this chapter, we have comprised various microorganisms which

116  Biofertilizers act as biofertilizers and also the nitrogen-fixing bacteria. First, many innovative methods, for example, growing of microorganisms, accumulation of these microorganisms, and additional appropriate conveniences for distribution, applying and framing these microorganisms for moving from greenhouse and laboratory to field test will cause. Then, the utilization of these microorganisms on a large scale would be vital to instruct the public, as there is a popular mythology that microbes are contributing mediators of ailment. Earlier, the usage of these microorganisms on a large scale, these misapprehensions have to be detached before applying to the environment. To combat with pathogenic diseases, several chemical pesticides, are used which leads loss of crops as well makes it unfit and possess ecological problems. Therefore, it is vital to eliminate these problems. For nourishing the developing populace, biofertilizers will act as a promising substitute and will enhance the development of plants and advance the yield. By the use of biofertilizers, we can advance the farming activities by making them cost-effective, easy to be done, and biodegradable accumulation of these fertilizers. In this chapter, we have also comprised the different nitrogen-fixing microbes and other substitutes for the easy making of biofertilizers. This study will help the reader choose microorganism and their respective biofertilizer to attain a high yield in farming areas.

Acknowledgments The authors gratefully acknowledge the support from the Ministry of Human Resource Development Department of Higher Education, Government of India, under the scheme of Establishment of Centre of Excellence for Training and Research in Frontier Areas of Science and Technology (FAST), for providing the financial support to perform this study vide letter No, F. No. 5–5/201 4–TS.Vll. Dinesh Kumar is also thankful DST, New Delhi, for financial support to this work (sanctioned vide project Sanction Order F. No. DST/TM/WTI/WIC/2K17/124(C)).

Abbreviations BGA PSM IAA PGP N-fixers

Blue-green algae Phosphate solubilizing microbes 3-Indoleacetic acid Plant growth-promoting Nitrogen fixers

N2 Fixation in Biofertilizers  117 K-solubilizer P-solubilizer P-mobilizers PSB VAM

Potassium solubilizers Phosphorus solubilizer Phosphorus mobilizers Phosphorus solubilizing bacteria Vesicular Arbuscular Mycorrhiza

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120  Biofertilizers 48. Wilson, L., Cyanobacteria: a potential nitrogen source in rice fields. Texas Rice, 6, 9, 2006. 49. Babalola, O.O., Glick, B.R., The use of microbial inoculants in African agriculture: current practice and future prospects. J. Food Agri. Environ., 10, 540, 2012. 50. Bashan, Y., De-Bashan, L.E., Chapter two-how the plant growth-promoting bacterium Azospirillum promotes plant growth—a critical assessment. Adv. Agron., 108, 77, 2010. 51. Duca, D., Lorv, J., Patten, C.L., Rose, D., Glick, B.R., Indole-3-acetic acid in plant–microbe interactions. Antonie Van Leeuwenhoek, 106, 85, 2014. 52. Glick, B.R., Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiol. Res., 169, 30, 2014. 53. Rodrigues, A. C., Bonifacio, A., de Araujo, F. F., Junior, M. A. L., Figueiredo, M. D. V. B., Azospirillum sp. as a challenge for agriculture. In Bacterial metabolites in sustainable agroecosystem. Springer, Cham. 29, 2015. 54. Fibach-Paldi, S., Burdman, S., Okon, Y., Key physiological properties contributing to rhizosphere adaptation and plant growth promotion abilities of Azospirillum brasilense. FEMS Microbiol. Lett., 326, 99, 2011. 55. Groppa, M.D., Benavides, M.P., Zawoznik, M.S., Root hydraulic conductance, aquaporins and plant growth promoting microorganisms: a revision. Appl. Soil. Ecol., 61, 247, 2012. 56. Richardson, A.E., Barea, J.-M., McNeill, A.M., Prigent-Combaret, C., Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms. Plant Soil, 321, 305, 2009. 57. Hungria, M., Campo, R.J., Souza, E.M., Pedrosa, F.O., Inoculation with selected strains of Azospirillum brasilense and A. lipoferum improves yields of maize and wheat in Brazil. Plant Soil, 331, 413, 2010. 58. Mehnaz, S., Azospirillum: a biofertilizer for every crop. In Plant microbes symbiosis: Applied facets. Springer, New Delhi. 297, 2015. 59. Soetan, K., Olaiya, C., Oyewole, O., The importance of mineral elements for humans, domestic animals and plants-A review. Afr. J. Food Sci., 4, 200, 2010. 60. Hariprasad, P., Niranjana, S., Isolation and characterization of phosphate solubilizing rhizobacteria to improve plant health of tomato. Plant Soil, 316, 13, 2009. 61. Kumar, V., Singh, P., Jorquera, M.A., Sangwan, P., Kumar, P., Verma, A., Agrawal, S., Isolation of phytase producing bacteria from Himalayan soils and their effect on growth and phosphorus uptake of Indian mustard (Brassica juncea). World J. Microbol. Biotechnol., 29, 1361, 2013. 62. Relwani, L., Krishna, P., Reddy, M.S., Effect of carbon and nitrogen sources on phosphate solubilization by a wild-type strain and UV-induced mutants of Aspergillus tubingensis. Curr. Microbiol., 57, 401, 2008. 63. Hariprasad, P., Niranjana, S., Isolation and characterization of phosphate solubilizing rhizobacteria to improve plant health of tomato. Plant Soil, 316, 13, 2009.

4 Organic Farming by Biofertilizers Anuradha1 and Jagvir Singh2* Department of Zoology, Raghuveer Singh Government Degree College, Lalitpur, U.P., India 2 Department of Chemistry, ARSD College (University of Delhi), New Delhi, India 1

Abstract

Biofertilizer is one of the best, simple, and inexpensive equipment of modern fertilizers. In today’s era, it is a gift to our agricultural science. Biofertilizer has been used in our agricultural sector since the earliest ages in India, which has also proved to be very useful today as a replacement for our traditional fertilizers. Domestic waste, dung, and green manure are in the form of traditional fertilizers. The effect of these fertilizers is less than chemical fertilizers, that is, they do not affect the environment as much as chemical fertilizers. Therefore, considering the purpose of crop development, farmers often try to use these chemical fertilizers in their crops. Since these chemical fertilizers used adversely affect our environment and pose a threat to life there. These fertilizers are mainly responsible for giving rise to pollution of water, air, and soil and lead to fatal diseases like cancer. Simultaneously, chemical fertilizers used can destroy the fertility power of the crop soil after a long time. Now, we have only one option biofertilizer which has proved to be helpful in dealing with the problems created and creating a healthy world. Since biofertilizer contains microorganisms that provide proper growth to the vegetative world as well as nutrition and development in physiology and regulation. It is prepared by the cooperation of living microorganisms in the making of these fertilizers. In this reaction, only those microorganisms are introduced which provide specific contribution to enhance the sustainable development and reproduction of the flora. Being an essential component of organic farming, biofertilizers play an important role in creating soil fertility and stability, reducing the negative impact of the environment and saving life on earth by improving the ecosystem.

*Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (121–150) © 2021 Scrivener Publishing LLC

121

122  Biofertilizers Keywords:  Biofertilizer, seedling treatment, Setts and Tuta treatment, cyanobacterial biofertilizer, mycorrhiza biofertilizers, vermicompost, organic farming, weed management

4.1 Introduction Biofertilizers are a type of organism that enhances the nutritional quality of the soil [1, 2]. These are the main sources of bacteria, fungi, and cyanobacteria [3, 4]. The glands at the roots of the dicotyledonous plants are formed by the symbiotic association of Rhizobium [5]. These bacteria stabilize atmospheric nitrogen and convert it into an organic form, which plants use as nutrients [6, 7]. Other bacteria such as Azospirillum and Azotobacter live in the soil. It can also stabilize atmospheric nitrogen. Thus, the nitrogen content in the soil increases [8]. It was known to people for a long time that pulses increase the fertility of the soil. But the scientific demonstration of this thing was achieved only after half of the 19th century. Knees are formed for nitrogen fixation in the roots of plants of Dahlan clan itself. The pulses clan is divided into three subscales: Mimosidi, Sijalpinidi, and Papilionidi. Knees are formed for nitrogen fixation in the roots of 90% of mimosidi and 23% of cajalpinidi and 97% of the members of papillonidi [9]. The commercial history [10]. of biofertilizers began in 1895 when the nitrogenous product started as Rhizobium culture in collaboration with two scientists “Mr. Knob and Mr. Hiltner”. This was followed by the discovery of Azotobacter and Indigo Green Algae and other microorganisms. Azospirillum and Vesicular Arbuscular Mycorrhiza (VAM) are recently discoveries. The first legume Rhizobium symbiosis in India was studied by Indian scientist Dr. NV Joshi. Its first commercial production started in 1956. During the ninth 5-year plan of the government of India, the Ministry of Agriculture started realistically promoting it and creating awareness among the people through the “National Project for the Use and Development of Biofertilizer” [11]. Friendly fungi (such as mycorrhiza) establish symbiotic relationships with plants. Many members of the genus Glomus make mycorrhiza [12]. In this symbiosis, fungi absorb phosphorus from the soil [13] and send it to the plants. Tactile plants from such relationships exhibit many other benefits such as immunity to root-bearing pathogens, salinity and tolerance to drought, and growth and development [14]. Many problems are associated with the increased use of chemical fertilizers in farming. As a result, the emphasis is being laid on organic farming and the use of biofertilizers [15, 16]. The availability of biofertilizer in

Organic Farming by Biofertilizers  123 India has also started recently and Indian farmers are using them in their crops. Its use has reduced the nutrient replenishment and dependence in the agricultural land here compared to chemical fertilizers. Stacked chemical fertilizers increase the yield of crops, but the continued use is deteriorating soil fertility and structure, so the use of chemical fertilizers as well as biofertilizers makes the fertile land increasingly fertile. The use of biofertilizers provides the soil with nutrients and soil, thereby increasing the yield. Farmers have started using chemical fertilizers and poisonous pesticides for the production of their crops, which are harmful to both the health and soil of humans [17]. Along with that the environment is also getting polluted. To stop all these things, if the farmer uses organic methods instead of chemical methods, then these problems can be overcome to a great extent.

4.2 Biofertilizers Nitrogen, potassium, and phosphorus are the most essential elements for any crop [18], and if they are deficient in plants or crops, then fertilizer is helpful in fulfilling them. If this fertilizer is a chemical compound, then it has a negative impact on the environment of the place where it is used. These hazardous chemical fertilizers can be reduced by the use of biofertilizers. Biofertilizers are fertilizers in which microorganisms work to reach nutrients to crops or plants through some chemical reactions and this type of farming is called organic farming. Biofertilizer has proved worthwhile in interdisciplinary fields [19] due to its multi-utility as shown in Figure 4.1. Researchers have identified and used various natural bacteria for sustainable crop production in what is known as biofertilizer or “bacterial fertilizer”, which is a type of microorganism containing live fertilizer. Biofertilizers can give better results by using chemical fertilizers as a supplement. In fact, biofertilizers are mixtures of particular microorganisms and any moisture-bearing substances. Biofertilizers are prepared by mixing specified amounts of special microorganisms in a moisture-laden dusty substance (like charcoal and lignite). It is often available in the market in the name of “culture”. Actually, biofertilizer is a natural product. They can be used in various crops and for the partial supply of sulfur. Their use does not have any side effect on the land; rather they improve the physical and biological properties of the land and help in increasing its fertility. Biofertilizers have an important role in organic farming. The effect of bacterial manure is gradual. About two to three billion

124  Biofertilizers

Suppress soil-borne pathogenic diseases in crop

Supply mixture of nutrients

Enhanced crop productions

Biofertilizer

Improved soil health

Maintenance of symbiotic relationship

Maintain microbial consortia in soil

Figure 4.1  Multifunctional areas in which biofertilizer is used extensively and they expand organic farming.

microorganisms are found in 1-g soil of our field, which consists mainly of bacteria, fungi, fungi, and protozoa. The first legume Rhizobium symbiosis in India was studied by Dr. NV Joshi. Its first commercial production started in the year 1956. These biofertilizers can be classified as follows, considering microorganisms as the basis. The use of biofertilizers and throughout the year demand in Indian states [20] is shows in Table 4.1 in a listed manner.

Organic Farming by Biofertilizers  125 Table 4.1  Collection of biofertilizers quantity (tones) in different states of India in a statistically listed manner. S. no.

State-wise production of biofertilizer in India, 2011–2012

Quantity (Tones)

1.

Maharashtra

8,743.69

2.

Uttar Pradesh

8,695.08

3.

Karnataka

5,760.32

4.

Tamil Nadu

3,373.81

5.

Madhya Pradesh

2,309.6

6.

Delhi

1617

7.

Tripura

1,542.85

8.

Andhra Pradesh

1,126.35

9.

Haryana

914.41

10.

Kerala

904.17

11.

Punjab

692.22

12.

West Bengal

603.2

13.

Orossa

590.12

14.

Pondichary

509.45

15.

Gujarat

276.34

16.

Uttraakhand

263.01

17.

Rajasthan

199.78

18.

Bihar

75

19.

Assam

68.33

20.

Nagaland

13

21.

Jharkhand

8.38

22.

Himachal Pradesh

1.29

126  Biofertilizers

4.2.1 Benefits of Biofertilizers Following are the main benefits from biofertilizers: 1. O  rganic fertilizers can be made at a low cost and the method of making them is simple. 2. The use of them increases soil fertilizer power and promotes sustainable agriculture production. 3. The use of biofertilizers increases the physical and chemical properties of the soil and increases the ability to hold water, so the bearing land can also be made fertile [21]. 4.  Nitrogen depletion in the soil can be accomplished by the use of bacteria such as Rhizobium, Azotobacter, and Azospirillum [5]. 5. Cyanobacteria increase the amount of organic substance in the soil by secreting protein, amino acid vitamin, and stabilize dinitrogen. 6. The use of mycorrhiza biofertilizer increases the surface area of the roots. VAM is very important for phosphate nutrition in plants. The capacity of absorption of water and mineral salts from the ground increases, due to which the production increases. 7. Biofertilizers are produced by natural resources. Therefore, being environment friendly, they do not pollute the environment.

4.2.2 Method of Biofertilizer Application Biofertilizer is produced by living microorganisms. Therefore, caution should be exercised in their use so that microorganisms are not adversely affected [22]. There are generally four methods of using them.

4.2.2.1 Seed Treatment The amount of biofertilizer is used depending on the seed size of the crops sown by the seed. Generally, 200-g biofertilizer is sufficient for the treatment of 10–15 kg of medium sized seeds which can make 400–500 ml solution in a glass of water. Then, pour the solution on 10–12 kg of seeds and mix well by hand and place it on shady place on clean sack/paper to dry for a short time (10–15 minutes). Sow immediately after this.

Organic Farming by Biofertilizers  127

4.2.2.2 Seedling Treatment Transplanting crop is used for sowing. The roots of their infant plants are sown immediately after dipping in a solution of biofertilizers for 10–15 minutes. To make the solution, 1–2 kg biofertilizer is mixed in 5–10 L of water in a large vessel.

4.2.2.3 Setts and Tuta Treatment This method is used in sugarcane and crops like potato. Normally, in an acre of land, 2–4 kg of biofertilizer solution is prepared in 20–40 L of water. In this solution, Selts (Pade) or tuber (Tuta) are treated by immersing them for 10–15 minutes or this solution is filtered and sprayed with a pump.

4.2.2.4 Soil Treatment For short duration crops, 3–5 kg of biofertilizer is blended with 60–100 kg of the same field soil or indigenous manure. After sprinkling the water overnight and spraying it over the standing crop in the field, the crop is watered. If treatment is to be done in crops for long duration, then soil treatment is done according to the above method. Just take biofertilizer quantity up to 6–7 kg and take soil or desi fertilizer of 100–120 kg.

4.2.3 Precautions During Application of Biofertilizers The following precautions should be taken in using biofertilizers. 1. A  void chemical fertilizers and pesticides at the time of their use and storage. 2.  If using insecticidal drugs, do it before treatment with biofertilizers. 3. While using biofertilizer in any crop, keep in mind that phosphate solubilizing biofertilizer must also be used along with that crop specific nitrogen fixation biofertilizer. 4.  Store biofertilizers in a room where the temperature is 20°C–28°C. 5. Make storage in a place where the sunlight does not fall directly. 6. Use it only till the last usage date mentioned on their packet. 7. Rhizobium biofertilizer is crop specific. Therefore, use that Rhizobium biofertilizer in the crop specified on the packet.

128  Biofertilizers

4.3 Classification of Biofertilizers Technically, biofertilizers have three types of basis of their work which are tabulated in Table 4.2. The biofertilizer is further divided into three parts according to microorganisms and these are the following.

4.3.1 Nitrogen Fixer Bacteria Bacterial biofertilizers are beneficial for farming and agricultural operations; hence, they are used. Some bacteria stabilize atmospheric nitrogen (N2) and give it to plants. About 79% of nitrogen is found in the atmosphere. This nitrogen is converted into nitrogen compounds by various physical and chemical changes that the plants receive. This reaction is known as fixation of nitrogen [23, 24]. When it rains and lightning flashes and the atmosphere reacts with nitrogen, oxygen to form nitrogen oxides which dissolve in water and reach the soil with rainwater in the form of nitrate ( NO3− ) which is absorbed by plant roots [25]. Some biotic bacteria such as Azotobacter and Clostridium [26] are found in the soil, which converts atmospheric nitrogen to ammonia (NH3). Nitrates are lost by many methods of nitration ( NO3− ) reduction and nitrate ( NO3− ) assimilation in soil, and this process is called denitrification. For example, nitratesnitrites–oxides of nitrogen–nitrogen (N2) gas–ammonia (NH3) compost or pile manure has very low denitrification. This entire process is known as the nitrogen cycle as shown in Figure 4.2.

Table 4.2  Based on the three types of biofertilizers and their major function, they are classified in a listed manner [27]. S. no.

Biofertilizers

Their work

1.

Obligate Aerobes

Microorganisms carry out their mechanism in the presence of oxygen such as Azotobacter.

2.

Obligate Anaerobes

Such microorganisms maintain their mechanism in the absence of oxygen.

3.

Facultative Anaerobes

Such microorganisms generally carry out their activities in the presence of oxygen, but also function smoothly in its absence.

Organic Farming by Biofertilizers  129 Atmospheric Nitrogen (N2) 79%

Nitrogen fixing convert N2 into NH3

Denitrifying bacteria NO3- convert into N2

Nitrifying Bacteria NH3 convert into NO2- & NO3-

Figure 4.2  Nitrogen convert to ammonia which used by plants and nitrifying bacteria and convert ammonia to nitrite and nitrate and nitrite which further used by denitrifying bacteria and convert into nitrogen. Now, this nitrogen free to atmosphere and this cycle is known as nitrogen cycle [28].

In addition, some bacteria such as Bacillus Radicicola, Rhizobium, Gram, Pea, Arhar, and Sam are found in root glands of roots which are called symbiotic bacteria. Symbiotic bacteria convert nitrogen into ammonium compounds and collect in the original glands that the plants receive. Leguminous plants are harvested from the ground to obtain organic fertilizer by the original glands, but the root including their root glands (root nodules) is left in the ground which after some time rot and are used as fertilizer beneficial for other crops.

4.3.1.1 Commercial Applications In the modern era, the culture of the Bacterium Rhizobium is cultivated at a commercial level. For this culture, extract of yeast is a medium, whose composition is as follows in Table 4.3: Rhizobium Meliloti and Rhizobium are used in the US and other countries for bacterial manure. The culture of these bacteria is largely done on yeast extract mannitol medium. In India, too, bacteria use yeast extract mannitol medium for culture. Shake this medium in large-sized flasks with a shaker for several hours and then treat it with CaCO3 and autoclave by plugging a cotton plug into the flasks. After this, let the medium cool down

130  Biofertilizers Table 4.3  The production of yeast extract mannitol medium with their quantity in different dose [29]. S. no.

Composite

Quantity

1.

Mannitol

10.0 g

2.

Potassiyum hydrogen phosphate (K2HPO4)

0.5 g

3.

Magnesium sulfate (MgSO4)

0.2 g

4.

Yeast extract

1.0 g

5.

Sodium chloride

0.1 g

6.

Distilled water

1.0 L

and then add Broth. After this, it is dried in tray. Thereafter, they are filled in polythene bags and distributed to the farmers. In addition, other media are also used, in which Azotobacter Chroococum and Bacillus Megatherium are grown. In India, these bacteria also make manure at the commercial level.

4.3.2 Cyanobacteria as Biofertilizers Blue-green algae or cyanobacteria have unprecedented potential for nitrogen fixation [30]. Based on their experiments, scientists have made it clear that species of filamentous cyanobacteria that hold heterocyst, such as oloceira, tolypothrix, anabaena, anabaena, cylindrospermum nostoc, and mastigocladus, do nitrogen fixation in paddy fields. These include nitrogen in the presence of the nitrogenase enzyme. It has been observed that when these organisms die, they get ammonia (NH3) from their body which is converted into nitrates ( NO3− ) by nitrifying bacteria. Several important works have been done on blue-green algae named Tolypothrix in Japan and Aulosira Fertillissima in India, and it has been clarified that 30 quintals of rice is grown by growing these algae in paddy fields which percentage yields are high. Phalaenopsis and Spirulina are being used as fertilizers in also India. Lyngbya and other cyanobacteria secrete antibiotics that destroy Pseudomonas and Mycobacterium. Apart from this, other members also have the property of destroying the larvae of mosquitoes and they are used as a larvicide.

4.3.2.1 Commercial Applications In the modern era, blue-green Bacteria such as Anabena, Tolypothrix, Aulosira, Nostoc, and Plectonema culture are cultured at a commercial

Organic Farming by Biofertilizers  131 level in laboratories which is used as a biofertilizer. Tray is used for preparing culture, which is 9 inches in length, 6 inches in width, and 3 inches in depth. The algae that are to be cultured are kept in a tray with pure soil and put 10 kg of soil and 200 g of superphosphate and then 2 to 2.5 inches in the tray. Fill the water up to an inch, the soil settles in the bottom. After that, spraying the sawdust over the water-filled in the tray, keep it in the sunlight. After a week or two, blue-green bacteria (cyanobacteria) grow and float on the water surface. They are carefully collected and filled in polythene bags and sent to the markets. From where farmers buy them and use them as fertilizer in their fields. This fertilizer is very cheap. It is estimated that fertilizer of 30 rupees is sufficient for one hectare of land. Efficient strains for the production of cyanobacteria are cultured in open tanks by selecting efficient strains. Water-filled mineral elements such as molybdenum (Mo) and phosphate (PO4) are added in plenty in the tanks. After this, the growth of cyanobacteria is allowed, and finally, the surface of the water is taken out and dried in the sun and its powder is made and mixed into the carrier. This mixture is packed in polythene bags and distributed to the farmers, which are then opened and spread by the farmers in the cultivated soil which acts like fertilizer. It has been observed that the application of cyanobacteria to rice fields does not require nitrogen fertilizers and other crops benefit from cyanobacteria residues. In cyanobacteria, the production of heterocyst rice increases by 10% to 30%. Scientists estimate that 25 kg per hectare using cyanobacteria fertilizer, nitrogen (N2) can be replenished.

4.3.2.2 Factors Affecting Cyanobacteria Biofertilizer The tabulated factors in Table 4.4 are given with their affecting xyanobacteria biofertilizers.

4.3.3 Mycorrhiza as Biofertilizers According to Frenk, mycorrhiza (Mycorrhiza, Mycos = Fungus, Rhiza = Root) means fungal origin [31]. In high-grade plants that spend their lives like a dead animal, their roots have many fungi that help in the absorption of organic substances in plants such as Monotropa and Neotia. Fungi do not have a specific relation to plant roots. Many species of fungi are found on the same plant root, or fungi of the same species are found on the roots of many plant species. Mycorrhizas are of the following types.

132  Biofertilizers Table 4.4  Tabulated factors directly affect the functioning of cyanobacterial biofertilizer [32]. S. no.

Factors

Actions

1.

Light

It is very important for photosynthetic species.

2.

Temperature

It is favorable for growth and consider to be from 32°C to 35°C.

3.

pH value

It is well increased between 6.0 and 7.5. Growth at pH less than 6.0 is adversely affected.

4.

Humidity

It grows in stagnant clean water at a rapid rate; it goes into dry states in dry states.

5.

Oxygen

It is essential need for every species.

4.3.3.1 Ectotrophic Mycorrhiza In this, fungus hyphae form a layer or mantle on the surface of the root of the nutritious plant. The nutritive plants on the epiblema of the root of the plant enter the hyphae and form a net. Most of the fungal fibers which are spread on the surface of the root enter the soil containing organic matter, which by secretion of some enzymes make organic matter soluble and absorbs them [33]. In this way, the absorbed organic material reaches the fungal fibers located in the root cells by which the plant nourishes. Root follicles are not found on the epithelium of the roots of such plants. Examples can be found in coniferous pines, oak in phagaceae, and birch in beech or betulaceae. Some plants such as Monotropa and Sarcodes are found in the roots of total Saprophyte Mycorrhiza. Their roots are completely surrounded by the mantle of fungi and this fungus for plants absorbs complete nutrition. These plants lack chlorophyll. Many forest trees such as Salix, Eucalyptus, Cedrus Deodara, Picea, and Popix also have ectotrophic mycorrhiza attached to them, causing roots. The morphology changes and the root hairs also do not develop. In this way, the relationship between nutrients and fungi is very beneficial because plants with mycorrhiza have an increased ability to absorb mineral salts.

4.3.3.2 Endotrophic Mycorrhiza In this type, the fungus does not form a layer on the root of the nutritious plant root but spreads on the surface of the root. Some of these fungal

Organic Farming by Biofertilizers  133 fibers enter the soil and some fungi penetrate deep into the cortex cells of the root and lie in the intercellular spaces [34]. Fungi get sugars and vitamins from the host plant. There is no harm to the plant by fungi. This fungus absorbs organic materials, minerals, and nitrogenous substances with the help of hyphae from the soil and delivers nutrients to the plant like tuber of green orchids [35]. It has been proved by experiments that if the nutritious plant and the fungi found in it are developed separately, the growth of both slows down very much. In this regard, fungi get sugary vitamins (Biotin and Thiamine) and amino acids from the nutritious plant. The fungus helps in increasing the absorption surface of the roots and helps in collecting the nutrient unit area in the plant. It has been observed that the seeds of the green orchids do not germinate well until they are infected with a fungus, it is estimated that the infant seedling fungi. They absorb their nutrition like potassium, phosphorus, nitrogen but these plants do not have any important function when the plants mature. These fungi also secrete beneficial auxins. The fertilizer stored in the dung heap is not nitrified, but fertilizer starts to nitrify quickly when applied in fields. Hemorrhoids have high organic content and lack of air. Hence, nitrification is not possible.

4.3.3.3 Changes in Mineral Compounds Phosphorus in cow dung or shit matter is mostly organic or inorganic form carbonic phosphorus which is not available to plants, often turns into inorganic phosphorus. Potassium is found mostly in urine which is easily found in the form of salts or in the soluble state [36]. The part of the potassium is converted into potassium carbonate which neutralizes the acids produced by the breakdown of carbohydrates. The salts of insoluble calcium, magnesium, and potassium are converted into soluble states after dissolution which is absorbed by plants.

4.3.3.4 Manure Value and Its Importance An average grade of cow dung manure normally prepared for cultivation contains 0.5% nitrogen (N2), 0.25% phosphorus pentoxide (P2O5), and 5% potassium oxide (K2O). Excess micronutrients like nitrogen, phosphorus, and potassium are also found in cow dung. The crops grown in the first year receive 30%–40% nitrogen, 20%–25% phosphorus, and 50%–70% potash from this manure. Nutrients are gradually obtained by this fertilizer to the plants, and thus, the effect of cow dung manure lasts for many years in the soil. Good

134  Biofertilizers rotten cow dung manure can be given in small plants without any harm. Phosphoric acid and potash are present in sufficient quantity in cow dung manure. The amount of calcium in the soil of the farm is increased by this manure, due to which the physical state of the soil improves. Dissection of living matter gives colloidal humous which increases the water-holding capacity of sandy soils and makes the spongy of the clay. This fertilizer leaves water slowly, due to which water plants keep getting for a long time. Some such nitrogenous substances are found in cow dung manure, which is helpful in the growth of plants.

4.3.4 Azolla as Biofertilizer Azolla is a low-grade plant found in floating floats in clean water. Species of this are A. carolinii, A. filiculoides, A. maxicana, A. microphylla, A. nilorica, A. pinetta, and A. rubra. A cavity is found on the dorsal surface of Azolla, in which the blue-green algae Anabanea Azolle living as symbiotic with Azolla is found [37]. Its utility is for nitrogen fixation. Example Using a 10-ton Azolla plant, 40 kg of biologically socialized nitrogen, soil Table 4.5  Tabulated factors and their influences directly affect the functioning of Azolla biofertilizer [38]. S. no.

Factors

Influence

1.

Soil and Scur

Azolla Nursery, putting single super phosphate is beneficial and this fertilizer adversely affects its growth.

2.

Temperature

20°C–32°C temperature is favorable for its growth.

3.

Soil pH

The organism cycle of Azolla ranges from 3.5 to 10.0 pH value but the pH value favorable for nitrogen fixation is 6.0.

4.

Biological Factors

On the growth of A. pinnata, it is very sensitive to various types of larvae, especially Lepideptera and Diptera class insects.

5.

Light

Light tolerance varies in different species. Excessive light generally adversely affects nitrogen fixation.

6.

Salinity

With the increase in salinity, the growth of Azolla also slows down and its growth stops at about 1.3% salinity.

Organic Farming by Biofertilizers  135 is obtained. Azolla is used as a green manure in rice crops in many parts of the world. Azolla is a member of the class Teridophyta, with numerous alternating branches on the rhizome attached to the lateral branches. Growth in Azolla generally occurs throughout the year but is more intense during the winter season. Factors affecting Azolla are listed in Table 4.5. Soil-based biofertilizers of blue-green algae are being commercially produced in many parts of India. Trough method in open soil, pit method, and field method are three major methods of production. VAM is used in forestry agriculture. It is found in soil. Hence, it is isolated from the soil. It can be isolated from the root zone of the plant (Rhizosphere). Its primary spore fungal network, infective roots, can be used as primary inoculum. The initial strains thus isolated can be cultured in pots with vascular host plants [39].

4.3.5 Vermicompost Vermicompost is a technique for efficient cycling of animal waste (sewage), crop residues, and agro-industrial litter [40]. The process of converting organic matter into compost is mainly of the microbial level [41]. Earthworms have an important role in the conversion of organic wastes to vermicompost. Vermicompost can be prepared from all types of organic residues [42]. Example: Agricultural residues: Dried organic waste such as Sorghum husk, which feeds on paddy straw after feeding cattle, dried leaves, pigeon pea, peanut rind, and wheat bran peel or husk.

4.3.5.1 Method of Vermicompost Garbage released from sugarcane factories. Cover the bottom of the cement ring with a polyethylene sheet. (Or use the sheet to cover the part you are using.) Spread a layer of organic waste (15–20 cm) above the sheet. Sprinkle phosphate element stones (rocks) on top of organic elements (2 kg). Prepare cow dung solution (15 kg) and pour it over the mixture in the form of the fold. Fill the ring completely, evenly, with the contents of these folds. Apply cow dung or soil coating on top of this material. Allow this material to rot for 20 days. After this period has passed, put earthworms over them. They will get holes on their own. With which they will enter the folds of this finished material. Cover the ring with gauze or sacks so that birds do not eat earthworms. Sprinkle this entire mixture with water at intervals of three days for 2 months. By doing this, the body temperature of earthworms is correct and the right amount of vapor is available

136  Biofertilizers to them. When this compost is ready, then it is black in color, is light in weight, and emits a fragrant smell [43]. After a period of 2 months (or whenever compost is ready), remove the ring and put the finished material on the floor in the shape of a cone. Leave it without stirring for two to three hours. Until the earthworms gradually reach its bottom. Separate the top of this pile. Filter the bottom of the pile so that the earthworms can be separated. They can be used again in the preparation of making more worm manure. Fill this prepared compost in bags and preserve it in a cool place [44].

4.4 Organic Farming The method of cultivation in which crops are produced without or with little use of chemical fertilizers and pesticides is called organic farming. Its main purpose is to increase the production of crops along with maintaining soil fertilizer strength [45].

4.4.1 Objectives of Organic Farming The organic farming is mainly used to save the fertilizer of soil from and to prevent the use of chemical substances in the food which indirectly take every day. Providing such nutrients to crops, which are insoluble in soil and crops and effective on microorganisms. Recycling using organic nitrogen and using organic manure and organic materials. Preventing sprinkling of weed, diseases occurring in crops, and medicines for destroying the kit, so that it does not harm the health. In organic farming, crops, as well as care of animals, which includes their habitat, their maintenance, their catering is also taken care of. The most important objective of organic farming is to secure its impact on the environment as well as the protection of wild animals and natural life.

4.4.2 Benefits of Organic Farming The most important thing in farming is two things: first is the farmer and second is the land of the farmer and the adoption of organic farming, both of them are of great benefit. By adopting organic farming, the fertile capacity of the land increases, as well as increasing the irrigation gap for crops. If the farmer does not use chemical fertilizer in farming and uses organic manure, then the cost of planting for his crop is also low. The crop production of the farmer increases, which also gives him more profit. The use of

Organic Farming by Biofertilizers  137 organic manure also improves the quality of the land [46]. With the use of this method, the capacity of water retention in the land increases and the evaporation of water are also lacking. Nowadays, our environment is also becoming very polluted and the use of organic methods for farming also benefits our environment greatly.

4.4.3 Benefit for Environment The water level of the land increases, as well as there is a reduction in pollution caused by soil, food, and water in the land by preventing the use of chemical substances. The use of animal dung and waste to make manure reduces pollution and reduces mosquitoes and other dirt caused by it, which prevents diseases [47]. If we look at the international market, then there is also a high demand for the products produced by organic farming. By doing organic farming, crop production increases, which also increases the income of farmers. In an agricultural country like India, it is very important that farmers use organic methods of farming so that the production of crops is large. The problem of this will be solved as well as the physical level of the farmers will also improve. The most of the land farming in India is based on rainfall, and nowadays, the rainfall is not getting as per the time, which also makes agricultural land barren. If farmers adopt organic farming, then this problem can also be overcome.

4.4.4 Methods of Organic Farming Organic farming is primarily a combination of ecological and modern technical knowledge of farming, along with the use of traditional methods of farming. We can study the methods of organic farming in the agroecology field. In the traditional farming method, farmers can use synthetic pesticides and water. It uses soluble artificial fertilizers, whereas, in organic farming, the farmer opposes the use of natural pesticides and fertilizers [48]. In organic farming methods, farmers mainly use crop rotation, organic manure, biological kit control, and mechanical farming, etc. These methods also require the use of natural environments to increase the production of crops such as nitrogen levels in the soil. To correct this, planting fruits, encouragement of natural insect predators, rotating crops, etc., are included.

4.4.5 Techniques for Organic Farming It does not matter how much progress a country makes. Agriculture has always been an area which has made man a place to meet food, his basic

138  Biofertilizers needs, and also as a business. As the population has increased drastically, problems have arisen in front of the agriculture industry [49] to meet the growing demand, for which different technologies have been adopted to deal with it. Many new technologies are being introduced to harvest and harvest the best quality crops in organic farming [50].

4.4.5.1 Crop Diversity Crop diversity is encouraged in organic farming, according to which many crops are produced at one place.

4.4.5.2 Soil Management Soil management is an important part of soil management; through its use, we can increase the quality of the land. To do this, we need to pay attention to soil type and soil characteristics.

4.4.5.3 Weed Management Weed means weedless vegetation that grows automatically in the midst of crops or plants and uses the nutrition received by the crops themselves, which are also disposed of in organic farming. In this way, the use of organic farming increases the production many times, as well as the environment does not get hurt and chemical-free food is also available to eat, which also does not harm the health [51]. There are many such examples, which have increased the production of crops many times already. This is the reason that educated youth are also leaving jobs and turning to farm and earning many times more profit. Organic agriculture refers to an agricultural method in which most of the elements are obtained from natural sources, rather than using chemical elements. Many people still view organic farming [52] with surprise. In fact, since the discovery of these chemical elements, the aspirations of farmers have increased. Indiscriminate pesticides and chemical substances are being used for maximum yield of the crop, but their overuse has reduced soil fertility. Our agriculture has become dependent on these chemical elements. Therefore, scientists have taken a step toward organic farming which has also been successful to a great extent. Organic fertilizers are used in organic farming by not using any kind of chemical pesticides [53, 54]. Nowadays, organic farming

Organic Farming by Biofertilizers  139 is also being done indoors. “Organic agriculture” is a biological system in which soil health does not have negative effects on the ecosystem as well.

4.5 Traditional Agriculture vs. Organic and Inorganic Farming In traditional farming, we use many types of chemical and pesticides [55] from crop bon to crop harvesting but also to the preparation of the field before sowing the seeds [56]. It could sprout easily and get out. Along with the soil, seeds are also treated (if the seed is not hybrid). Similarly, when seeds are germinating, many types of weeds are also growing at the same time. The problem of weeds is overcome by hand in organic farming and with the help of traditional agricultural weeds. Although it is a lot of hard work, it does not cause any harm to the soil, neither to humans nor to the environment. Similar farmers are using chemical fertilizers and pesticides many times from the time the crop grows to the cutting [57]. Due to this, the fertility of the soil is gradually decreasing, while on the other hand, what happens in organic farming is that, in organic farming, without using any kind of pesticides and chemical fertilizers, native manure made from animal dung as well as natural cultivating by resorting to adaptation cycle [58]. This is a big difference between organic and traditional farming. Now, we know what the benefits of organic farming are. After all, why should someone do organic farming?

4.5.1 Problems Created by Traditional Farming • Decrease soil fertility. • Nitrate flows into rainwater and contaminates drinking water sources. • Need to more fuel and tillage. • Affects human health. • Biodiversity is being destroyed by doing the same farming again and again. • Animals are also being given poisonous fodder. • Spraying of poisonous material affects the health of humans as well as soil health.

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4.6 Reasons for Doing Organic Farming Organic farming is a type of agriculture that avoids the use of synthesized fertilizers, pesticides, growth controllers, and food products of cattle, etc. Organic agriculture is entirely dependent on the cultivation of crops, plant residues, animal manures, leguminous plants, green manures, farm organic wastes and biofertilizers, mechanical farming, and mineral providing rocks [59]. To maintain soil productivity, plants have to control nutrients and biological pests, control of weeds, control of pests, and other pests. All types of agricultural products like grains, meat, milk, eggs, and fiber such as cotton, jute, and flowers can be obtained [60]. In this way, bio-agriculture helps in creating a sustainable lifestyle for the next several generations. Bio-agriculture prepares healthy soil by properly caring for the living components of the soil, in which the microorganisms [61] found in the fields play an important role, they are helpful in the transformation and transfer of nutrients [62]. The use of this system not only strengthens the composition of the land but also develops its ability to hold water. Such farmers maintain the fertility of the land through the intervention of certain crops, fertilizers, and organic matter. This system produces healthy plants, which are safer than pests and insects. The first policy of bio-farmers is to protect the plants from the control of pests and diseases through good nutrition and operation. Bio-farmers use cover crops and the cycling of plants with intelligence in such a way that the ecology of the farm is changed, and their habitat destroys weeds, pests, and disease-causing organisms. The weeds are controlled by methods of transmission such as crop rotation, technical ploughing, removal of weeds in the hands, as well as covering crops, mulch, weed burning, and other management methods. To control the bacteria that attack plants, these bio-farmers use bacteria found in the soil, beneficial to insects and birds. Bio-farmers rely on birds keeping a check on the variety of soil organisms, beneficial insects and the number of pests [63]. When the number of pesticides is highly increased, farmers protect crops using a variety of methods such as birds hunting pests, obstructing the reproductive system, using nets and inhibitors [64]. The following reasons for organic farming are discussed in detail.

4.6.1 To Save Soil Health Soil is the place where crops grow. When chemical fertilizers and pesticides were not born, farmers used compost from all types of garbage, including

Organic Farming by Biofertilizers  141 dung, which increased soil fertility. But now, all kinds of chemical fertilizers and pesticides have come on the market. Farmers use it, which increases the crop of the farmer. But with the gradual decrease in soil fertility, the farmer is moving completely on organic manure and chemical fertilizers which is a very bad sign [65].

4.6.2 To Preserve Nutrients As the number of chemical fertilizers increased, the nutrient content in the food grains and vegetables produced continued to decrease. Today, the situation is such that many vegetables and grains including brinjal have become such that more harmful elements have been made from nutrients [66]. Consumption of food made from crops produced using the use of terrible chemical fertilizers, and pesticides are spreading various diseases among the people [67]. It is difficult for the common man to get them treated. So, if organic farming is not done instead of traditional farming, then the nutrients in the food grains will not be saved.

4.6.3 To Reduce the Cost of Agriculture All types of chemical elements and pesticides are used in traditional agriculture [68]. In this, farmers have to spend a lot of money to prepare a crop. While organic farming will be done, it will not cost so much money. Instead of various types of expensive chemical fertilizers, compost is made from all the waste litter and waste including cow dung, which costs less money.

4.6.4 To Prevent Hazardous Elements in Animal Products What really happens is that a large part of the crop that the farmer grows is used as animal feed. When animals eat those four, many different types of dangerous elements are born inside them. Then, humans eat products like milk, meat, and eggs obtained from the same animals [69]. Through these milk and eggs, these elements enter into man’s body. They cause all kinds of diseases. Research published in the US claimed that more than 90% of the chemical elements that reach the human body come only through milk and meat.

4.6.5 To Protect the Environment Spraying of all types of pesticides is affecting the environment badly. It is claimed that when farmers spray pesticides, more than 90% of it gets

142  Biofertilizers into the air, whereas less than 10% of the crop is planted. In this way, when the 90% part mixes in air, the pollution level in the environment increases. There is a considerable influence on people living in that area. So, doing organic farming will help in keeping the environment clean.

4.6.6 Natural and Good Taste Once people taste food obtained from organic agriculture, and they never forget its taste because the crops and vegetables have improved flavor. One thing that is worth noting here is that the farmers doing organic farming always pay attention to quality and not quantity [70].

4.7 Advantage of Organic Farming Some of the major benefits are as follows.

4.7.1 Good Nutrition Getting good nutrition is important for every human being. In fact, nutrients play the most important role in the physical development of a human being. Many times, the complete development of children is not done due to lack of nutrients.

4.7.2 Good Health Chemical fertilizers and pesticides used in farming reach every part of our crops, when they are in use, all kinds of harmful substances enter the human body and humans suffer from all kinds of diseases. If we adopt organic agriculture, then we and our future generations will get a good health.

4.7.3 Freedom From Poison All kinds of toxic chemical elements are used in traditional agriculture. Due to which humans, animals and soil all face many problems. If organic farming is done then you will get rid of the poison [71].

Organic Farming by Biofertilizers  143

4.7.4 Less Money Common people have the impression that the value of crops derived from organic agriculture is high, but it is not so. Rather, the cost of crops and vegetables derived from organic agriculture is low because it also costs less.

4.7.5 Great Taste Who does not like a good taste? Every man wants his food to taste good and if he gets it for less money than it will continue to grow.

4.7.6 Environmental Safety Environmental protection is an issue on which all of us need to think deeply. It is impossible to stop the contaminated water and the smoke coming out of the big factories but through organic farming one can contribute toward the environment.

4.8 Disadvantages of Organic Farming Above, we read about the benefits of organic farming. When it has so many benefits, why is not everyone doing it? This is because there are also many disadvantages of organic farming, which are discussed as follows.

4.8.1 Lack of Information Farmers in India are not aware of large-scale organic farming. The government is also doing nothing to make farmers aware of this. In such a situation, it is important that we make this information available to farmers [72].

4.8.2 Lack of Outline Organic farming requires a lot of equipment, which is not available in our country right now. For this, the government should try to provide financial assistance to farmers who do organic farming [73, 74]. More and more people will move toward this.

144  Biofertilizers

4.8.3 Making More Money in the Beginning When you start organic farming, it costs a lot, like seeds, machines, and fertilizers that are very expensive for farming, due to which many farmers do not do so.

4.9 Conclusion Biofertilizers are plant-derived nutrients extracted from organisms such as algae, bacteria, and fungi that have no harmful effects on the land and the environment. Organic farming system is a type of agriculture in which artificial inorganic fertilizers, pesticides, growth controllers, and livestock feeds are avoided. Foods grown from the system of organic farming are free from harmful chemicals and the use of unnatural flavors and preservatives. Crop rotation, polyculture (multi-agriculture), and appropriate soil management systems are an integral part of a sustainable farming system, along with protective plants to maintain soil vapor levels. Vermicompost is an arrangement in which the remaining waste is fertilized by an easy method which helps to make agricultural produce healthy as well as our environment. This method is so simple that it can be prepared in your backyard, in a corner of your school grounds, or even in the corner of a public park. They are nurturing us by benefiting us from many types of organic agriculture. It has proved useful in many ways by increasing its production, avoiding environmental pollution, avoiding the harmful effects of pesticides, and saving money. With the new technology, biofertilizers are being prepared according to the crops and their crop cycles, which we should encourage them. Fertilizer chemical reactions mainly depend on several environmental factors. The ability of the earth to retain water using biofertilizers, according to its other nitrates, phosphates, and even calcium, and molybdenum levels, which helps in protein synthesis in rhizobia and alkalinity, salinity, and agricultural soil acidity, affecting all reactions, reaches the peak of progress along with the farmer.

Acknowledgement The author is grateful to Dr. H.S. Bhargav for critical reading of the chapter.

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Organic Farming by Biofertilizers  147 32. Hari, M., Perumal, K., Booklet on Bio-fertilizer (phosphabacteria). Shri Annm Murugapa Chettiar Research Centre Taramani Cheninai, 1–6, 2010. 33. Bhat, M.I., Rashid, A., Rasool, F., Mahdi, S.S., Haq, S.A., Bhat, R.A., Effect of Rhizobium and V A mycorrhizae on green gram under temperate conditions. Res. J. Agricul. Sci., 1, 2, 113–116, 2010. 34. Karandashov, V., Bucher, M., Symbiotic phosphate transport in arbuscular mycorrhizas. Trends Plant Sci., 10, 1, 22–29, 2005. https://doi.org/10.1016/j. tplants.2004.12.003PMid:15642520 35. Lovelock, C.E., Wright, S.F., Clark, D.A., Ruess, R.W., Soil stocks of glomalin produced by arbuscularmycorrhizal fungi across a tropical rain forest landscape. J. Ecol., 92, 278–287, 2004. https://doi.org/10.1111/j.0022-0477.2004.00855.x 36. Choudhury, M.A., Kennedy, I.R. Prospect and potentials for system of biological nitrogen fixation in sustainable rice production. Biological Fertilizer and Soil., 39, 219–27, 2004. https://doi.org/10.1007/s00374-003-0706-2 37. Miranda, A.F., Biswas, B., Ramkumar, N., Singh, R., Kumar, J., James, A., Roddick, F., Subudhi, S., Bhaskar, T., Mouradov, A., Aquatic plant Azolla as the universal feedstock for biofuel production. Biotechnol. Biofuels, 9, 221, 2016. https://doi.org/10.1186/s13068-016-0628-5 PMid:27777623 PMCid:PMC5069886 38. Babalola, O.O., Glick, B.R., The use of microbial inoculants in African agriculture: current practice and future prospects. J. Food Agric. Environ., 10, 540-549, 2012. 39. Hodkinson, B.P., Allen, L.L., Forrest, B., Goffinet, B., Serusiaux, E., Andresson, S., V. Miao, J.-P. Bellenger, F., LutzoniLichen-symbiotic cyanobacteria associated with Peltigera have an alternative vanadium-dependent nitrogen fixation. Eur. J. Phycol., 19, 11–19, 2014. https://doi.org/10.1080/09 670262.2013.873143 40. Gupta, P.K., Vermicomposting for sustainable agricultur, pp. 11–14, Bharat Printing Press, Jodhpur, India, 2005. 41. Bhatnagar, R.K. and Palta, P.K., Vermiculture and vermicomposting, pp. 101, Kalyani Publication, 1998. 42. Mall, A.K., Dubey, A., Prasad, S., Vermicompost: An inevitable tool of organic farming for sustainable agriculture. Agrobios Newsletter, 3, 8, 10–11, 2005. 43. Vasanthi, D. and Kumaraswamy, K., Efficacy of vermicompost to improve soil fertility and rice yields. J. Indian Soc. Soil Sci., 47, 268–272, 1999. 44. Devi, M., Organic farming: Scope and importance. Agrobios Newsletter, 6, 4, 14, 2007. 45. Remesh, P., Organic farming research in M.P. Organic farming in rain fed agriculture, pp. 13–17, Central institute for dry land agriculture, Hyderabad, 2008. 46. Bot, A. and Benites, J., The importance of soil organic matter: Key to drought resistant soil and sustained food production (Vol. 80). Food & Agriculture Org, 2005.

148  Biofertilizers 47. Brodt, S., Learning about tree management in rural central India: A local global continuum. Human Org., 61, 1, 58–67, 2002. https://doi.org/10.17730/ humo.61.1.7tcb 9htj7ee2ag1m 48. Swathi, V., The use and benefits of bio-fertilizer and biochar on agricultural soils unpublished B.Sc. thesis. Department of Chemical and Biological Engineering. Chalmers University of Technology Goteborg Sweden. 20–24, 2010. 49. Board, N.I.I.R., The Complete Technology Book on Bio-Fertilizer and Organic Farming. National Institute of Industrial Re, 2004. 50. Mahimaraja, S., Dooraisamy, P., Lakshmanan, A., et al., Composting technology and organic waste utilization. J. Sci., 1, 3, 332–560, 2008. 51. Singh, A.K., Rural Marketing: Indian Perspective. New Age International, 2007. 52. Adesemoye, A.O., Kloepper, J.W., Plant-microbes interactions in enhanced fertilizer use efficiency. Appl. Microbiol. Biotechnol., 85, 1, 1–12, 2009. https:// doi.org/10.1007/ s00253-009-2196-0 PMid:19707753 53. Vessey, J.K., Plant growth promoting Rhizobacteria as bio-fertilizers. J. Plant Soil, 225, 43, 571–86, 2003. https://doi.org/10.1023/A:1026037216893 54. Sinha, R.K., Valani, D., Chauhan, K. et al., Embarking on a second green revolution for sustainable agriculture by vermiculture biotechnology using earthworms. Int. J. Agric. Health Safety, 1, 50–64, 2014. 55. Jackson, M.L., Soil Chemical Analysis, pp. 183–192, Prentice Hall of India Private Limited, New Delhi, India, 1973. 56. Son, T.N., Thu, V.V., Duong, V.C. and Hiraoka, H., Effect of organic and bio-fertilizers on soybean and rice cropping system. Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki, Japan, 2007. 57. Carter, O.G., The effect of chemical fertilizers on seedling establishment. Anim. Prod. Sci., 7, 25, 174–180, 1967. https://doi.org/10.1071/EA9670174 58. Kannaiyan, S., Biofertilizers for sustainable crop production. Biotechnology of Biofertilizers, pp. 9–49, Narosa Publishing House, New Delhi, India, 2002. 59. Mishra, D.J., Singh, R., Mishra, U.K., Kumar, S.S., Role of Bio-Fertilizer in Organic Agriculture: A Review. Res. J. Recent Sci., 2, ISC-2012, 39–41, 2013. https://doi.org/10.1016/S2212-4268(12)60010-4 60. Sundar, I., Sustainable agriculture and sustainability of Indian agriculture in the context of globalisation. Int. J. Environ. Pollut., 18, 5, 455–462, 2002. https://doi.org/10.1504/ IJEP.2002.002339 61. Gupta, K., Barat, G.K., Wagle, D.S., Chawla, H.K.L., Nutrient contents and antinutritional factors in conventional and nonconventional leafy vegetables. Food Chem., 31, 105–116, 1989. https://doi.org/10.1016/0308-8146​ (89)90021-6 62. Kuhnlein, H.V., Micronutrient Nutrition and Traditional Food System of Indigenous Peoples, 94 pp., FAO, Rome, 2003.

Organic Farming by Biofertilizers  149 63. Arun, K.S., Bio-fertilizers for sustainable agriculture. Mechanism of P-solubilization. Sixth edition, pp. 196–197, Agribios Publishers, Jodhpur, India, 2007. 64. Raja, N., Bipesticides and biofertilizers: ecofriendly sources for sustainable agriculture. J. Biofertil. Biopestic., 3, 112–15, 2013. 65. Chun-Li, W., Shiuan-Yuh, C., Chiu-Chung, Y., Present situation and future perspective of bio-fertilizer for environmentally friendly agriculture. Annual Rep., 1–5, 2014. 66. Youssef, M.M.A. and Eissa, M.F.M., Biofertilizers and their role in management of plant parasitic nematodes: A review. Biotechnol. Pharm. Resour., 5, 1, 1–6, 2014. 67. Santos, V.B., Araujo, S.F., Leite, L.F., et al., Soil microbial biomass and organic matter fractions during transition from conventional to organic farming systems. Geodderma., 170, 227–31, 2012. https://doi.org/10.1016/j. geoderma.2011.11.007 68. Radwan, M.A., El-Wakil, H.B., Osman, K.A., Toxicity and biochemical impact of certain oximecarbamate pesticides against the terrestrial snail, T. pisana (Müller). J. Environ. Sci. Health, 27, 759–773, 1992. https://doi. org/10.1080/03601239209372811 69. Mfilinge, A., Mtei, K., Ndakidemi. Effect of Rhizobium inoculation and supplementation with phosphorus and potassium on growth leaf chlorophyll content and nitrogen fixation of bush bean varieties. Am. J. Res. Commun., 2, 10, 49–87, 2014. 70. Khosro, M. and Yousef, S. Bacterial bio-fertilizers for sustainable crop production: A review. APRN J. Agric. Biol. Sci., 7, 5, 237–308, 2012. 71. El-Shahaat, M.S., Aly, Nagda A., Eshra, E.H., Mesbah, H.A., Ghoneim, Emtiaz I., Toxicity of certain copper fungicides and other pesticides to terrestrial snails. J. Agric. Sci. Mansoura Univ., 34, 5, 5501–5507, 2009. 72. Aktar, W., Sengupta, D., Chowdhury, A., Impact of pesticides use in agriculture: their benefits and hazards. Interdiscip. Toxicol., 2, 1–12, 2009. https:// doi.org/10.2478/ v10102-009-0001-7 PMid:21217838 PMCid:PMC2984095 73. Anonymous, IFOAM Basic Standards for Organic Production and Processing, 68 pp. International Federation of Organic Agriculture Movements (IFOAM), Victoria, Canada, 2002. 74. Rai, S.N. and Gaur, A.C., Chraracterization of Azotobacter spp. and effect of Azotobacter and Azospirillum as inoculant on the yield and N-uptake of wheat crop. Plant Soil, 109, 131–134, 1988. https://doi.org/10.1007/ BF02197592

5 Phosphorus Solubilizing Microorganisms Rafig Gurbanov1,2*, Berkay Kalkanci3, Hazel Karadag3 and Gizem Samgane3 Department of Molecular Biology and Genetics, Bilecik Şeyh Edebali University, Bilecik, Turkey 2 Biotechnology Application and Research Center, Bilecik Şeyh Edebali University, Bilecik, Turkey 3 Department of Biotechnology, Bilecik Şeyh Edebali University, Bilecik, Turkey 1

Abstract

The phosphorus pollution of the chemosphere and biosphere has become a serious environmental issue. The main reason for this pollution may be considered as the extensive and unconscious use of chemical fertilizers and animal feeds containing phosphorus in agricultural and farming activities. On the other hand, phosphorus is an essential element of our ecosystem that needs to be supplied externally for all organisms. Nevertheless, the organisms especially plants cannot uptake the majority of the soil or aquatic phosphorus for their growth; therefore, it needs to be converted into soluble forms. Many microbial species can catabolize both organic and inorganic forms of phosphate into the soluble phosphate and provide the rhizosphere with sufficient phosphorus. These phosphorus solubilizing microorganisms are promising as effective biological approaches to address environmental phosphate pollution and polluted fecal wastes of animal farms. As a component of the rhizosphere, they can be applied to the soil, sprouts, and seeds to provide the crops with usable phosphorus. In this chapter, brief literature review on phosphate pollution, phosphate-solubilizing microbial species, and their enzymes are provided. Moreover, the role of these microbes in plant growth, the application methods in agriculture, and the factors affecting microbial survival are mentioned. Keywords:  Agricultural plant, arbuscular mycorrhizal fungi, enzymes, solubilization, pollution, inert phosphate

*Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (151–182) © 2021 Scrivener Publishing LLC

151

152  Biofertilizers

5.1 Phosphorus Pollution Being a non-renewable geological resource, phosphorus is a key element for the production of nutrients and fuels that cannot be replaced by any other chemical element [1]. It is vital for all forms of life, as it directs the basic cellular energy cycles that make living cellular systems to work [2]. Since it is one of the most important nutrients necessary for photosynthesis, farmers continually supplement the soil with phosphorus to enhance crop production. It also plays a role in reducing the risks associated with metal contamination of soils; precipitation of soluble metals in the form of poorly soluble phosphate minerals in contaminated soils can significantly reduce bioavailability [3]. Different phosphate compounds are cycling in between the rocks, water resources, soils, sediments, and living organisms in the form of mineral and organic phosphorus [4]. Over time, rain and weathering cause rocks to release phosphate ions and other minerals. The released inorganic phosphate can be dispersed into soils and water and taken up by plants [4]. Consumption of plants by animals also makes phosphate to be transported to other organisms. Phosphate is included in bioorganic molecules like DNA in plants and animals, and eventually, organic phosphate is returned to the soil when the organism dies or degrades [5]. The phosphorus circulates in between the earth’s lithosphere, hydrosphere, and biosphere [6]. The extraction of phosphorus from the biosphere and hydrosphere is not applicable, whereas the phosphate rock in the lithosphere is a good source of phosphorus [6]. The phosphate rock is usually used as a raw material in the production of phosphate fertilizer [7]. The phosphate pollution is increasing in the crop and food production as well as waste management chains, because of the low-cost phosphate fertilizers [8].  One of the main causes of phosphate pollution is urban wastewater originating from fertilizer flow and sewage from agricultural areas. Although phosphorus is generally found to be a limiting nutrient for freshwater habitats, its concentrations in wastewater systems may exceed levels that algae and macrophytes can utilize which in turn can lead to phosphate toxicity [9, 10]. Phosphorus pollution caused by intense animal farming generates excessive phosphorus accumulation in the soil and eutrophication creating environmental concern [11]. Eutrophication is characterized by enriching the aqueous environments with various organic compounds [12]. It is one of the most common environmental problems of inland waters and needs to be solved urgently in developing countries, especially in the protection of water resources [13]. Deterioration of water quality from scattered food sources occurs for various reasons [14]. First,

Phosphate-Dissolving microbes  153 the agricultural inputs of phosphorus in commercial fertilizer and animal feed supplements often exceed agricultural output [15]. Second, excessive animal density can enhance fertilizer production that exceeds both soil storage capacities and regional requirements of crops [16]. In many cases, excessive fertilization of the soil promotes leakage of phosphate into moving and downstream water ecosystems [17]. Such excess phosphorus in the soil can last for a thousand years, facilitate the accumulation of phosphate in downstream lakes, and change the mechanisms that regulate lake structure and function [14]. Before the 1990s, the eutrophication formation was essentially attributed to point and non-point discharge of wastewater rich in phosphorus [18]. While measures to address the eutrophication problem focus on reducing external resources, a delay in recovery has been found for many lakes after the reduction of the external phosphorus load [18]. Therefore, the release of phosphorus from sediments is considered to be the main process responsible for this delay [19]. According to lake studies in Europe and North America, this process lasts typically 10–15 years following external phosphate reduction [19]. The release of phosphate from sediments can contribute to most (up to 80%) of total phosphate input in some lakes, and becomes the main driving force of primary production, especially when bottom waters become anoxic in the summertime [20].

5.2 Phosphate Solubilization The soil has always been a volatile environment and is a biological resource of intense microbial life, which is primarily influenced by its maternal matter’s molecular structure and the activity of the organism it maintains [21]. Phosphorus required for intracellular needs relies on the average amount of phosphate in the field as well as on its solubility, which is ascertained through various natural transformations and biochemical reactions [22]. Ground phosphate is generally categorized as organic and inorganic phosphorus [23]. The reactions of fixation and immobilization taking place in the ground convert phosphate toward variants that are not beneficial for crop activity [24]. It is worth recalling that large amounts of the administered phosphate fertilizers are settled in the land, making it unusable for seed intake [4]. Phosphorus can be found as inorganic phosphorus produced by the decomposition of the bedrock or as organophosphate acquired from rotting plants, animals, or microbes [25]. Primary crystals including apatite, hydroxyapatite, and oxyapatite signify the elemental sources of phosphate [26]. The insolubility is the principal fundamental

154  Biofertilizers characteristic of the above crystal varieties [27]. These can, however, be kept solubilized and serviceable to microbes and plants within certain scenarios [29]. The phosphate is an indispensable part of the living organism’s metabolic tasks [28]. It has an established function in a variety of cellular events including hereditary trait transmission and metabolic pathway management [29]. The cell can take several forms of phosphate, but the − largest part is absorbed in HPO2− 4 or H 2 PO 4 forms [30]. A process called mineral phosphate solubility is the transformation of inorganic unserviceable phosphorus into serviceable substances HPO24− or H2PO−4 for seed consumption [31]. Mineral phosphorus can also be connected to the exterior of poorly soluble and assimilable iron, aluminum, and manganese hydrated oxides [30, 32]. Organic phosphate derivatives in certain ecosystems can pass around 50% of overall phosphate [33]. Some of these phosphate compounds are high molecular-mass items. Following biochemical transformation to dissolved ionic phosphorus Pi , HPO24− H2PO−4 , or small organic phosphorus, these could be assimilated by organisms [34]. Based on the utilization rate of known high-quality rock phosphate reserves, phosphorus is expected to be depleted in this century [35]. Eventually, the manufacture of phosphorus-based fertilizers would allow small-grade rock phosphates to be extracted at significantly higher rates, thereby raising operating expenses of phosphate fertilizers [1]. The development of new non-acid methods that can be applied to raw materials is necessary to enhance the technical and ecological efficiency in fertilizer production [36]. Some of the latest non-acid rock phosphate control approaches are the primary application of phosphates to the land as a phosphate supply [36]. However, the straightforward introduction of phosphorus as a compost is restricted, resulting in low resolution due to its structure [37]. The design of the phosphorus form through mechanical action alters the geometry and widens that bioavailability [37]. Tricalcium phosphate, aluminum phosphate, iron phosphate, and other insoluble phosphate forms can be turned onto bioavailable phosphorus through the biological activity of certain microscopic species living in diverse surroundings [38–41]. In this context, it has been found that soil microorganisms are generally more effective for dissolving phosphate for plants from both inorganic and organic sources and mineralizing complex phosphate compounds [42–44]. Phosphate-solubilizing microorganisms were previously used to produce soluble phosphate fertilizers from phosphate rocks [45]. Phosphate-solubilizing microorganisms serve a significantly essential function in the reversal of crop phosphorus shortage [46]. They do adapt inert phosphate as well as a huge chunk of bioavailable phosphorus [47]. Besides, through the decay of phosphate-rich

(

(

)

)

Phosphate-Dissolving microbes  155 biomaterials, they can release soluble inorganic phosphorus into the soil, hence taking an active role by dissolving and making phosphorus usable for plants [48]. The decomposition of natural phosphate is thus primarily equivalent to the availability and conduct of phosphorus solubilizing microorganisms [49]. In natural soil systems, phosphate-solubilizing microorganisms consist of a wide community of fungi and bacteria communicating with land near plant roots, called rhizospheres [49]. They are found in nearly every habitats, but the quantity differs based on climate and land parameters [50]. Many researchers have documented shreds of evidence on the solubilization of inert phosphorus by microorganisms [51, 52]. Among these, the most accepted ones are bacteria and/or fungi producing small organic acids [53–55]. Various microbial organic acids are characterized, as well. Many phosphorus solubilizers like organic acids produced by bacterial culture or fungi are known to chelate ore ions or lower pH to dissolve phosphorus [56]. Eventually, the acidification of microbial microenvironments causes the discharge of phosphorus ions from minerals by replacing Ca2+ with H+ [57, 58]. Therefore, an efficient and low-cost microbe-mediated strategy that can process and utilize lowgrade rock phosphates and/or natural phosphorus resources can be developed in the near future [59].

5.3 Microbial Mechanisms of Phosphate Solubilization Plant phosphorus deficiency is a common phenomenon worldwide, resulting in strong demand for phosphate-based chemical fertilizer applications [60]. Worldwide phosphate fertilizer yield is around 10%–25% [61] and the bioavailable phosphorus concentration reaches one milligram per kilogram of soil [62]. Different types of microbes assist the plant growth by turning phosphate from inert form to plant-usable form [63]. They are promising as the best environmentally friendly option to provide plants with cheap phosphorus. Microbial phosphate solubilization mechanisms are largely varied in nature, and much of the earthly cycle of inert organic and inorganic soil phosphates is assigned to bacteria and fungi [64]. However, it has been suggested that bacteria are predominant and more effective than fungi in this process [65]. The main processes used by microorganisms for soil phosphorus solubility can be summarized as secretion of mineral solvent compounds, enzyme-mediated biochemical phosphate mineralization, and phosphate assimilation by releasing phosphorus from more complex substrates [66].

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5.3.1 Organic Phosphate Solubilization Depending on soil characteristics organic phosphate can make up 4%–90% of total soil phosphorus content [67]. Organic phosphate solubilization also called phosphate mineralization has an imperative character in the phosphate availability of the horticulture [40]. Organic phosphorus compounds (nucleic acids, phospholipids, and sugar phosphates) cannot be directly used by the plant; therefore, they need to be converted to mineral form by enzymes [68]. These compounds are easily mineralized as they are accessible for microbes and their enzymes [69] and mineralization mainly depends on their chemical nature [70]. This process occurs by the action of bacterial enzymes such as non-specific acid phosphatases, phytases, lyases, and phosphonatases cleaving carbon-phosphate bonds [71]. By hydrolysis of ester bonds in high molecular weight organic phosphate, phosphatases cause the discharge of phosphate ions. According to their optimum pH, they are categorized as acidic, neutral and alkaline phosphatase enzymes [72]. They are secreted by both microorganisms and plant roots. However, it is very difficult to distinguish phosphatases according to production sources [73]. The phytases decompose phytate or myo-inositol phosphate substances [74]. As the biggest supply of inositol, phytate is the main component of phosphorus accumulated in rhizomes and spores and is also a key soil constituent of organic phosphorus [4]. The main element in controlling soil phytate mineralization is microbial species [75]. Although the plants’ ability to acquire phosphorus from the phytate is exceptionally restricted, the perimeter of the microorganisms in the rhizosphere offers plants the opportunity to extract phosphorus directly from the phytate [75]. Phosphonatases and lyases cause enzymatic hydrolysis of the ester links in phosphoenolpyruvate and phosphonoacetate and turn them into phosphorus ions and hydrocarbons for intake [76]. The availability of organic phosphate compounds can be a limitation for plant nutrition. Since phosphorus is highly reactive, it may interact with other metallic elements in the rhizosphere region and become unusable for plants delaying their growth as well as crop yields [65]. Therefore, the ability of bacterial enzymes to perform the desired function in the rhizosphere is a very important consideration for plant nutrition [65].

5.3.2 Inorganic Phosphate Solubilization The competence of organic microbial acids to dissolve inert phosphorus depends on the acidity level of the environment. Indeed, a decrease in pH

Phosphate-Dissolving microbes  157 has been reported because of organic acids synthesized during the development of phosphorus solubilizing microbes in a culture environment [77, 78]. The delivery of organic acids through direct oxidation occurring on microbial membranes decreases the medium pH (acidification). Organic acids produced from phosphate-solubilizing bacteria include gluconic, formic, 2-ketogluconic, citric, oxalic, lactic, isovaleric, succinic, glycolic, and acetic acids [79]. Organic acids responsible for phosphate solubility are fermentation products of organic carbon sources (glucose) or microbial metabolic products of oxidative phosphorylation [80]. There was a clear positive association between the production of organic acid and the index of phosphate solubilization [81]. When inorganic phosphate is applied to the soil, it interacts with other metal ions, making phosphorus unusable for crops by the generation and release of insoluble salts such as calcium, iron, and aluminum phosphates [82]. Organic acids produced by microorganisms initiate chelation reaction, and phosphorus is separated from the metal part and becomes available for plant uptake [82]. The chelating nature of hydroxylic and carboxylic functional side chains in these acids make the release of phosphate-bound metals, thus converting phosphorus into soluble forms [83]. Being powerful chelating agents of iron, calcium, and aluminum ions, humic, 2-keto-gluconic, and fulvic acids are efficient in the solubilization of inorganic phosphate from any of these metal substances [84]. Microorganisms liberate humic and fulvic acids during the degradation of plant matter [85, 86]. The variety of organic acids produced by different phosphate-solubilizing bacteria are presented in Table 5.1. In addition to organic acids, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and carbonic acid are also known to be effective in phosphate solubility [34, 87]. Even so, the input of synthetic acid to the liberation orthophosphates remains to be less effective than that of natural acid [88]. Synthetic acids are released by nitrification and sulfuroxidizing bacteria during the fermentation of nitrogenous or inorganic sulfur residues [74]. Moreover, the reaction between hydrogen sulfide and iron phosphate causes the formation of iron sulfate and phosphorus is released at the same time. Thus, the generation of hydrogen sulfide is considered as one of the phosphate solubilization mechanisms [71]. Given the role of phosphorus in plant growth and development, phosphatesolubilizing bacteria is likely to be used as an effective biofertilizer to improve the overall performance of crops, especially in areas with phosphate deficiency. Due to the environmental hazards and increasing costs of chemical fertilizers, the use of these bacteria in sustainable agricultural practices can be advantageous.

158  Biofertilizers Table 5.1  Various organic acids produced by phosphate-solubilizing bacteria*. Producer

Acid

Reference

Enterobacter sp. Fs-11

MA, GA

[89]

Pseudomonas trivialis BIHB 769

GA, 2-KGA, LA, SA, FA, MA

[90]

Pseudomonas poae BIHB 808

GA, 2-KGA, SA, CA, MA

[90]

Pseudomonas sp. BIHB 751

OA, GA, 2-KGA, FA, MA

[90]

Enterobacter sp. Hy-40

OA, GA, MA, LA, CA, SA, FuA

[91]

Arthrobacter sp. Hy-505

OA, GA, LA, CA

[91]

Azotobacter sp. Hy-510

OA, GA, TA, LA, SA, FuA

[91]

Enterobacter sp. Hy-402

OA, GA, TA, CA, SA, FuA

[91]

Rhodococcus erythropolis CC-BC11

GA

[92]

Bacillus megaterium CC-BC10

CA, LA, PA

[92]

Arthrobacter sp. CC-BC03

CA, LA

[92]

Arthrobacter ureafaciens CC-BC02

CA

[92]

Serratia marcescens CC-BC14

CA, GA, SA, LA

[92]

Phyllobacterium myrsinacearum CC-BC19

GA

[92]

Delftia sp. CC-BC21

SA

[92]

Chryseobacterium sp. CC-BC05

CA

[92]

*Citric acid (CA), Gluconic acid (GA), Formic acid (FA), Fumaric Acid (FuA), Succinic acid (SA), Lactic acid (LA), Malic acid (MA), Oxalic acid (OA), and 2α-ketogluconic acid (2-KGA).

5.4 Phosphate-Solubilizing Bacteria Various numbers of bacterial species have phosphorus dissolving properties [93]. Besides, the concentration of these bacteria in the rhizosphere soil is higher than in non-rhizosphere soil [93]. They play an important role in the growth and yield of plants by dissolving phosphorus in the soil [93]. Due to these roles, the use of phosphate-solubilizing bacteria instead

Phosphate-Dissolving microbes  159 of chemical fertilizers in agriculture is also important for environmental sustainability [93]. Various bacteria that have phosphate solving features have been identified in different studies (Table 5.2). In a study, most of the bacteria which were isolated from the rhizosphere have demonstrated the generation of carboxylic acid (gluconic) and from modest to superior (400–1,300 mg L−1) phosphorus solubility [49]. These isolates also encouraged the productivity of pea crops cultivated in land under soluble phosphate limiting conditions [49]. Rhizosphere-associated bacterial species with phosphate dissolution ability are reported to support the growth of plants. For this reason, their Table 5.2  Different types of bacteria solubilizing phosphorus. Main phosphate-solubilizing bacterial species

Reference

Bacillus cereus

[94]

Bacillus circulans

[40, 95, 96]

Bacillus megaterium

[40, 92, 94–97]

Bacillus polymyxa; Bacillus subtilis

[40, 94, 95, 97, 98]

Bacillus pulvifaciens

[99]

Bacillus coagulans; Bacillus fusiformis; Bacillus pumilus; Bacillus chitinolyticus

[40]

Bacillus sircalmous

[95]

Bacillus thuringiensis

[94]

Thiobacillus ferrooxidans

[40]

Pseudomonas canescens

[81]

Pseudomonas putida

[40, 98–101]

Pseudomonas calcis

[40]

Pseudomonas fluorescens

[40, 97–101]

Pseudomonas striata

[40, 95, 97, 98]

Pantoea agglomerans

[102]

Rhizobium meliloti

[40]

Rhizobium leguminosarum

[82, 97, 103]

Mesorhizobium mediterraneum

[104]

160  Biofertilizers use in agriculture as biofertilizers or bio-control agents has been the focus of many studies [105]. Phosphorus efficiency of plants can be increased with inoculations of phosphate-solubilizing bacteria in all agricultural lands. In this, many agriculturally valuable crops including corn [106], soybean [107], wheat [107, 108], rapeseed [109], mung bean [110], and tomatoes [111] have demonstrated increased performance and yield. The plant-promoting features of phosphate-solubilizing bacteria under different environmental stresses such as heavy metal contamination [112, 113], pesticides [112, 114], osmotic stress [115], phytopathogens [106], high-temperature [116, 117], and salinity [118] have also been documented. Today, the biofertilizer market is worth around $2 billion, whereas the total fertilizer market is around $245 billion [119]. Moreover, phosphate-solubilizing bacteria–based biofertilizers represent just 14% of the global fertilizer market [119]. Phosphate-solubilizing bacteria are being used more commercially in agriculture day by day [120]. In this manner, phosphate-solubilizing bacteria have a lot to pick up from this market [119]. On the other hand, there is a need to comprehend their constraining components and underscore the incredible chances of their utilization [119].

5.5 Phosphate-Solubilizing Fungi Because of the constantly increasing human population and decreasing fertile soil, available agricultural areas are used more than necessary. Besides, highly productive plant varieties are developed with the development of modern agricultural techniques. However, these plant varieties need more nutrients, including phosphorus. For this reason, farmers extensively use chemical fertilizers that are low cost but cause serious environmental pollution. As a result of the use of chemical fertilizers, most of the soil phosphorus cannot be used by plants. Therefore, there is a need for new eco-friendly methods alternative to chemical fertilizers. Similar to bacteria, phosphorus solubilizing fungi can be an alternative way to solve this problem. These fungi not only augment the soil phosphate content but also have a vital function in the overall plant development and the continuation of environmental sustainability. Over the last 30 years, various fungi capable of dissolving phosphate have been identified (Table 5.3). Besides, the phosphate solubilization potential of these fungi has been investigated [121]. The most firefighting of these fungi is Aspergillus and Penicillium species [122]. Qualitative analysis of the phosphorus solubilization abilities of fungi strains is accomplished by calculating the zone of a dissolved

Phosphate-Dissolving microbes  161 Table 5.3  Different types of fungi solubilizing phosphorus. Main phosphate-solubilizing fungal species

Reference

Aspergillus awamori

[40, 95, 97, 126]

Aspergillus clavatus

[81]

Aspergillus niger

[40, 81, 98, 126–129]

Aspergillus candidus; Aspergillus parasiticus; Aspergillus fumigatues; Aspergillus rugulosus

[127, 129]

Aspergillus flavus

[40, 126]

Aspergillus foetidus; Aspergillus nidulans; Aspergillus wentii

[40]

Aspergillus terreus

[40, 126]

Aspergillus tubingensis

[128]

Aspergillus sydawi; Aspergillus ochraceus; Aspergillus versicolor

[126]

Penicillium bilaii

[96, 97]

Penicillium citrinum

[130]

Penicillium digitatum; Penicillium lilacinium; Penicillium balaji; Penicillium funicolosum

[40]

Penicillium oxalicum

[98]

Penicillium simplicissimum; Penicillium rubrum

[127, 129]

field in Pikovskaya’s solid media supplemented with insoluble phosphate [123]. However, this analysis does not give definitive results. Therefore, the qualitative analysis should be done in a liquid medium. Unlike bacteria, phosphate solubilization abilities of fungi strains are not lost even in subcultures. Moreover, fungal hyphae can travel long distances in the soil [124] and secrete more organic acids than bacteria [125]. Roots are an important region for the uptake of micro minerals and exudation of organic compounds. It also functions as an energy and carbon source for the root microflora. The root perimeter, where the microbial activity is high, is called a rhizosphere. It was reported that the number of microbial cells in the rhizosphere is higher than in the other parts of the soil [131]. The interplay between the microorganisms and plants regulate

162  Biofertilizers the biogeochemical mineral recycling, thus maintaining the structure of the microbial community in the rhizosphere [132]. All microbes that can reproduce without damaging the host plant tissues are called endophytes [133]. In plant-microbe interactions, endophyte organisms (bacteria and fungi) live without creating a pathological condition in plant roots, while others live by establishing a symbiotic relationship with the roots by forming the structures called mycorrhiza [134]. These infectious species are thought to enter the roots with natural wounds formed during seedling growth [135]. Colonization of endophytic microbes in plant roots can be local, between the cells or systematic. The endophytic microenvironment protects microbes against environmental stress factors. Endophytic microbes reproduce in host plant tissues and secrete certain chemicals that provide disease resistance. They also help the host plant to make more use of mineral substances in the soil. Some endophytic fungi can solubilize phosphorus in the soil and support the growth of the plant at low phosphorus concentrations [136–138]. Endophytic fungi also support the plant growth in environmental stress conditions by enhanced production of bioactive metabolites and plant growth hormone gibberellins [139]. They provide the mineralization of insoluble nutrients (especially phosphorus) [139] and organic substances [137]. Previously, the root-isolated endophytic fungus strain enhanced the plant maturation by producing growth hormone and solubilizing soil phosphorus [140]. On the other hand, mycorrhiza (fungiroot) is known as symbiotic structures established between some fungi and roots in the plant’s rhizosphere. Therefore, mycorrhiza has a more specialized structure than endophytic fungi. Mycorrhiza is classified into different types according to the structural relationship between plant and fungus. If the fungal hypha has infected the plant roots, then it is called endomycorrhiza; if it has not, then it is called ectomycorrhiza. It is called ectoendomycorrhizae if it exhibits both properties/mixed behavior. The five known subgroups of endomycorrhiza exist being arbuscular, ericoid, arbutoid, monotropoid, and orchid. Among them, the most debated and agriculturally important ones are arbuscular mycorrhizae infecting both cultivated and wild crops [141]. All these different symbiotic relationships support the root system with water and mineral supply, helping the plant to withstand drought stress and various diseases [142].

5.5.1 Phosphate-Solubilizing Fungi as Plant Growth Promoters There has been an increase in the use of arbuscular mycorrhizal fungi to increase agricultural production over the past few years since it supports

Phosphate-Dissolving microbes  163 plant growth and provides the production of many industrial metabolites [143, 144]. Arbuscular mycorrhizal fungi account for only 0.1%–0.5% of the total fungal population in the soil [124]. They support plant development with several mechanisms. These mechanisms are as follows: a)

Solubilization reactions and the formation of macronutrients b) Production of biocontrol metabolites like siderophores and other antibiotics c) Biological protection against various plant pathogens d) The production of plant growth hormones like auxin and gibberellin. Today, arbuscular mycorrhizal fungi are used as an inoculant to support plant growth [55]. High efficiency and sustainability are the main factors in good quality microbial inoculants. Under laboratory conditions, fungi with better phosphorus solubilization abilities are selected and plants that can be suitable carriers for these fungi are identified. Then, phosphate solubilization ability and its suitability in the carrier plant are analyzed. As a result of these tests, suitable biological materials are stored at 28°C for about 3 months. At present, Penicillium bilaiae and Penicillium radicum are commercially available fungi at large scale and shown to be effective as phosphate-solubilizing agents for crops [145]. Numerous studies have shown the efficiency of phosphate solubilization by both single and composite fungal cultures on the growth parameters of various plants [130, 146–152]. The effect of composite inoculum of different arbuscular mycorrhizal fungi on growth and seed production of chickpea was investigated [130]. As a result of this study, it was shown that the fungi-inoculated group increased the plant height, weight, and the number of seeds compared to the non-inoculated group [130]. These fungi have also been shown to improve the production of grain and common bean crops. [146, 147]. Previously, the inoculation of arbuscular mycorrhizal fungi caused a significant improvement in crop production cultivated in soil containing rock phosphate and superphosphate [148]. Penicillium oxalicum P4 strain isolated from a chalky soil increased the live weight of the plant upon the external addition of rock phosphate [149]. Aspergillus niger P85 strain did not increase the live weight of the plant but increased the phosphate accumulation inside the plant [149]. Besides, the production of organic acids has been determined due to fungal inoculation [149]. Aspergillus aculeatus P93 strain increased the dry weight of the corn grown in calcareous soil and the total amount of phosphorus

164  Biofertilizers inside the plant [150]. Phosphate-solubilizing fungus strain magnified the total production of cardamom plant and chlorophylls in its leaves [151]. It has also been determined that this fungus strain produces indole acetic acid, ammonia, hydrolytic enzymes, and siderophores [151]. The biomass and the phosphorus content of the wheat sprouts increased considerably because of the inoculation with Penicillium aculeatum in biochar-refined semi-sterile soil environment [152]. This study is promising in the production of biological composts containing Penicillium fungi, to complement the fertilizer worth of phosphate-rich charcoals [152].

5.5.2 The Methods of using Phosphate-Solubilizing Fungi in Agriculture The widely used method is to treat the plant seed with the desired fungal inoculant [153, 154]. In this application, the seeds are treated with desired fungi in a liquid culture medium [155]. Then, fungi attached to the seed surface are detected and the seeds with the presence of fungi are accepted as grafted [155]. However, the number of live inoculum on the grain may be insufficient, and/or the applied chemical processes may negatively affect the survival of the fungi [155]. There is also a method that is based on the treatment of moist plant parts with the fungus before planting it in the field [156]. Another preferred method is based on the direct treatment of the soil suitable for the desired plant with the soil-specific fungal culture [155]. The advantages of this method are listed below [155]: a)

Increases the likelihood of more fungal population solubilizing phosphorus per unit area in soil. b) Ensures that the interaction of inoculum with chemical substances diffusing from the chemically treated seeds is kept to a minimum. Thus, reduces the negative effects of chemicals on microbes. c) Faster than the seed inoculation techniques due to the absence of seed mixing step. d) Dry and desiccated ambient conditions are better tolerated compared to carrier-based inoculants [155]. Today, two approaches are accepted in all the applied microbial inoculation techniques [157]. The first is the monoculture method, in which one particular phosphorus solubilizing fungal inoculum is used [157].

Phosphate-Dissolving microbes  165 The second is the multi-culture method in which different fungi species are mixed in the natural field and/or pot conditions [157]. The ecosystem in which the microorganisms communicate with plants is continuously influenced by abiotic and biotic variables that affect their growth, interactions, and development. These factors are important both ecologically and economically as they significantly affect the yield and production of the products. Therefore, it is necessary to determine how fungal populations and their phosphate solubilization activities are impressed by environmental stresses. The obtained information can be used to estimate the suitability of particular fungal inoculum for use in various agroecological regions. Many other factors affect the colonization, development, and survival of fungi strains. The most important ones are age and life cycle of the plant, the composition and structures of root secretions, and the number of environmental pollutants such as pesticides and heavy metals. Nevertheless, gene transfer by using molecular approaches can increase growth-promoting abilities and host spectrum of phosphatesolubilizing fungi [158].

5.6 Bacteria-Fungi Consortium for Phosphate Solubilization One of the alternative ways to increase productivity in phosphate solubilization is to inoculate different species supporting plant growth by using various mechanisms [159–161]. In this context, studies on the synergistic effect of the bacteria-fungi consortium are progressing. This consortium demonstrated better performance especially in nutrient-poor soils [160, 162–166]. Besides, they are used together in many other applications such as increasing the yield of crop plants [167], improving fruit quality [168, 169], minimizing the consumption of chemical composts [170], and enhancing phytoremediation [164]. The synergistic consortium of phosphate-solubilizing bacteria and fungi attracts the attention of researchers, as documented in Table 5.4. Unfortunately, too much speculation has also been made regarding this synergistic effect [171]. However, co-inoculation has proven to be more effective than individual inoculation for plant growth [87, 167–169, 171–189]. In this synergistic effect, fungi absorb the insolubilized phosphorus from the soil and transform it into the soluble form [190, 191]. Whereas the bacteria are thought to increase the usability of the absorbed phosphorus by plants [190, 191].

Alfalfa Medicago sativa L.

Maize Zea mays L.

Fun neliformis mosseae

Funneliformis mosseae

Rhizophagus fasciculatus

Claroideoglomus Etunicatum; Funneliformis mosseae; Rhizophagus intraradices

Glomus mosseae

Pantoesstewarti

Pseudomonas fluorescens

Bacillus polymyxa

Bacillus subtilis

Bacillus polymyxa

Onion Allium cepa L.

Grey-haired Acacia Acacia gerrardii

Kindal tree Terminalia paniculate; Terminalia tomentosa

Common reed Phragmites australis

Funneliformis mosseae

Pseudomonas fluorescens

Experimental

Fungi plant

Bacteria

Co-inoculation significantly increased the biomass, height, and yield of the onion plant compared to uninoculated controls and the individual inoculation.

Co-inoculation under salinity stress significantly increased the shoot and dry root weight, nodule count, and leghemoglobin compared to uninoculated controls and individual inoculation.

Co-inoculation has been observed to increase the growth, dry weight, and phosphorus absorption of the plant compared to the individual inoculation of these microorganisms or uninoculated controls.

Co-inoculation of soil containing phosphate rock has been observed to significantly increase the grain yield, phosphorus, and nitrogen content in the corn plant.

Co-inoculation has been observed to increase the biomass and phosphorus absorption in Medicago sativa L. plant.

Co-inoculation of soil with limited phosphorus and 1% bio-coal increased the biomass, root colonization, and phosphorus absorption in Phragmites autralis plant.

Effect

Table 5.4  Synergistic effects of arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria on plants.

[196]

[195]

[189]

[194]

[193]

[192]

Reference

166  Biofertilizers

Phosphate-Dissolving microbes  167

5.7 Conclusions Phosphorus pollution is one of the most important environmental concerns since the available environmental phosphate cannot be degraded for the utilization of high eukaryotes. Based on the current scientific and technological developments, it seems that phosphorus solubilization through the application of efficient microbial species and/or their enzymes is the best and probable the only eco-friendly option to apply in agricultural and land protection processes. When a tremendous diminish in currently available and cultivable lands is considered, efficient utilization of these croplands (sustainable crop production) is also a necessity confronting our ecosystem. The application of these bacteria and/or fungi as a part of biological fertilizers is a promising approach as well; however, bioprospecting of novel microorganisms, genes, proteins/enzymes, metabolites, and cell-surface biomolecules with high performances is crucial to get things done. Moreover, high-throughput bioengineering research can pave the way for new opportunities and success of field applications. Recombinant strains with high-solubilization capacities operating at certain safe and predefined circumstances should also be developed based on the knowledge obtained from bioprospecting studies.

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6 Exophytical and Endophytical Interactions of Plants and Microbial Activities A. Mbotho1, D. Selikane1, J.S. Sefadi2* and M.J. Mochane1† Department of Life Sciences, Central University of Technology, Bloemfontein, South Africa 2 Department of Physical and Earth Sciences, Sol Plaatje University, Kimberley, South Africa 1

Abstract

Associations between living organisms have existed from as long as life began. Living things have had to find ways to co-exist and compete for resources required for survival. These associations have led to evolution and ultimately life as is known to many science communities and elsewhere. Plant-microbe interactions are some of these associations between living organisms, involving plants and microorganisms. Some of these interactions have proved to be advantageous to both the symbionts involved, whereas some are purely parasitic, benefiting one organism at the expense of the other. In certain instances, the interaction does not benefit or harm the other organisms especially where an organism is only using other as habitat without taking anything or addition in the process. The associations between plants and microorganisms have led to some of the greatest human discoveries, mainly in the agricultural sector. These biological processes are imitated forming compounds which improve plant health, including the manipulation of microbial biochemical processes to improve crop yields through production of biofertilizers and biopesticides. This chapter will discuss these plant-microbe interactions, how they occur, their effect toward the microorganisms and plants involved, and, additionally, how they are used in biotechnology and agriculture. Keywords:  Plant-microbe interactions, beneficial and pathogenic microbes, bioremediation, biofertilizers

*Corresponding author: [email protected] † Corresponding author: [email protected]; [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (183–210) © 2021 Scrivener Publishing LLC

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184  Biofertilizers

6.1 Introduction Plant-microbe interactions are the relationships that exist between microorganisms and plants. These relationships involving microbes and plants may yield favorable or harmful effects determined by the nature of participants involved. Plants interact with microbes throughout their lifespan, as early as from germination of the seeds (in seed-producing plants) up until the death of the plant. Microbial associations to plants can be through the shoot system (a system consisting of organs which typically grow above the ground, namely, leaves, flowers, fruits, stems, and buds) or root system (a network system off all the roots of a plant or an underground structure, whose main purpose is to provide anchorage of the plant to the soil while also absorbing nutrients and water). Microbes can interact in an exophytic way, growing on the surface outside the plant such as on the reproductive system (flowers, fruits, and seeds) as well as other external plant organs, including leaves, roots, and stems, or endophytically, growing within the plant [1]. Most microbial activity occurs in the rhizosphere which is an area encompassing the roots of the plant host as well as the surrounding environment, including soil and organisms that reside in the soil. The rhizosphere is believed to contain microbial densities that are 100 times larger by comparison than the densities present in topsoil. The microbiome of the rhizosphere is largely affected by the environment, ecological diversity, the plant host, and its root exudates [2, 3]. Due to their autotropic nature, plants are responsible for sustaining all life forms on earth directly or indirectly so. An autotrophic nature referred to as producers in the food chain which create their nutrients and energy through a process called photosynthesis (a phenomenon carried out by plants and certain organisms pertaining the harvesting of light energy and converting it to chemical energy which is used to drive activities of the organism). They interact with neighboring plants, microorganisms, as well as invertebrate herbivores around their surrounding area. This responsibility has led plants to be innovative since they are sessile (immobile). They have evolved mechanisms in order to regulate the overall outcomes of their interactions with microbes [3]. These mechanisms include the use of chemical signaling which is mediated by the root system of the plant. Immune signaling pathways are mediated by pattern recognition receptors of the host cells which detect danger and allow the plant to release chemicals that attract beneficial microbes while simultaneously repelling pathogens. The signaling pathway, however, comes at a cost for the plant; a significant amount of its carbon is lost which in extreme cases can

Exophytical and Endophytical Interactions  185 subsequently lead to pocket-sized growth and a low yield [4–6]. Chemicals which are secreted by the plant’s root system are referred to as exudates. These exudates are synthesized continuously by the roots; upon accumulation, they are secreted into various compounds onto the surrounding soil of the rhizosphere. Compounds include enzymes, water, hydrogen ions, mucilage, as well as carbon-containing primary and secondary metabolites [3, 4]. Plant-microbe associations affect both the plant host as well as the microbial symbiont. Plant phenotype and ecology are largely affected by the symbiont microbes found in the environment as well as the competition for soil resources. Plant-associated microorganisms form complex interactions which affect plant ecology and/or diversity [3, 6]. Current advances in genomic technology present evidence that indicates the significant impact that the plant’s genotype has over the composition of phylosphere, endosphere, and rhizospheric microbial populations. Recent studies on plant exudates suggest that the secretion of root exudates is a regulatory biocontrol system which is aimed at selecting a specific soil bacterial community according to the plants’ specifics [3]. The roles of each of the participants in plant-microbe interactions have been recognized with regard to ecosystem function. Modern technology has made research concerning associations of microorganisms with plants easier, thus broadening possibilities and opportunities for future investigations. However, the mechanisms involved in plant-microbe associations and how they operate in detail is still an unclear subject [3]. An understanding of plant-microbe interactions is essential as these interactions greatly affect plant development and physiology. The presence or absence of a particular microbe can determine the survival or destruction of that plant. It is, however, important to note that other factors besides the host as well as the symbiont such as environmental factors, residing organisms as well as soil composition play a big role in these plant-microbe interactions. It is, however, important to note that other factors besides the host as well as the symbiont such as environmental factors, residing organisms including soil composition are key role-players of these interrelationships. This chapter will discuss the beneficial and pathogenic (harmful) interactions in connection with plants and microbes, microorganisms involved, plants affected, as well as the result thereof of these interactions.

6.2 Beneficial Interactions Beneficial interactions with regard to microorganisms and plants are connections that are convenient for the plant. Relationships of this kind

186  Biofertilizers are said to have a mutualistic nature, where the interaction is beneficial for both parties involved (i.e., the plant host and microbial symbiont). In these interactions, the presence of the microbe provides the plant with advantageous factors that would otherwise not be available in the absence of that microbe, with the plant, in turn, providing sustenance in the form of carbohydrates to the microbial symbiont. Kafle et al. [1] stated that, according to recent evidence, fatty acids were observed to directly cross the interface of mycorrhiza to the microbe partner, whereas previous beliefs assumed that hexoses were used to transfer carbon to the fungal symbiont. The factors provided by the microbe to the host plant differ with respect to the plant, microbe, and overall conditions surrounding the plant (soil composition, environment, and/or climate) [6]. Some of the benefits provided by the microbe to the plant include a tolerance to abiotic (salinity, drought, temperature and heavy metals, etc.) and biotic (fungi, bacteria, viruses, parasites, insects, weeds, and other plants) stresses, production of certain elements such as production of phytohormones which improve plant nutrition ultimately leading to optimal growth and thus high yield, as well as protection against pathogenic microorganisms [6, 7]. The interactions formed by beneficial microorganisms with plants also have a great potential for the development into agricultural agents such as microbial fertilizers (biofertilizers) and biopesticides, biocontrol agents, use in rhizoremediation, and bioremediation that can aid in the improvement of crop nutrient efficiency and stress tolerance [1]. Manipulation of beneficial plant-microbe interactions contributes to a broader comprehension in relation to ecological roles of microbial populations for sustainable agriculture [2]. A few examples of the beneficial plant-microbe interactions include various microorganism including fungi and bacteria belonging to different genera, some of which are discussed further in this paper [4].

6.2.1 Arbuscular Mycorrhizal Fungi One of most ancient and probably the first interaction of plants and microorganisms was the colonization of ancestral filamentous fungi of the mycorrhizae. The interaction encapsulated the facilitation of nutrient and fluid consumption by the mycorrhizal constituent to the plant host, which was met with a subsequent action of the plant host of providing photosynthetically derived carbon to the mycorrhizal symbiont [8]. Mycorrhiza is classified under two groups, that is, endomycorrhiza and ectomycorrhiza

Exophytical and Endophytical Interactions  187 [4]. Endomycorrhiza have roots resembling those of normal plants in terms of color, size, and shape with the exception of hyphae which penetrates into the cortical cells of plant roots forming arbuscules or vesicles. In certain cases, the endomycorrhiza may possess both the arbuscule and the vesicles and therefore is referred to as vesicular arbuscular mycorrhizae (VAM) [2]. Ectomycorrhiza, on the other hand, metabolizes plant carbohydrates, namely, mannitol and trehalose for the plant as many plants are incapable of these metabolisms. Ectomycorrhiza also produce protease enzymes which cause protein degradation of leaf litter. Furthermore, nutriment of soil is absorbed by ectomycorrhiza using extra radicle hyphae, delivering it to the plant via branched arbuscules [2]. In today’s world, up to 90% of living plants form symbiotic interactions with mycorrhizal fungi of which about 80% of these plant-fungi interactions are with the AMF group [8]. Previous studies by Kafle et al. [1] stated that it is about 65% of all known species that form plant-microbe associations with AMF. The 15% difference between these studies can be attributed to the differences in years that the studies were conducted by different researchers as well as continued research which uncovers more evidence of interactions of AMF with plants. Arbuscular mycorrhizae fungi (AMF) is a fungus under the subphylum Glomeromycota consisting of no more than 350 species. AMF has coexisted with plants for more than 400 million years, remaining morphologically unaltered. The symbiosis is suspected to be the most important symbiosis on earth; this is, however, under discussion. The evidence of this symbiosis suggests that the relationship between plants and AMF played a critical role in land plant evolution [1]. AMF forms interactions with several plants including all legumes as well as some agronomically important crops (see Table 6.1). Agronomically important crops include wheat, corn, and rice. The symbiosis of AMF with host plants is beneficial for both the parties involved. AMF colonizes the host plant’s roots, therefore transferring nutrients which are crucial for plant development. The host plant in return transfers about 20%–25% of its carbohydrates which were derived photosynthetically to the fungal symbiont [1, 2]. The establishment of arbuscular mycorrhizal symbiosis is characterized by the onset of a suitable root colonization by hyphae belonging to AM, accompanied by development of appressorium together with cortex penetration where formation of arbuscules takes place. Prior to the onset of colonization, the exchange of continuous signals is assumed to take place between the symbionts in preparation and as a means of establishing the colonization [2, 4]. Table 6.1 shows the benefits conferred by mycorrhizal fungi unto the plant hosts they associate with, mainly crops.

188  Biofertilizers Table 6.1  The impact of mycorrhizal fungi on crops [2]. (Open Access) Mycorrhizal fungi

Crop host

Benefit to plant

References

Glomus spp.

Helianthus annuus

Enhances root growth and yield of sunflower.

[9]

Hymenoscyphus ericae

Vaccinium corymbosum, Calluna vulgaris

Improves the growth and improves amino acid uptake.

[10, 11]

Acaulospora scrobiculata

Pongamia pinnata

Improves seedling growth.

[12]

Scutellospora heterogama

Passiflora alata

Stimulates plant growth and inhibit nematode infection.

[13]

Scutellospora fulgida

Asclepias syriaca

Improves biomass, foliar P, and trichome density.

[14]

Glomus mosseae

Solanum lycopersicum

Improves growth and resistance against bacterial wilt.

[15]

Glomus clarum

Cucumis sativus

Improves yield and P and Zn uptake.

[16]

Glomus etunicatum

Citrus aurantifolia

Improves growth via increase in chlorophyll contents, photosynthesis rate.

[17]

Glomus intraradices

Zea mays

Improves maize seedlings tolerance to low temperature stress.

[18]

(Continued)

Exophytical and Endophytical Interactions  189 Table 6.1  The impact of mycorrhizal fungi on crops [2]. (Open Access) (Continued) Mycorrhizal fungi

Crop host

Benefit to plant

References

Glomus fasciculatum

Wedilia chinensis

Improves total biomass and nutrition uptake.

[19]

Scutellospora spp.

Zea mays, Glycine max

Improves the root and shoot growth.

[20]

Archaeosporatrappei

Plantago lanceolata

Increases in growth rate resulting in endurance to actions of herbivores.

[21]

Rhizophagus fasciculatus

Solanum lycopersicum, Capsicum annuum

Improves fruit yield, biomass, P accumulation in shoots.

[22]

Rhizophagus clarus

Capsicum annuum

Improves nitrogen and phosphorus uptake.

[23]

Funneliformis mosseae

Morus alba

Improves growth via increase in chlorophyll contents, photosynthesis rate, and stomatal conductance.

[24]

6.2.2 Plant Growth-Promoting Microorganisms Pathogenic nature of some microorganisms makes plant-microbe interactions a battleground of competition for resources between different microbes. With the fight against pathogenic microorganisms which bring about destruction of plants as well as enormous agricultural losses, solutions to plant growth and development producing high yields of crops and protection against pathogens are very crucial for agricultural sustainability. Fortunately, diverse microbes inhabiting the rhizosphere possess growth and activity inhibition of some soil-borne pathogenic

190  Biofertilizers organisms [4]. These microorganisms with this capability are referred to as plant growth-­promoting microorganisms (PGPMs). An example is that of plant growth-promoting rhizobacteria (PGPR) which can activate a plant to defense mode through induced systemic resistance (ISR) which is in charge of reducing the activity of harmful microorganisms [25, 26]. Figure 6.1 shows the plant’s action toward various biotic and abiotic strains with ISR activation to assist in counteracting the effects of the pathogenic attack. The most studied rhizobacteria equipped with ISR induction abilities are Pseudomonas and Bacillus species [27]. Archived records which describe the undertakings of microorganisms that generate health and growth of plants are present and include bacteria (a)

Healthy state

(b)

Infection

(c)

Disease suppressiveness

(d)

Nutrient Deficiency

ISR

Pathogens

Beneficials Pathogens

Beneficials

Pathogens

Beneficials

Beneficials

Figure 6.1  Plants react differently to varying environmental pressures and alternatively adjust their microbiome to suit the stressor. (a) Displays a plant at optimal conditions with sufficient nutrients (seen as green pentagons) and a systematic production of exudates (shown by red arrows) to maintain this homeostatic state relating to the present pathogenic and beneficial microbes. (b) Reveals the transformation of exudates as a result of pathogenic (red microorganism) infection leading to the formation of stress-induced exudates (indicated by blue arrows) which hinder growth of pathogens, simultaneously allowing beneficial microbe growth and activating ISR to further promote resistance to leaf infection. (c) During a state of disease suppressiveness, microbes attracted by stress-induced exudates are assisted further which represses disease. (d) In conditions of nutrient deficiency, the plant alters the roots metabolomic outline to induce production of nutrients or encourage colonization by beneficial microbes which will aid the plant in dealing with the issue. The variance in font size is to indicate the quantity of the microbes under the distinct conditions. (Adapted from [8]. Open Access).

Exophytical and Endophytical Interactions  191 and fungi such as Burkholderia and Trichoderma, respectively. Recent evidence reveals that the bacterium Bacillus subtilis FB17 is recruited exclusively by the plant roots’ secretions of L-malic acid, additionally excluding other species of Bacillus. Selective behavior is symbolic of individual rhizobacterial target signals which are only compatible with the said bacteria allowing it to be the only one to colonize the host [4]. The selective behavior also indicates the plants’ autoregulatory mechanisms which mediate and control root colonization and furthermore its extent, perhaps as a measure of defense (Figure 6.1) as well as control of carbon costs lost to the microbial symbiont [1] Rhizobacteria take up rhizospheric residence, whereby they exert constructive outcomes on plants [28]. PGPRs colonize plant roots and provide the plant with many benefits including producing plant growth stimulating compounds which is expressive of their given name as plant growth-promoting bacteria [26, 27]. The reactions of PGPRs have similarities with the reactions of legume-rhizobia signaling mechanisms which include flavonoids participation. Compounds exuded by roots of PGPRs are involved in modulating the interactions between plants and PGPRs. However, the procedures of releasing the precise signals by roots to recruit distinct bacterial species are still a mystery [4]. Plant growth-promoting rhizobacteria greatly influence soil fertility maintenance [29] which stimulates growth, simultaneously increasing and improving crop yields. The beneficial aspects of PGPRs make them suitable for a role as biofertilizers. The use of PGPRs as biofertilizers would minimize the need for chemical fertilizers that are hazardous to human, animal, and plant health [1, 6, 7, 26, 30]. The reactions of PGPRs with plants yield various compounds, all of which are essential for plant growth. These compounds include phytohormones (responsible for seed germination, flowering, sequence of leaves and fruits, etc.), ammonia which is produced through nitrogen fixation, and siderophores. PGPRs are also involved in reactions such as the solubilization of phosphate, iron availability, as well as the ISR mechanism [6, 26] as shown in Figure 6.1. In addition to its potential use as a biofertilizer, PGPRs also possess biocontrol capabilities which are due to its synthesis of compounds such as hydrogen cyanide (HCN) which acts as a biocontrol agent for weeds. PGPRs also synthesize antibiotics as well as produce lytic enzymes (chitins, proteases, lipases, etc.) that lyse portions of the pathogenic cell walls, thus retarding their growth [26]. Some examples of PGPRs as well as the nutrients they make available to the plant they form a symbiosis with are shown in Table 6.2. Some of the bacteria in Table 6.2 are also endophytes that fix nitrogen.

192  Biofertilizers Table 6.2  Rhizobacteria that promote plant growth and the nutrients they make available to the plant [26]. (Open Access) Nutrient

PGPR (Bacteria)

References

N, P, K, Fe

Rhizobium leguminosarum

[31]

Bradyrhizobium japonicum UCM B-6018

[32]

N, P, Fe

Pseudomonas aeriginosa BS8

[33]

N, P

Bacillus mucilaginosus

[34]

N, Fe

Pseudomonas strain GRP3

[35]

Pseudomonas fluorescens C7

[36]

Azospirillum spp.

[37]

Pseudodomonas alcaligens PsA15

[38]

Mycobacterium phlei MbP18

[39]

Azospirillum lipoferum, Azospirillum brasilense

[40]

Klebsiella pneumonia, Pantoea agglomerans

[41]

Azotobacter spp.

[42]

Azotobacter chroococcum

[43]

Streptomyces spp.

[44]

Microccocus spp.

[45]

Achromobacter spp.

[46]

Bacillus spp., Burkholderia spp.

[42]

Bacillus megaterium

[47]

Pseudomonas alcaligenes

[48]

Pseudomonas aeruginosa

[49]

Bacillus edaphicus

[50]

N

P

K

(Continued)

Exophytical and Endophytical Interactions  193 Table 6.2  Rhizobacteria that promote plant growth and the nutrients they make available to the plant [26]. (Open Access) (Continued) Nutrient

PGPR (Bacteria)

References

Zn

Serratia spp.

[51]

Pseudomonas aeruginosa

[52]

Flavobacterium spp.

[53]

Pseudomonas spp. PsM6, P. jessenii PjM15

[54]

Acetobacter diazotrophicus

[55]

Rhizobia spp.

[56]

Pseudomonas sp. Z5

[57]

Note: N, Nitrogen; P, Phosphorus; K, Potassium; Fe, Iron; Zn, Zinc.

6.2.3 Rhizobia The soil is very limited in certain nutrients or elements. These limited nutrients are, however, crucial for plant development, including growth. It is in these instances that plant-microbe interactions come in handy. Nitrogen is one of the elements necessary for plant growth but is very limited in the soil. Fortunately, there is a group of microorganisms with the ability to transform atmospheric dinitrogen to nitrogen-containing composites which are utilized by plants for plant growth. These microorganisms are called nitrogen-fixing microorganisms (N-fixing microbes) or diazotrophs. In most cases, bacteria are responsible for nitrogenfixation; however, some archaea can also fix nitrogen. Some of these N-fixing microbes are said to be free-living as they can fix nitrogen independently, whereas some can only fix nitrogen in symbiotic associations. Nodule bacteria known as rhizobia are among the most important beneficial microorganisms alongside AMF. Rhizobia form a symbiotic relationship with leguminous plants where it lessens atmospheric dinitrogen (N2) into ammonia (NH3) in a process known as nitrogen-fixation (N-fixation) [1, 8]. Rhizobia associate with plant roots, with the process of N-fixation occurring inside the root nodules, it is then secreted to host plant while the leguminous plant in return provides the rhizobia with dicarboxylates [2, 8].

194  Biofertilizers

6.2.4 Endophytes Endophytic microbes reside within the tissue of plants. The endophytes can be bacterial or fungal. The associations between these endophytes can be very beneficial for the plant host from growth-inducing metabolite and phytohormone production, an improved nitrogen nutrition in diazotrophic endophytes (see Table 6.2), biosynthesis of 1-aminocyclopropane1-carboxylate (ACC) deaminase, phosphate solubilization to inhibition of pathogenic microbial growth [4]. The capability of endophytes for plant growth promotion and can occur directly or indirectly with the former employing nitrogen fixation, formation of plant regulators including the plant hormones auxins, gibberellins, and cytokines, hindrance of ethylene production by ACC deaminase, as well as the changing of sensory mechanisms of sugar in plants. Indirect mechanisms concern the obstruction of pathogenic activity [4]. One of the examples of beneficial endophyte interactions with plants involves transformations of chemical processes and biosynthesis operations of trehalose in plants which provide a tolerance of abiotic stresses [4].

6.3 Pathogenic (Harmful) Interactions Pathogenic interactions are plant-microbe interactions which are harmful to the plant. In these associations which can be classified as parasitic interactions, only one-party benefits, whereas the other suffers. The parasitic microbe will not only compete with beneficial microbial symbionts for resources provided by the host plant but will furthermore cause disease onto the plant ultimately bringing about destruction of that plant either by significantly exhausting plant resources leading to growth inhibition and low yield or by extremely damaging plant structure which eventually kills the plant. Since the dawn of civilization, plant diseases have plagued crops leading to severe agricultural losses as well as causing significant effects on the well-being of humans. Infection causing plant diseases are still the major causative agents of human suffering and large economic losses [5]. Pathogenic microbes can infiltrate the plant through the rhizosphere as well as directly through the plant tissue above the soil (phyllosphere). According to Dou and Zhou [5], plants are inhabited by diverse numbers of microscopic organisms some of which are capable of causing adverse infections leading to diseases. A small portion of pathogens possess the ability to successfully invade plant hosts, causing diseases. Plants have, however, evolved defense mechanisms to counteract the invasion

Exophytical and Endophytical Interactions  195 of pathogenic attacks such as the hypersensitive response (HR), referring to a quick defense response that is induced by plants when invaded by pathogens where plants recognize and repel invasion through this phenomenon [5, 57]. The response of HR leads to a localized apoptosis of the initial infection site which then inhibits a further spread of the disease. The HR response leads to restriction of pathogen growth through a selective apoptosis of the infected cell, a process known as programmed cell death (PCD) [57]. Furthermore, actions of PCD also trigger a nonspecific resistance which is called systemic acquired resistance (SAR). SAR ensures that resistance from pathogens in maintained for a significant period thus protecting the plant from various pathogens [5, 57]. Although plantassociated bacteria possess abilities or rather mechanisms to assist the plant in protecting itself through ISR [25], some microbes are not so easy to fight or even manipulate genetically, such as oomycetes [58]. There are a number of pathogens that affect plants, in addition to oomycetes, there are fungi, bacteria, and viruses, all of which have disastrous effects upon their interactions with plant life.

6.3.1 Oomycetes Oomycetes are one of the major plant pathogens together with fungi. They are classified under the kingdom Protoctista. Oomycetes are similar to fungi in that they form filamentous growth when in their vegetative state, producing mycelia with the formation of spores which they use for asexual and sexual reproduction. The sexual reproduction of oomycetes occurs through the formation of oospores resulting from the hyphae of male antheridia contacting the female oogonia, whereas asexual reproduction results from the production of zoospores due to a cytoplasmic cleavage formation of sporangia. The sporangia which give rise to zoospores allow the dispersal of oomycetes by wind and water where zoospores swim in the water to ultimately reach plant surface where they attach themselves through with the use of adhesion molecules [59]. Contrary to some fungi, oomycetes are a challenge since they are not easily controlled using genetic manipulation [58]. Similarly, to fungi, oomycetes have different modes of colonization of the plant host. Colonization by oomycetes can be biotrophic, necrotrophic, or hemibiotrophic. Biotrophic colonization involves close associations with living plants where the oomycete grows and reproduces within the living plant tissue by absorbing the plant’s nutrients. Necrotrophic colonization, on the other hand, does the opposite of biotrophic colonization in that the oomycete kills the plant tissue prior to colonizing it and thereafter feeds on the dead plant tissue.

196  Biofertilizers The last mode of oomycete colonization known as hemibiotrophy which is the amalgamation of the above-mentioned colonization lifestyles where the onset of colonization is biotrophic; however, as the interaction prolongs, overtime the host plant cells eventually die leaving the oomycete to feed on the dead cells, thus changing its lifestyle from a biotrophic one to a necrotrophic one [58, 59]. Among the oomycetes which have been cause for many plant diseases, some have been found to cause damage in a wide range of plants. The oomycete Phytophthora is one such plant pathogen. Phytophthora is the most studied genus of oomycetes, affecting various plants including potatoes, cocoa, soybean, and even some species of forest trees found in regions of California and Australia [58]. More examples of oomycete interactions are shown in Table 6.3. A brief analysis of Table 6.3 suggests that all species of the Pytophthora genus are hemibiotrophic, which shows their versatile lifestyle to survive both in living and dead plant cells; this versatility has allowed the Phythophtora to invade a broad spectrum of Plantae. The downy mildew, on the other hand, Table 6.3  Examples of oomycetes, the plants they affect, as well as their mode of colonization or lifestyle [59]. (Open Access) Host organ affected

Lifestyle (Colonization)

Quercus agrifolia, Notholithocarpus densiflorus

Phloem, Inner bark

Hemibiotrophic

P. parasitica

S. lycopersicum, S. tuberosum, C. annuum

Root, Leaves

Hemobiotrophic

P. sojae

G. max, G. soja, Lupinus spp.

Roots

Hemibiotrophic

P. palmivora

M. truncatula, N. benthamiana

Roots, Trunks, Buds and Leaves

Hemibiotrophic

P. infestans

Potato, tomato and wild tobacco

Shoots

Hemibiotrophic

P. capsici

C. annuum

Stems and fruit

Hemibiotrophic

Oomycete

Plant host

P. ramorum

(Continued)

Exophytical and Endophytical Interactions  197 Table 6.3  Examples of oomycetes, the plants they affect, as well as their mode of colonization or lifestyle [59]. (Open Access) (Continued) Host organ affected

Lifestyle (Colonization)

Most annual and herbaceous perennial species

Roots

Hemibiotrophic

Pythium ultimum

Zea mays, G. max, S. tuberosum, and Triticum spp.

Roots

Necrotrophic

A. candida

A. thaliana, other Brassicaceae plants

Leaves

Biotrophic

Aphanomyces euteiches

M. truncatula, Pisum sativum, M. sativa

Roots

Biotrophic

H. arabidopsidis

A. thaliana

Leaves

Biotrophi

H. parasitica

Capsella bursapastoris, Brassicaceae, and A. thaliana

Leaves

Biotrophic

Peronospora manshurica

G. max

Leaves

Biotrophic

Plasmopara viticola

Vitis spp.

Leaves

Biotrophic

Oomycete

Plant host

P. cinnamomic

Note: A., Arabidopsis; C., Capsicum; G., Glycine; H., Hyaloperonospora; M., Medicago; N., Nicotiana; P., Phytophthora; S., Solanum.

in the families Peronosporaceae and Albuginaceae such as Hyaloperonospora Arabidopsis, Hyaloperonospora parasitica, and Plasmopara viticola, including Albugo candida which are the causative agents of white rusts, all exhibit the obligate biotrophic lifestyle, as they can only survive in living plant cells. From Table 6.3, only one organism seems to live a necrotrophic lifestyle, namely, the Pythium ultimum which suggests that either there are not many oomycetes that colonize plants in this form or there has not been enough research done on this particular group of oomycetes.

198  Biofertilizers

6.3.2 Fungi Fungi are one of the major plant pathogens, with some research suggesting that they may possess the most diverse ecologically and economically relevant threats. Most fungal pathogens affecting plants are found in the phyla Ascomycota as well as Basidiomycota. Fungal plant pathogens are responsible for large amounts of agricultural losses. They cause losses in the quality of crops, fruits, and plant material in general. They use conserved proteins in their infection processes. It stands to reason that an understanding of theses conserved proteins would therefore be very crucial in controlling fungal diseases [60]. Fungi, much like oomycetes, have different modes of colonization, namely, biotrophic, necrotrophic, and hemibiotrophic, these colonization modes differentiate between various fungi species based on their lifestyle associations with the plant host as well as the way in which they feed from the host plant [58, 60]. The lifestyle categorization is the same as that of oomycetes with a biotrophic lifestyle that entails the intimate interaction of the fungus with the plant host where the fungus has established itself in the hosts living cells and simultaneously feeding from live tissue belonging to the host plant. The same case applies with necrotrophic lifestyle where the fungus destroys the host plant and thereafter feasts on the dead cells of the plant. This thus suggests that the hemibiotrophic relationship of fungi with plants will also resemble that of oomycetes, with the fungus beginning a biotrophical relationship with the plant till a point where the plant is killed by the gradual infection imposed by the fungus whereby the fungus will then convert its lifestyle to a necrophytic one by consuming the dead cells of the plant host. The pathogenic fungi form filamentous growth as well as spores for reproduction. The spores are carried by wind, water, and insect vectors, and upon reaching the plant surface, they firmly attach to plant surface to avoid being washed away [60]. Plant pathogenic fungi examples include the biotrophic fungi Cladosporium fulvum which causes tomato leaf mold as well the Ustilago maydis which is also a biotrophic fungus which causes smut disease on maize and teosinte. A unique property of these biotrophic fungi is that unlike other fungi as well as oomycetes, they resemble endophytes in that they do not produce haustoria. The pathogenic fungi, however, can produce specialized infection structures such as appressoria, which penetrate the host cell. Peroxisomes play a critical role during these processes by facilitating the full functions of virulence protein [60]. Infection of the fungal specie Erysiphe cihoracearum results in a disease on A. thaliana which is characterized by white powder-like spots on leaves and stems. Magnaporthe grisea/oryzae is a fungal specie that

Exophytical and Endophytical Interactions  199 has catastrophic implications for rice as it causes gray lesions to form which completely spread across the shoot area causing rice blast [61]. Colletotrichum higginsianum is another economically important fungus which causes a disease that forms black lesions on cruciferous vegetation such as cabbage (Brassica) and radish (Raphanus) with the addition of A. thaliana n. The C. higginsianum pathogen is said to be adapted for A. thaliana [62].

6.3.3 Bacteria Bacteria are small single-celled organisms which were only found to be pathogenic to plants, causing plant disease as little as over 100 years back. Plant bacterial pathogens are responsible for serious plant diseases, however not as severely as fungus and viruses. Compared to fungi and viruses, pathogenic bacteria cause less economical costs and damage to plants. It should be noted, however, that bacteria are capable of causing severe economically damaging diseases which start from very minor symptoms such as spots, eruptions, and swelling that can be observed on the surface of the plant such as on the leaf or the flesh of fruits on plant leaves and fruits, going to severe rotting of bulbs which is indicative by the exudation of putrid smells and ultimately to the death of the infected plant [63]. Plant bacterial pathogens which cause bacterial diseases can occur in any plant leading to distortions of leaves and shoots called fasciation due to hormones as well as a rapid multiplication of the plant’s cells leading to enlargements observed in the areas of convergence of the soil, stem, and roots referred to as crown gall [63]. The development of a pathogenic infection due to bacteria may follow various routes with speculations that the whole ordeal merely happens passively due to accidental cases. It is assumed that pathogenic bacteria can enter the plant through natural openings which include stomata, hydathodes, or lentils. In some cases, however, wounds or abrasions to the plant hosts roots, stem, or on leaves due to feeding of insects as well as by seed immersion into inoculum can introduce the bacteria to the plant [2, 63]. Entry of bacteria into the plant host is followed by attachment of the bacterial pathogen, with the adhesion to the host plant facilitated by hair like polymeric organelles called Pili. After colonization has been established, the onset of disease symptoms can be observed in the form of wilts, spots, blights, crankers, and galls [2]. The majority of infection causing bacteria can be those belonging to the Actinobacteria or the Proteobacteria phyla [2, 21]. Virulence factors are the determinants of the severity of disease which is measured by the type

200  Biofertilizers of virulence factor utilized by the bacterium. Virulence factors are ranged from type I to type IV depending on the bacterial specie. Virulence factors include secretion systems, extracellular enzymes, polysaccharides, plant hormones, as well as toxins [2]. Early associations of pathogenic bacteria with plants were observed in apples and pears which were infected with fire blight disease in 1878. The disease is now well known and widespread and is caused by a bacterial plant pathogen called Erwinia amylovora. E. amylovora which to this day remains a big problem as it significantly decreases production of nutritious fruit [63]. Some of the examples of plant pathogenic bacteria include X. oryzae pv., the rice bacterium that is a causative agent of chlorotic stripes which give rice leaves a water-soaked appearance. The bacterium pathogen also affects Arabidopsin leaves, causing lesions [64]. Another Xanthomonas specie, namely, Xanthomonas albilineansis, utilizes secondary metabolites in the form of toxins which affect the host plants’ physiology and biochemistry ultimately causing leaf scald disease of sugar cane. The released toxin acts as the effective pathogenicity factor. In certain cases, Xanthomonas secrete exopolysaccharides (EPS) as opposed to using plant hormones. The EPS provides virulence for the Xanthomonas with, observations of previous studies with Xanthomonas mutants which lack EPS show that virulence in theses mutants is ultimately lost [2]. Clavibacter michiganensis  subsp.  sepedonicus is a bacterium that infects potatoes causing a disease called potato ring rot. It is famous for its resistance which allows it to survive on machinery and packaging material [63]. Pseudomonas syringae is another bacterial pathogen; however, P. syringae is the first pathogen proven to infect the Arabidopsis plant and result in disease signs [65]. Pseudomonas syringae infections cause small water-soaked chlorotic lesions on the Arabidopsis plant. An alternative pathogenic attack to the plant host is that observed in Erwinia carotovora which secretes extracellular proteins that generate a chemical deterioration of the host plants’ cellular membrane, thus breaking the first line of defense rendering the plant defenseless and susceptible to effective infection by the bacterial pathogen [2].

6.3.4 Viruses Viruses are very small organisms that are known to infect all living things including plants, animals, bacteria, and fungi. Viruses are obligate parasites which means their survival is solely depended on its host, there is yet to be a virus known to be capable of living without a host. Although

Exophytical and Endophytical Interactions  201 small viruses have evolved means to survive, they carry nucleic acids of their genetic information that encodes three proteins which upon finding a specific host they inject into the host for reproduction and replication into the host [66]. Plant-viral interactions are extremely detrimental to the plant. They cause serious diseases which amount to catastrophic losses of crop yield and quality leading to economic losses worldwide. Virus caused diseases in plants pose a difficult challenge as they are very difficult to cure, as a result, attempts to try and cure the plant eventually lead to death of that plant [66, 67]. Some examples of plant-virus interactions include Cassava brown streak virus (CBSV or Potyviridae), which is a virus that causes cassava brown streak disease. CBSV or Potyviridae is a great challenge for the production of cassava and can be rated as the most serious inhibitor of Table 6.4  Ranking of the 10 significant viruses, listed my order of importance [67]. (Open Access) Rank

Virus

Disease caused

Reference

1

Tobacco Mosaic Virus (TMV)

Mosai disease of tobacco

[68]

2

Tomato Spotted Wilt Virus (TSWV)

Spotted wilt disease of tomatoes

[69]

3

Tomato Yellow Leaf Curl Virus (TYLCV)

Tomato yellow leaf curl disease

[70]

4

Cucumber Mosai Virus (CMV)

Cucumber mosaic disease

[71, 72]

5

Potato Virus Y (PVY)

Potato tuber necrotic ringspot disease

[73, 74]

6

Cauliflower Mosaic Virus (CaMV)

Cauliflower mosaic disease

[75]

7

African Cassava Mosaic Virus (ACMV)

Cassava mosaic disease

[76]

8

Plum Pox Virus (PPV)

Sharka disease

[77]

9

Brome Mosaic Virus (BMV)

Brome mosaic disease

[78, 79]

10

Potato Virus X (PVX)

Potato mosaic disease

[80]

202  Biofertilizers cassava growth. Single-stranded DNA viruses such as the ones of the Geminiviridaes are among some of the most important viruses which have deleterious outcomes on the economy as they constitute to billions in financial losses. The tomato yellow leaf curl virus (TYLCV) is responsible for a vast majority of catastrophic tomato disease in the world, ranking number 3 on the list (Table 6.4) of the most important plant viruses causing plant death. TYLCV and the African cassava mosaic virus (ACMV) rated number 7 on the list of the viruses most associated with plant pathology (Table 6.4) are made even worse by their efficient transmission by whitefly vectors [67]. Tobacco mosaic virus (TMV) is regarded as a leading virus in terms of its effective colonization of plants causing enormous plant losses. It is rated number 1 (Table 6.4) on the list of viruses ranked according to their associations with plant pathology. Intensive research has been conducted on TMV in an attempt to understand the mechanisms involved so that a control of TMV-induced diseases on tobacco can be established. Figure 6.2 shows the tobacco leaves of the Nicotiana tabacum cv. Turk plants as well as the Nicotiana tabacum cv. Glurk leaf which were infected with TMV. The leaf shown on Figure 6.2a indicates the systemic infection

(a)

(b)

Figure 6.2  (a) Shows a spread-out infection by TMV on the leaf of N. tabacum cv. Turk plant which is identifiable by formation of patterns on the leaf. (b) Shows the death of cells of the N. tabacum cv. Glurk plant, which is specified by dark brown spots on the leaf due to TMV infection. (Adapted from [67]. Open Access)

Exophytical and Endophytical Interactions  203 of  TMV characterized by the formation of mosaic on the leaves of N. tabacum cv. Turk plants. Figure 6.2b shows the infection of Nicotiana tabacum cv. Glurk leaf by TMV. Symptoms of infection are clearly visible on the leaf demonstrated by the formation of brown spots. This figure shows the symptoms of infection by TMV in different tobacco plants. The spread of disease in the plants would account for the large plant losses implicated by the virus. More examples of viruses which infect and cause disease of plants are listed in Table 6.4 together with how they are ranked according to their influence in plant pathology.

6.4 Conclusion Plant-microbe interactions are one of the oldest relationships to be formed on earth. The very existence of these interactions could be the basis for all life forms that exist on this planet. With millions of years that have passed, these interactions are only evolving more and yet still much is not known about the full mechanisms they carry out. It is evident that there is a lot of research work to be done about the full mechanisms that take place in these associations. Certainly, a full understanding of these interactions together with the processes that lead to their respective outcomes would yield ground-breaking innovations or improvement in the agricultural sector as well as the planet at large. As it is, research in the processes such as the use of some of these plant-microbe interactions to create biofertilizers and biopesticides are benefiting plants and the environment as a whole. These biological processes made possible by researching these plant-microbe interactions and prevent the use of chemical and toxic pesticides which are causing more soil pollution and water contamination. They get leached onto the earth and penetrate below ground thus disturbing the soils natural microbiota, polluting underground water and, moreover, causing loss of biodiversity. The use of bioremediation as a means to rid the environment of toxins is a promising venture which is made possible by the continuous study of these interactions between plants and microorganisms. Pharmacological and molecular studies provide evidence of the basic regulatory mechanisms which suggest operations between plants and microscopic organisms could be effective as biocontrol agents. Processes associated with beneficial plant-microbe interactions give promise to a better world where the use of toxins and the presence of them thereof in the environment is significantly reduced due to use of

204  Biofertilizers bioinoculants which are eco-friendly, therefore having no adverse effects on crop development. Pathogenic interactions, on the other hand, provide perspective on how these processes they undergo may be used to give rise to solutions on how to control or at least minimize or counteract pathogenic organisms on plant life. The use of pathogenic pathogens could prove very effective as biopesticides which act against other pathogens including insects. Plants show defense mechanisms against pathogenicity which with continued research could give interesting solution in better understanding pathogenic pathways. In addition to HR, SAR, and ISR, plants possess self-defense mechanisms consisting of chemical and biochemical substances which some are pre-existing such as toxic exudates which defend against pathogenic spores. From the majority of research studies so far, it would seem that the model plant genus Arabidopsis is affected largely by a broad spectrum of pathogenic organisms. It seems almost all above discussed pathogens have the capability to invade this genus. With the many defense mechanisms that plants have evolved since the dawn of civilization, it is a wonder that this genus has yet to develop stronger means to defend itself from the many pathogens that seem to effortlessly be able to break through its defenses. This evidence, however, could also be a consequence of intensive research of the model plant or even the versatility of this plant to host different species which makes the plant ideal for research purposes. More research into plant-microbe associations is promising a better understanding as well as opportunities to produce biological substances which will also improve the current climate challenges caused by the excessive use of toxic chemicals.

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210  Biofertilizers 74. Tribodet, M., Glais, L., Kerlan, C., Jacquot, E., Characterization of Potato virus Y (PVY) molecular determinants involved in the vein necrosis symptom induced by PVYN isolates in infected Nicotiana tabacum cv. Xanthi. J. of Gen. Virol. 86(7): 2101–2105, 2005. 75. Love, A.J., Laird, J., Holt, J., Hamilton, A.J., Sadanandom, A., Milner, J.J., Cauliflower mosaic virus protein P6 is a suppressor of RNA silencing. J. of Gen. Virol. 88(12): 3439–3444, 2007. 76. Tiendrébéogo, F., Lefeuvre, P., Hoareau, M., Harimalala, M.A., De Bruyn, A., Villemot, J., Traore, V.S.E., Konate, G., Traore, A.S., Barro, N., Evolution of African cassava mosaic virus by recombination between bipartite and monopartite begomoviruses. Virol. J. 9(1): 67, 2012. 77. Rodamilans, B., Rodamilans, San León, D., Mühlberger, L., Candresse, T., Neumüller, M., Oliveros, J. C., García, J. A., Transcriptomic analysis of Prunus domestica undergoing hypersensitive response to Plum pox virus infection. PloS one. 9(6), 2014. 78. Jeżewska, M., Trzmiel, K., Zarzyńska-Nowak, A., Detection of infectious Brome mosaic virus in irrigation ditches and draining strands in Poland. European J. Plant Pathol. 153(1):285–292, 2019. 79. Ni, P., Vaughan, R.C., Tragesser, B. Hoover, H., Kao, C.C., The plant host can affect the encapsidation of brome mosaic virus (BMV) RNA: BMV virions are surprisingly heterogeneous. J. mol. biol. 426(5):1061–1076, 2014. 80. Cong, Q.Q., Wang, J., Liu, J., Lan, Y.F., Guo, Z. K., Yang, J.G., Li, X.D., Tian, Y.P., Evaluation of Potato virus X mild mutants for cross protection against severe infection in China. Virol. J. 16(1): 36., 2019.

7 Biofertilizer Formulations Sana Saif, Zeeshan Abid, Muhammad Faheem Ashiq, Muhammad Altaf* and Raja Shahid Ashraf Department of Chemistry, Government College University Lahore, Lahore, Pakistan

Abstract

The growing human population has increased the fertilizers demand for sustainable agriculture. Recent advances suggest the use of fertilizers based on microorganisms to avoid the hazardous effects of chemical fertilizers. Biofertlizers are the formulations containing living or latent microorganisms supported on a suitable carrier material that is easy-to-use as well as environmentally safe. A complete formulation strategy includes several crucial steps such as choice of particular microbe, selection of suitable carrier material, and formulation process. In addition, some additives and sticking materials are also added. The microorganisms like bacteria, algae, fungi, and actinomycetes have been reported extensively. Depending upon the nature of inoculum, four different kinds of formulations are prepared. In case of carrier-based formulations, carrier materials such as peat, wheat bran, talc, and vermiculite are in extensive use. To avoid the unwanted effects of powder formulations, peat prills are used in granular formulations. While the liquid formulations are prepared to get ease in handling and enhance the effectiveness of formulation. Moreover, cell immobilization is the latest formulation minimizing all the limitations of already existing formulation types. This chapter presents and explains all the steps involved in formulation strategy, emphasizing their use for better quality control. Keywords:  Biofertilizers, biofertilizer formulations, types of formulations, inoculants, inoculant preparation

*Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (211–256) © 2021 Scrivener Publishing LLC

211

212  Biofertilizers

List of Abbreviations PGPM Plant growth-promoting microorganisms ACC 1-aminocyclopropane-1-carboxylate PGPR Plant growth-promoting rhizobacteria IAA Indole-3-acetic acid ECMF Ectomycorrhizal fungi ENMF Endomycorrhizal fungi AMF Arbuscular mycorrhizae

7.1 Introduction 7.1.1 Evolution of Biofertilizers The role of conventional agriculture in food supply for human beings cannot be ignored. However, the continuous increase of the human population demands sustainable modes of productivity. The human population has replaced the land available for food production. It imparts pressure on agricultural resources to meet the growing need for food. Therefore, there is an urgent need to adopt some novel technologies that could fulfill the food demand of 7.6 billion people, and in 2050, this number is expected to touch approximately 10 billion. It is a big challenge to supply food to this increasing population. It could only be possible using pesticides or chemical fertilizers or both [1]. Since the Third Agricultural (Green) Revolution, the chemical fertilizers are in use tremendously. Chemical fertilizers are the compositions containing various macronutrients (K, N, S, and others) and micronutrients (zinc, iron, copper, and bismuth) that, when applied to the soil or on a plant, can supplement it and augment fertility of the soil and thus crop growth [2, 3]. The availability and sufficiency of these nutrients are vital for sustainable agriculture [4]. Commonly known nitrogen (N), phosphorus (P), and potassium (K) fertilizers, also called NPK fertilizers, exhibit a high production demand that provides N (NH3, (NH2)2CO, and NH4NO3), P ((NH4)2HPO4, superphosphates), and K (potash, sulfate of potash, KCl, KNO3, kieserite) in compound forms. However, increasingly high use of chemical fertilizers rendered the soil degraded, highly polluted, and inert toward food production and also caused severe environmental hazards [5]. Since plants cannot take up these hazardous chemicals, they start to accumulate in the soil and ultimately in groundwater. Such chemicals conduct the eutrophication of the water bodies, soil acidification, weakening plant roots, and also disease incidence [6]. Moreover,

Biofertilizer Formulations  213 nitrates can cause a severe disease, i.e., “acquired methemoglobinemia”, also called Blue Baby Syndrome [7]. The excessive use of chemical fertilizer caused harsh abiotic stress, which has consequences of abrupt temperature variations. It is inevitable to use chemical fertilizers as the food demand is rising continuously in the world. However, considering the adverse effects of chemical fertilizers for prolonged use, the organic farming has been appeared as an alternative in terms of growing healthy food, environment-friendly, and thus long-term sustainability [8]. In this regard, biofertilizers are forms of organic farming that are being used. Biofertilizers are natural or man-made substances containing living or inactive strains of efficient microbes that supplement the plant by colonizing the rhizosphere or directly to the roots when applied to targeted crops, roots, seeds, or soil [9]. Biofertilizers help plants by providing a continuous source of micro and macronutrients. Sometimes, these are also termed as “bio inoculants or microbial inoculants”. In 1896, the very first commercially available biofertilizer, “Nitagin”, was produced by Nobbe and Hiltner in the USA. At that time, this organic fertilizer did not seek much attention. Nevertheless, the Ministry of Agriculture and Farmers under the Ninth Plan (1997–2002) started a real effort to set up the National Project on Development and the use of Biofertilizers (NPDB) [10]. Since then, it is inevitable to use biofertilizers for plant growth and productivity due to their unique features toward less environmental effects, high plant growth, and thus more productivity.

7.1.2 Biofertilizers: A Sustainable Approach Chemical fertilizers have undoubtedly led to critical environmental contamination to fulfill the need for food for the continuously growing population. Moreover, severe damage to friendly insects and natural microbial habitat has occurred. Nonetheless, this indiscriminate use of such chemicals has made the crops less resistant to pathogens; hence, more prone to diseases [11, 12]. Considering all the hazardous effects, biofertilizers are proved to be a safe substitute for the chemical fertilizers that minimize ecological disturbance. It has been proved that biofertilizers increase the contents of essential amino acids, nitrogen fixation, vitamins, and proteins and thus elevate the yield of about 10%–14% [13]. The term “biofertilizer” also called “microbial inoculant” is often wrongly used for products of animal manure and chemical fertilizers that are organically supplemented and intercropping [14]. However, microbial inoculant is a substance containing living or latent microorganisms supported on a carrier material that can supplement the targeted plant when applied to either roots, soil,

214  Biofertilizers or plant surface [15]. Unlike chemical fertilizers, biofertilizers do not provide nutrients directly to the plant. Either they colonize the rhizosphere or directly to the roots, stimulate their growth, make nutrients available, protect plants from ecological strains, and enhance its tolerance against heavy metals, temperature variations, salinity, and drought [16]. Moreover, they make nutrients available by mineral solubilization and increasing the concentration of essential nutrients in plant roots [17]. So, the microbial processes are accelerated in the soil, which augments the fertility of the soil by assimilating the nutrients and enhancing their extent of availability. Plants uptake 17 nutrients for growth, while only three or four are those added back to it. Continuous removal of nutrients through the soil makes it insufficient for growth. Biofertilizers are the substances that restock the nutrients and keep the soil a buffer of nutrients. Biofertilizers do not have any hazardous effects on soil composition, plant health, or environment. Table 7.1  Comparison of benefits and limitations of biofertilizers [19]. Advantages

Limitations

◦◦ Reduced use of chemical fertilizers and environmental pollution

◦◦ Slow process than chemical fertilizers

◦◦ Uptake and validity of nutrients is increased

◦◦ Difficult to store as these are sensitive toward changes in temperature and humidity

◦◦ Releases doping substances

◦◦ It is hard to find a retailer selling product in remote areas

◦◦ Secretion of antibiotics resistant to plant diseases

◦◦ Biofertilizers can only complement chemical fertilizers not could replace them

◦◦ An economical approach for poor farmers

◦◦ Availability of biofertilizers can reduce due to shortage of either a particular strain or growing medium

◦◦ Complex organic materials are transferred to simpler ones ◦◦ Increased root proliferation as growth-promoting hormones are released ◦◦ Environment-friendly

Biofertilizer Formulations  215 So, they are frankly used as nutrient materials to increase plant growth and productivity. Every 1 g of biofertilizer contains nearly 10 million of strains so anyone can get desirable results even with a small amount of biofertilizer [18]. It is a good practice of sustainable agriculture by approaching biofertilizers, containing potential microbes that elevate plant growth by multiple means and keep the atmosphere healthy. Table 7.1 presents the benefits and limitations of biofertilizers.

7.2 Biofertilizer Formulations Biofertilizer is a preparation that contains living or inactive microorganisms while formulation authorized easy handling, significantly long storage time, and also the efficacy of biofertilizer. Biofertilizer formulation functions as a carrier substance for microorganisms to deliver them from production site to the field. Hence, bioformulation exhibits a carrier state for live or latent bio inoculants that are supplied to the targeted site, either soil or directly plant [20]. The success of inoculant technology mainly depends upon two factors: the type of microbial strain and the formulation of inoculum. Practically, potential success of biofertilizer is determined through the formulation strategy [21]. A formulation may be varied according to the type of soil, the interest of application, available resources, and type of plant. The complete formulation strategy demands a selection of strain, selection of a suitable carrier for carrier-based inoculants, and then formulation strategy.

7.2.1 Selection of Strain The production of suitable inoculants comprises a critical process of strain selection. Therefore, the selected organism as strain must be competitive and highly efficient toward indigenous populations present in the soil [22]. Since it is preferable to use a combination of strains than a single one, both must be compatible with developing an efficient inoculum [23]. Recent studies declared the use of the following type of strains.

7.2.1.1 Microbial Strains The living or latent microorganisms containing biofertilizers are termed as “bio inoculants”. Various inoculants are being used commercially for the biofertilizers. Depending upon the type of microorganisms; these are bacteria, fungi, algae, and actinomycetes that function as nitrogen fixers or solubilizers for various soil nutrients [24]. While based on function, it

216  Biofertilizers could be N2 fixers, P solubilizers, phosphorus and K mobilizers, and symbiotic mycorrhizae. The microbial strains that are mainly used for the formulations are known as plant growth-promoting microorganisms (PGPMs). The nature of PGPM used for the formulation depends upon the nature of the crop, environmental conditions, and targeted land.

7.2.1.1.1 Bacterial Strains

Soil is an efficient buffer of nutrients. It naturally contains a range of bacterial communities that can be pathogenic, beneficial, or maybe neutral. Under varying conditions, these bacterial species compete for niches or desired nutrients with the plant [25]. Beneficial microorganisms can have more than one type of mode of action. Either they form a symbiotic relationship with roots or in free form in the soil [26]. Whatever the form is, bacteria have a magical capacity to store the nutrients and colonize in the rhizosphere. Active plant roots exudate which attract the microbes and an association results that enhance the availability of nutrients in the specific root area [27]. Moreover, they also secrete growth-promoting Indole-3acetic acid (IAA) that helps general growth promotion. These substances enhance resistance against abiotic stresses and plant pathogens [28–30]. By mean of action, these beneficial microbes can be distributed into two separate classes, which are health and growth promoters of plants. Plant growth promoters nourish the plant by providing nutrients and growth-enhancing substances to plants when pathogens are not present. The category includes nitrogen fixers, growth promoters, phosphorus solubilizers, and mobilizers [31, 32]. While plant health promoters sustain growth in abiotic stress and the presence of a pathogen. Either they minimize the hazardous effects of pathogens or block the pathogen directly [33, 34]. Bacterial strains commonly show the possible mechanisms of growth promotion that help plants with nutrient uptake and their utilization. Nitrogen fixation is followed by growth-prompting bacteria named as rhizobacteria (PGPR) and many endophytic bacteria that may live in symbiosis or nonsymbiosis. Many plants especially Azospirillum are involved in the excretion of phytohormones. Both physiological and morphological changes of inoculated plants undergo excretion of IAA. Ethylene expositions have been induced by the reduced growth rate. The PGPR can lower the ethylene level and hence enhance the growth rate. Bacteria hydrolyze the precursor of ethylene that is 1-aminocyclopropane-1-carboxylate (ACC). Products of hydrolysis are commonly used as a nutrient source for the bacterium. Novel mechanisms include synthesis of lump chrome, phytase degrading compounds, volatile compounds production, and mineral reduction through phenazines. All these possible mechanisms enable organisms to

Biofertilizer Formulations  217 help the plants nutritionally. Figure 7.1 shows a few possible mechanisms adopted by growth-­promoting substances and Table 7.2 presents an overview of plant inoculation by different bacterial strains.

7.2.1.1.2 PGPR

The rhizosphere is a repository comprising a specific volume of soil with an excess of the bacterial population surrounding plant roots influenced by the root exudates [36]. It is an ecological niche that is a hub of physical, chemical, and biological activities around plant roots. Rhizosphere commonly contains 100–1,000 times higher bacterial population than the bulk soil. Bacteria exhibit metabolic versatility to stimulate and utilize the root exudates efficiently. Moreover, microbial species covered 15% of the plant root surface that belongs to different bacterial species [37]. About 5%–30% photosynthetic products of plants (in sugar form) are secreted into the rhizosphere through plant roots that are consumed by microbial populations [38]. Subsequent metabolic activities rendered the rhizosphere, a mineral nutrient source that is transported and then plants uptake them. The organisms colonizing rhizosphere include algae, fungi, protozoa, actinomycetes, and bacteria. However, bacteria have proved itself the most abundant microbe in the rhizosphere due to its unique features to enhance growth. Beneficial rhizosphere bacteria can be found in two different forms. Either

N2 Fixation

Production of phytohormones

Phytase degradation Possible mechanisms of growth-promoting substances

Mineral reduction through phenazines

Activity of ACC

Figure 7.1  Schematic of possible mechanisms adopted by growth-promoting substances [35].

Mung Bean, Chickpea

Chilies

Chickpea

Wheat Cotton

Rice plant

Rice plant

P. fluorescens

Bacillus sp. Pseudomonas sp.

Pseudomonas aeruginosa Z5 Azotobactor

Streptomyces philanthi

Streptomyces aurantiogriseus

Targeted crop

Bradyrhizobium

Bacterial strain (PGPR)

Produce IAA and solubilize Phosphorous Increases amount of Phosphorous in soil

Fabaceae

Poaceae Malvaceae

Poaceae

Overcome the sheath blight disease

Constrain growth of pathogenic fungi

Increase in chitinase, peroxidase, β-1

Solanaceae

Poaceae

Produce IAA, having ACC-deaminase activity

Mode of action

Fabaceae

Family

Table 7.2  An overview of plant inoculation using different strains.

Cure rice leaves

Improve yield

Increase plant growth, improve cotton yield

Increase germination, proline content, and length of root and shoot

Increase germination, no. of flowers, reduce fruit rotting

Enhance plant growth, nodulation

Result

(Continued)

[53]

[52]

[51]

[50]

[49]

[48]

References

218  Biofertilizers

Make microbial communities

– –

–

Almost all terrestrial plants

Spinach

Cucumber

Azotobactor vinelandii

Azotobactor chroococcum

Rhizobium Laguerre

Bacillus mucilaginosus

Cucurbitaceae

Amaranthaceae

Nitrogen fixation

Poaceae

Rice plant

Bacillus, Enterobacter

Produce IAA, enhance growth and germination

Produce indole acetic acid, solubilize phosphate

Nitrogen-fixing bacteria

Nitrogen fixation, phosphate solubilization

–

–

Rhodopseudomonas palustris

Mode of action

Family

Targeted crop

Bacterial strain (PGPR)

Table 7.2  An overview of plant inoculation using different strains. (Continued)

Improve yield

Promote growth, enhance number and size of leaves

Improve plant growth

Improve plant growth

Improve plant health and growth by symbiosis

Effective in the growth of various plants

Result

[59]

[58]

[57]

[56]

[55]

[54]

References

Biofertilizer Formulations  219

220  Biofertilizers a symbiotic relationship with roots is present or are free-living in the soil [26]. The PGPR is a group of heterogeneous bacteria, occurred in the rhizosphere, at plant roots surfaces or in particular relationship with roots that ultimately improve and promote the plant growth characteristics either qualitatively or quantitatively [39]. Schroth and Kloepper [40] used the term “PGPR” for the first time for beneficial microbes found in the rhizosphere. Moreover, they also can control phytopathogenic microorganisms. PGPR serves as an active component in biofertilizer formulations [41]. PGPR interacts with plants in two different ways. One is the symbiotic relationship, where bacteria are present in plant bodies and live inside by exchanging metabolites directly. While the other is free-living rhizobacteria, living outside the plant body [42]. In host plants, symbiotic bacteria mostly dwell in intercellular spaces but certain bacteria form a mutualistic interaction and penetrate the host plant. Besides, a few bacteria have the potential of integrating the physiology with that of a host, emerging specialized structures. Rhizobia, a class of mutualistic bacteria, establish a mutualistic symbiotic relationship with legume family that fix atmospheric nitrogen in particular root structure called nodules and make it available for the plant to utilize. PGPR can have two modes of action: one is direct and other is indirect mechanism. In the direct mechanism, microbes function directly within the plant that includes biofertilization, rhizoremediation, growth stimulation, and stress control in plants. While indirect one is a biological control mechanism that acts outside the plant by minimizing the effect of diseases that include antibiosis, installation of systemic resistance, and nutrient and niche competition [43]. Direct mechanism includes biological N2 fixation, P solubilization, and phytohormone production (Figure 7.2). In biological nitrogen fixation, root/legume symbiotic bacteria possess the specificity for plant roots that ultimately form root nodules, particularly in Rhizobium. Free-living bacteria fix nitrogen but do not possess specificity, e.g., Azospirillum, Azotobacter. Organic phosphorus is mineralized by bacteria into inorganic forms that are utilized by plants. The primary mechanism involved in the organic acid secretion due to sugar metabolism acids released acts as Chelator of Ca2+ accompanying the inorganic phosphate production. For example, Pseudomonas and Bacilli production of IAA, a crucial auxin necessary for plant growth, Cytokinin production, and Gibberellins are secreted that influence developmental processes including flowering, seed germination, and fruit set. While indirect mechanisms, as shown in the Figure 7.3, include siderophore production, production of chitinase and glucanase, antibiotics production, induced systematic resistance, plant stress modulation, and ACC deaminase production. Siderophore is below 1-kDa molecular

Biofertilizer Formulations  221

Direct Mechanism

Biological Nitrogen Fixation

Phosphate Solubilization

Phytohormone Production

Figure 7.2  Types of direct mechanisms shown by growth-promoting microorganisms [44].

Siderophore production

Induced systematic resistance

Production of chitinase and glucanase

Indirect mechanism

ACC production

Antibiotics production

Plant stress modulation

Figure 7.3  Possible indirect mechanisms of PGPR [44].

222  Biofertilizers weight compounds that bind iron reversibly. Its soil concentration is 10−30. Pseudomonas genus bacteria are reported releasing pyochelin. PGPR secrets cell wall degrading enzymes like chitinase to effect soil-borne pathogens. Streptomyces spp. have a direct inhibitory effect to degrade fungal cell walls. ISR is a process where a plant is treated by PGPR provoke host defense as shown by a reduction in the incidence of disease or severity due to pathogens. Oxidative stress of plants is minimized by producing enzymes including peroxidase, catalase, glutathione reductase, and superoxide dismutase. ACC deaminase protects plants from the hazardous effects of ethylene under abiotic stress. Several credible PGPR from genera Acinetobacter, Azospirillum, Rhizobium, Agrobacterium, Burkholderia, Thiobacillus, Arthobacter, Pseudomonads, Azotobacter, Bradyrhizobium, and Frankia have been reported [15, 45]. The role of PGPR for enhanced plant growth includes nitrogen fixation for plants, plant growth regulators, abiotic stress tolerance, production of ACC-deaminase, chitinase, and glucanase that are protection enzymes against various plant diseases, volatile organic compounds (VOC), and siderophores production. However, their mode of action commonly varies by varying host plants. Stresses due to soil are major restrictions for sustainable production and can be of two types: biotic and abiotic. Biotic stresses are mainly due to plant pathogens such as bacteria, nematodes, and viruses, while abiotic stresses are due to the presence of heavy metals in soil, nutrients deficiency, temperature, drought, salinity, and so on [46]. Biofertilizer products containing PGPR have been reported and used worldwide, contributing to soil fertility and enhanced crop yield. Hence, PGPR potential has been lead to sustainable agriculture. A combination of PGPR with composts can enhance plant growth and plant biocontrol. Pseudomonas spp. and Bacillus spp. are PGPR that were reported as an effective biocontrol agent [47]. PGPR with sufficient densities in biofertilizers helps in creating the rhizosphere that is necessary for plant growth. PGPR increases the availability of nutrients and inhibiting the pathogenic activity. The high availability of nutrients could enhance the fertility of the soil, improve biocontrol effects, and extend the survival rate of microorganisms in the soil. Different bacterial strains used in different biofertilizer formulations are discussed.

7.2.1.1.3 Algal Strains

Among previously reported formulations based on microorganisms, including bacteria, prokaryotic cyanobacteria, and fungi, microalgae have been gained much attention. Due to their excellent potential for

Targeted crop

Rice

Corn

Corn

Roma Tomato

Spinach, Chinease Chives

Willow (Salix)

Algal strain

Chlorophyta

Chlorella pyrenoidosa, Chlorella vulgaris

Nostoc

Acutodesmus dimorphic

Chlorella fusca

Microsystis aeruginosa, Chlorella sp.

Biostimulant

Increased mineral content Increased chlorophyll content and enzymatic activity

Amaranthaceae Amaryllidaceae Salicaceae

Produce IAA

Rich growth hormones

Nitrogen fixation

Mode of action

Solanaceae

Poaceae

Poaceae

Poaceae

Family

Table 7.3  The study of algal strains in different formulations.

Enhance the growth and photosynthetic activity

Increased crop yield

Enhance floral production, plant growth

Enhanced sprouting

Enhance plant growth

Improve soil fertility

Result

[75]

[74]

[73]

[72]

[71]

[70]

References

Biofertilizer Formulations  223

Targeted crop

Soybean

Flowers, Ornamental plants

Above 80% of plants (mostly crops)

–

Fungal strain

Arbuscular mycorrhizal fungi

Trichoderma sp. Gliocladium sp.

Arbuscular mycorrhizal fungi

Aspergillus, Penicillium –

–

Asteraceae

Fabaceae

Family

Table 7.4  Effect of fungal strains on targeted crops.

Solubilize inorganic P with the help of organic acids, form phosphate enzymes

Form association with plants, enhance N availability

Plant pathogens

Control soil erosion, protect the plant from other microorganisms through the mycelial network

Mode of action

It helps in normal growth and maturity of plants

Improve plant growth

Plant protection

Assist plant protection and enhance food security

Result

[86]

[85]

[84]

[83]

References

224  Biofertilizers

Biofertilizer Formulations  225 improving crop yield and soil fertility, they are being used worldwide. The role of algal formulations as biofertilizers in agricultural systems is well known. However, algae have proved for reducing soil erosion by regulating the flow of water into the soil and then improving the soil fertility. Moreover, they have played a significant role in the wastelands and biocontrol of pests and pathogens [60]. The algal role in wastewater treatment and macrobiotic crusts is well studied [61]. The microalgae, especially cyanobacteria, are being used as a biofertilizer annually. Various scattered reports on using green algae are also available [62]. Microalgae are divided into Euglenophyta, Rhodophyta (red algae), Pyrrophyta, Chrysophyta, Phaeophyta (brown algae) and Chlorophyta (green algae). Both algae and cyanobacteria are photosynthetic organisms used as soil conditioners and hence biofertilizer strains. Only cyanobacteria can produce oxygen, hence photosynthetic prokaryotes. Since the 1950s, comprehensive research on algal industrial applications has been conducted, when the productivity of algal biomass and yields was studied [63]. The algae have an excellent ability to grow on those lands that are agriculturally non-productive and with minimum input of freshwater. Algae have main benefits as they can be cultivated in high inputs of agricultural runoff and wastewater, recovering the excess nutrients and regenerate water for further agricultural usage. Moreover, they can also be used for reducing greenhouse gas emissions when CO2 and NOx from industrial sources are sequestered [64]. Cyanobacteria are the simplest living autotrophic plants that are widely distributed in oceans and are capable of converting inorganic matter into food materials [65]. Cyanobacteria have unique features of water holding capacity, ability to fix atmospheric N2, adaptation to unfavorably extreme conditions and short generation time, collectively make them a good biofertilizer source for improving soil composition and physio-­ chemical properties [66]. Cyanobacteria secrete plant-growth-­promoting hormones in the form of secondary metabolites, regulate the nutrient transport from soil to the plant, and improve chemical properties and agglomeration of soil [67, 68]. The physiological properties and diverse morphology of cyanobacteria enable them as wide distributors in an ecosystem and tolerate the environmental stresses [69]. The Tables 7.3 and 7.4 cover some reports of the recent fungal strains used in the agricultural sector.

7.2.1.1.4 Fungal Strains

A fossil study record has indicated that mycorrhizae have been connected with plants since 400 million years ago or even more [76]. This has led to a theoretical assumption that these early symbiotic relationships were possibly the cause for the establishment of plants. Since the very first reported plant-fungal symbiotic relationship, all plants studied are found in a

226  Biofertilizers symbiotic relationship with fungi [77]. The fungi in a symbiotic relationship can express mutualism, commensalism, or parasitism, a relationship where it can cause a positive, neutral, or negative effect. Mycorrhizae are widespread non-pathogenic, a symbiotic association of fungi with plants can be natural or in cultivation environment where a bidirectional nutrient transfer occurred. Plants supply sugar to fungi which, as a result, helps in the acquisition of soil nutrients [78]. According to the functional and structural characteristics, mycorrhizae are distinguished into seven types [79]. Frank established mycorrhizae into endomycorrhizae and ectomycorrhizae, two large subdivisions. Ectomycorrhizal fungi (ECMF) in an association form a mantle network of hyphae in the plant roots. Endomycorrhizal fungi (ENMF) is classified into arbuscular mycorrhizae (AMF), monostrophic mycorrhizae, ericoid mycorrhizae, ectomycorrhizae, arbutin mycorrhizae, or orchid mycorrhizae. Each class differs in the intracellular hyphal establishment but featured by the occupation of root cells by fungal hyphae. AMF belong to phylum Glomeromycota that are obligate symbionts. AMF is especially significant for those plants that are deficient in phosphorus, as it supplies P through the rhizosphere. AMF form arbuscules, that are more variable than ECMF, as they mostly form a symbiosis with herbaceous plants [80]. ECMF are commonly Basidiomycetes and Ascomycetes. The mantle network is extended deep into the soil which is directly responsible for mobilization, absorption, and translocation of water and soil nutrients to the roots [81]. These two groups of mycorrhizae are reported for increasing drought, pathogenic tolerance, and heavy metal tolerance. So, they have a key role in sustainable agriculture and forestry [82].

7.2.1.1.5 Actinomycetes

The microbial population also contains actinomycetes other than algae, fungi, and PGPR. About 100 genera of actinomycetes exist in the soil. These are unicellular bacteria that make up a large part of the microbial population, especially under alkaline conditions. However, the studies related to actinomycetes as biofertilizers are limited [87]. As they have phosphate solubilizing and cellulolytic properties they can be used for improving soil quality. They are next to the bacteria in abundance but their activity is quite slow. They decompose various substances like fat, polysaccharides, cellulose, proteins, and organic acids slowly than bacteria and fungi. Dicko selected three strains of actinomycetes, and maize growth and its yield were checked to provide its formulations. The results were significant than the controls [88].

Biofertilizer Formulations  227

7.3 Types of Formulations The formulation of biofertilizer is a multistep followed process that consists upon one or more than one effective microorganisms in a specific carrier with additives such as sticking agents that help in storage and transport. Figure 7.4 illustrates the complete process of formulation preparation. As

Soil contains different microbial communities that make the survival of plants possible.

Strain of interest is selected from a range of microorganisms present in a culture.

Culture is purified and beneficial strain is obtained.

Scale-up production of beneficial strain

Carrier material is selected and sterilized for carrying microbial strain

Additives such as sticking materials as added

After selecting packing and storage materials, it is transported

Inoculation of biofertilizer to the field

Figure 7.4  Overview of complete formulation process.

228  Biofertilizers inoculants are prepared and stored below the optimal condition, they must have expanded shelf life. Moreover, the microorganisms should be either robust or shielded so they could survive in a significantly high number under harsh conditions. A good formulation enables the microbes with high persistence in soil, improving its activity to get maximum benefit by providing optimal conditions [89]. Table 7.5 summarizes features of a good bioformulation. There is no perfect formulation but an efficient formulation must consider the following characteristics.

Qualities of bioformulations

Table 7.5  Features of a good bioformulation. ◦◦ Carrier material must offer stability and protection to microbial cells during complete process [90] ◦◦ Formulation must enhance the activity of microbe in the field keeping in mind the preferences of use ◦◦ Should be able to shield the bacterial population from environmental unfriendly factors ◦◦ Easy to handle and apply while delivering to the field in an appropriate manner ◦◦ It should occupy a higher number of viable cells ◦◦ The inoculant should have sufficient mean life at given temperature [91] ◦◦ Same procedure must be applicable to the prepare inoculant and make the addition of nutrients possible [91] ◦◦ It should not be hazardous to the environment [91] ◦◦ Inoculum should be economical for commercially viable [92] ◦◦ It should improve soil fertility and resist soil pH change while in storage form [91] ◦◦ The inoculant must compete with soil organisms for nutrients as well as niches for survival particularly against grazing protozoa ◦◦ It should have the ability to protect from environmental extremes and should contain ecological competence when applied to soil [91] ◦◦ The release of the active ingredient in the entrapped formulation should be moderate ◦◦ It can be transported to fields using agrochemical machinery ◦◦ It should be suitable for a wide bacterial range [91] ◦◦ Polymer entrapped formulations may harm the microbes, so they should not contain any preservatives [93] ◦◦ In the absence of any protection, nutrient-poor environments often cause a high contamination level. The inoculant must not enhance this contamination level [91]

Biofertilizer Formulations  229 Depending upon the nature of the material used a formulation medium, four dispersal forms are generally used [91]: 1. 2. 3. 4.

Dry inoculants (mostly powders) Slurries (a suspension of powder in liquid substance) Granular Liquids

The success of the formulation depends on factors like target crop, environmental constraints, cost, ease of use, market availability, and growth yield. Bacterial inoculants mostly used peat as a carrier material. However, peat is not easily available in overall world, and use of peat rendered an adverse effect on atmosphere and ecosystem due to its process of extraction. This highlights an urgent need for developing new formulations using substitutes of peat so that effects could be minimized [94]. As shown in the Figure 7.5, many factors affect the effectiveness of inoculants such as growth phase of microbes, temperature, and dehydration rate. When bacterial culture mixes with a carrier like spores, active cells, flocculated cells, or cysts of different PGPR species, it is called the growth phase of microbes. This incorporation has affected the survival and efficacy of inoculants. Rhizobia in nonsterile peat have more injurious effects of high moisture than in sterile peat. Roughly, 40%–50% part is suitable for unsterilized peat while 60% for sterilized peat. Polyethylene of higher density is

Dehydration rate Storage temperature

Growth phase of microbes

Factors affecting the efficacy of inoculant formulation Sterilization

Moisture content

Packing material

Figure 7.5  Factors affecting the efficacy of inoculant formulation [23].

230  Biofertilizers mostly used as packing material as it allows high gas exchange, allows O intake and CO loss, and also has no effect of gamma radiations. The sterile carrier has more advantages over nonsterile carriers, but its production process has a high cost. To control field contaminants, it is necessary to sterilized it. The sterilization process includes two methods. First is gamma irradiation, and second is autoclaving.

7.3.1 Carrier-Based/Powder Formulations 7.3.1.1 Selection of Carrier Material Arid soils along semiarid ones are most prone to salinity, mild resistance for temperature variations, and low nutrient supply due to water deficiency, which poses an additional challenge for using inoculants [95]. The microbial inoculants critically suffer the problems for survival and colonization in the rhizosphere that ultimately affects the health of plant and growth yield [96]. Arid soils undergo poor microbial existence in the soil, inability of PGPM evolution, and disease mitigation [97]. However, improved formulation methods and use of carrier substances made the delivery of bio inoculants efficient in stressful conditions in agricultural systems [98]. The carrier material is like delivery vehicle that is employed to transfer living or latent microbes from an industrial production unit to the rhizosphere. For the sake of good quality inoculant, it is compulsory to be made by an efficient carrier. The characteristics of a good carrier material is having the capacity to provide the enough number of microbial cells in the particularly good condition of it at the right time [99]. A superior quality carrier must contain the following features: • High water retention, good capacity to hold water, and acceptable for maximum possible bacteria [100] • Absence of lump forming substances • Cost-effective • Adequate amounts of it must be available [101] • The carriers used for seed treatment must assure its survival as they are not instantly sown [102] • Seed coating carriers must exhibit a good adhesive ability to the seed [103] • Must be uniform both physically and chemically [91] • Nontoxic nature [101] • They are generally nonpolluting and biodegradable [104]

Biofertilizer Formulations  231 • They contain excellent pH buffering capacity and have neutral pH • Are supportive for growth promotion of bacteria and their survival [104] • Compliant to nutrients supplement [104] • Manageable to all operations like mixing and packaging [104] As the choice of a good carrier decides the physical form of inoculant, so there is no perfect and universal material suitable for all organisms. The carrier materials belong to various origins: organic, inorganic, or synthetic. Depending upon the origin, these are classified into further categories as illustrated in Figure 7.6: A mixture of carriers is a common practice where a combination of two carriers is used: a soil mixture, peat, compost, husk, and many others [105].

ic soil, and Soils (coal, inorgan clays) Chemical Composition based

Inorganic carriers (perlite, talc, vermiculite)

Plant waste materials (soybean meal, manure, cellulose, sawdusk)

Carriers Natural Sources based

Inert materials (ground rock phosphate, polyacrylamide gel)

Oil-dried bacteria Soils (coal, inorganic soi l, and clays)

Figure 7.6  Types of carrier formulation materials depending upon the composition and source [23].

232  Biofertilizers

7.3.1.1.1 Peat

Peat is the carrier material for microbial inoculants that are used most commonly all around the world. Due to its ability for bacterial protection, it is commonly used for manufacturing legume inoculants. The peat is preferred as a carrier for bacterial species in numerous studies where it was accepted that it remains unchallenged to be used as a carrier. After long time, peat is formed due to partial decaying of flora. It offers protective and nutritive conditions that are responsible for the evolution of microorganisms on the particle surface and in the crevices. As a suitable carrier material, peat must have high organic content, non-toxic, highly absorptive, good water holding capacity, easily sterilized, and economic to use [106]. It also exhibits high surface areas that abet the high growth of inoculants. Government agencies usually have the knowledge that how to control its quality. Moreover, the bacterial population is metabolically active, and in some formulations, it continues to grow in the storage period [20]. Peat is extensively used as carrier material due to high availability but its processing is much costly because several step process is required to make it useable [107]. • Inoculant Formulation The conventional ways to prepare inoculant involve inoculating neutral and nonsterile peat as a carrier, with a bacterial strain per gram of peat is 107 CFU, for high degree of strain in inoculant. An inoculant with a high number of viable cells can be produced by inoculation of 104 CFU/g of the carrier. In the inoculum, maximum bacterial growth occurs up to the density of 108 to 109 CFU/g in a serious competition with contamination [108]. The harvested crop is drained and sieved while inoculum preparation, to remove the rough and coarse material before slow drying to 5%. The drying stage is particularly significant since improper work can contribute the production of toxins. Air drying should prefer over oven drying as it avoids the production of contaminants. But in case of oven drying, temperature should never exceed 100°C. The extent of drying depends upon the type and particle size of peat but the content of moisture should not exceed sufficiently that the final product brings the desired moisture level. After drying, peat is grounded enough to be passed through 250-µm sieve. The pH level is adjusted between 6.5–7.0 by liming the carrier as it is usually acidic [109]. The carried is sterilized and liquid inoculum is added in sufficient quantity. For bacterial formulations, moisture in the range of 40%–55% is accepted. Finally, the inoculated carrier is incubated for bacterial multiplication for a particular period. Peat can be used as carrier material for ectomycorrhizal inoculants and AMF [23]. However, there are

Biofertilizer Formulations  233 some principle drawbacks to use peat as a carrier material. The quality of peat is source dependent and is highly variable that affects the inoculant effectiveness formulated by manufacturers and among different batches formulated by the same manufacturer [91, 110]. Peat inoculants are usually suffer severe contamination level that reduces the average shelf life of formulation [21]. Additionally, they contain a low tolerance level for temperature variations and release toxic components upon heat sterilization [111]. Sometimes, peat can reduce plant growth and create interfaces within the seed monitoring process [112]. Their availability has been restricted to a few countries. Seed air delivery of inoculant usually undergoes blowing off peat powder from the seed surface. All the downsides of peat had made researchers look for alternatives. But still, peat is in use for biofertilizer formulations. Several amendments have been made to increase the formulation effectiveness with many microorganisms. Peat found amendments by the use of chitin, pyrophyllite, a mycelium Aspergillus niger, and compost made from Agaricus bisporus. They resulted in improved strain growth and promoted germination of seeds when formulations were used for seed treatment increases plant growth and its yield [113].

7.3.1.1.2 Wheat Bran

Wheat bran has been used as a very strong carrier for mass growth of ectomycorhizae and phosphate-solubilizing fungi due to water preservation and high organic material [23]. While the Bacillus spp. genera and Pseudomonas spp. have been shown cellulose degradation, however, that certain phosphate solubilizing fungi are unable to replicate in the substrate due to the deficiency of cellulose enzymes required for degrading woody substances.

7.3.1.1.3 Talc

Talc is a silicate containing minerally hydrated magnesium. This, in the form of talcum powder, the softest material ever made, is used as a storage medium. Talc was commonly used as a carrier for Trichoderma viride, a biocontrol agent. It was noted that the formulation containing the PGPR mixture was used for the managing crop covering blight of crop (rice) and improved yield of grain. The PGPR was developed separately or in combination with formulations based on talc or wax [114]. Talc-dependent inoculants of PGPR Burkholderia cepacia and Bacillus atrophaeus inhibited the growth of Fusarium oxysporum gladioli, which developed in gladiolus for corm and dust applications. Corm yield rose to 150% by the absence of corm rot in the greenhouse and the least vascular wilt [115].

234  Biofertilizers

7.3.1.1.4 Biochar

Biochar belongs to the charcoal class that is generally produced by decomposition of organic contents under high temperature a limited supply of O2, a process called pyrolysis. It has been distinguished from charcoal due to its applications in the soil amendment [116]. Moreover, it also finds importance for improving the fertility of the soil and other ecosystem services such as carbon sequestration that mitigate climate changes [117]. However, it has also been reported that biochar is responsible for changing the biological community of soil [118]. If we have to prepare biochar at large scale using for carbon sequestration, it is a good opportunity to use biochar as a carrier material of PGPR to transfer them into agricultural lands [119]. Therefore, biochar is suitable as a carrier material substituting for rare, expensive, nonrenewable, and greenhouse causing carriers. Also, they contain a large internal surface area about 2–20 μM of pore space, providing a protected habitat for microbial growth within the internal spaces. The production process renders it a pre-sterilized medium that can absorb nutrients as well as growth factors [120]. • Biochar Formulation Increased plant production using biochar and bacterial inoculants has been documented by Glaser [121]. A recent study found carrier-based formulation of Azospirillum lipoferum as an inoculant with biochar from two separate sources (coconut shell and acacia wood) as carrier material, and a comparison to lignite was assessed. For both carriers, the average population value of the carrier from coconut source was 10.7 CFU g−1 within 180 days of inoculation. The sprout vigor index for green gram (CO3) was also strong for coconut-based biochar. The survival rate of Azospirillum lipoferum is also found to increase for 6 months of storage in the correct population relative to acacia wood-based biochar and lignite [122]. Another work reported the suitability of pinewood-derived biochar carriers pyrolyzed at 300°C and genetically engineered to regulate inoculum as green fluorescent protein markers were created. Selective plate count assay revealed that the addition of soil bacteria utilizing biochar as a carrier medium greatly improved the cell survival. However, the biochar carrier had not so strongly affected the overall bacterial population. Both of these therapies resulted in very similar colonization of bacteria on roots with root mass community density of 105 CFU g−1. Studies have shown that biochar has an extraordinary influence on the development of plant production, whereas inoculum has a marginal effect [119]. When living in the region, biochar does not decompose readily into the dirt, improves the survival of bacteria, and positively affects inoculated plants, relative to peat.

Biofertilizer Formulations  235

7.3.1.1.5 Vermiculite

Vermiculite is an effective inoculant and is used as an alternate to peat in the production of inoculants of bacteria. This is a hydrated aluminum silicate comprising magnesium alloy that exfoliates at elevated temperatures of 700–1,000°C. Without fermentation which is more costly and specialized incubation equipment, vermiculite can be generated for different bacterial inoculums, making it desirable for many formulations [108]. Vermiculite as a carrier finds many advantages: • The exfoliation process lowers the contamination level • It is inorganic: that sterilized by common sterilization processes, as sterilization at high-temperature yields toxic compounds or causes structural changes • It is highly economical and easily available [108] • It exhibits a multilamellar structure that provides proper aeration and site for microbial proliferation • Vermiculite is anti-crushing [123] • It functions as plant growth-promoting substance [124] • Its particle size of 45–80 mm provides the better capacity to hold water and permit the formulation to follow properly on the surface of seed. • At room temperature, the number of microbes does not change on seed surface for 1 day after inoculant preparation [108]

7.3.1.2 Sterilization of Carrier Both sterile and non-sterile carriers can generate inoculants by injecting targeted microbes. There is no question that a sterile container is more beneficial than a non-sterile form. However, the sterile product has other disadvantages, including expensive work, extra labor, and an aseptic process while packing. There are two forms of formulation used: first, for treatment of seeds, and, second, for direct soil use. The formulas can be powder treatment of seeds or granules for direct soil application on the basis of different delivery methods [125]. Strong formulations are commonly used, but have some disadvantages in terms of cost, labor and energy-intensive methods. They also suffer low shelf life, cluster forming through multiple phases, high rates of emissions, weak consistency and erratic field performance. Today, inoculant development involves creative strategies aimed at increasing inoculant performance, expanding shelf life, and developing a new generation of formulations. The best alternatives for powder

236  Biofertilizers formulations are the formulations that rely on liquids and alginates. Solid carriers can need to be modified, because customers are already involved in other formulations.

7.3.2 Granular Formulations To minimize the undesirable effects of powder-based inoculants, a new formulation is gaining much interest, particularly granular formulation. Peat prills, silica grains, tiny marble, or calcite material are used as starting materials to prepare granules. The granules are then loaded with the appropriate microorganism. Their granular size is varied, but there is a close correlation in mother culture between finished product quality and population density [103]. Granules are far more efficient than peat, because they are less fragile and simpler to use [89, 90]. The relationship between inoculants depending on peat and granular is rather complex. Some studies have shown that the granular formulation of rhizobia in N2 fixation or nodule formation is not equivalent to that of peat formulations [126, 127]. While several experiments have shown that granular formulations are superior in terms of N aggregation, biomass, nodule scale, nodule weight, and N2 binding to both peat and liquid formulations [105, 128]. In particular, granular formulations are used under soil stress conditions such as cold fields, wet fields, high acid stress, and moisture demand conditions. On the other side, there are also some drawbacks to this, as they are denser, so it is difficult to transport them to the fields. In contrast, the storage cost and the level of production are far higher.

7.3.3 Liquid Formulations Liquid inoculants are mainly established from aqueous (broth cultures), polymer-based suspensions, mineral oil, oil-in-water, or organic oils [129]. These are upgraded inoculants where the development of “no formulation” inoculants has occurred. These are microbial cultures amended with those substances that can improve stickiness, stability, and formulation’s dispersal abilities [130]. Liquid inoculants have been assisted because they are easier to handle, formulate, and apply both to the seeds and soil [90]. Peat has been the most popular carrier ever used; however, due to insufficient availability and growing depletion, scientists are searching for the best replacement for nearly all types of biofertilizers in the form of liquid inoculants. The new seeding machinery rapidly adopted liquid inoculants as they are conveniently sprayed onto the seeds and moved through the seed auger [131].

Biofertilizer Formulations  237 The liquid inoculants have been distinguished due to followings: 1. handling is quite an easy task; 2. they make possible the addition of sufficient nutrients in the inoculants like additives and cell protectants which stimulate cell, spore, or cyst formation that ultimately improves the performance; 3. exhibit greater shielding against environmental stress; 4. have increased effectiveness as compared to powder-based formulations. While at some level, they also behave as disadvantageous: 1. in some cases, liquid fertilizers exhibit limited shelf life; 2. low-temperature storage conditions; 3. a high production cost limits the use mostly within the developed countries while diminishes the use in other ones [22]; 4. those bacteria with poor survival such as Azospirillum sp. do not find any help for providing a protective environment: they are quite useless [132].

7.3.3.1 Inoculant Preparation Liquid inoculum is mostly produced through a simple fermentation process where fermentor is aseptically packed and stored without losing viability. It is highly economical as it does not require any processing and sterilization of carrier material that is often solid. Liquid formulation achieved no contamination as it is a complete sterilization process. It is often wrongly considered that liquid inoculum is a broth culture in a fermentor or a liquid suspension of some solid material inoculum. Instead, it is a medium containing nitrogen, carbon, and some vitamin sources that helps for microbial growth and various cell protectants. The additives exhibit such features that provide better adhesion, prevention from osmosis, stabilization of the product, increase rhizobial survival, and inactivation of seed coat toxins. Additionally, they also protect the inoculum when severe environmental conditions are present. When an inoculum has been prepared, it is dissolved in a liquid such as water, mineral, or any organic oil for inoculating the seeds by spraying the formulation on seeds before sowing or by dipping for some time. After

238  Biofertilizers drying, seeds are sown. This method avoids the loss of inoculum and ensures the coverage of seeds without any involvement of the seed inspection system [112]. Legumes are sown into the soil where the temperature is very close to 40°C. High temperature severely damages the rhizobial survival that affects its ability of nitrogen fixation, these additives protect these high temperature and desiccation conditions. Liquid cultures that contain cell protectants maintain high bacterial number and enhance the formation of cysts and spores that are resting cells, which provide resistance to unfavorable conditions thus increasing the survival of microorganisms.

7.3.3.2 Common Additives The choice of additives depends upon the ability for protection of microbial cells in storage as well as on the seed surface when extreme temperature conditions and desiccation are present. Polymers with high molecular weights having good water solubility, complex chemical, and nontoxic nature are generally good additives [93]. Commonly used additives are sucrose, glycerol, sodium alginate, carboxymethyl cellulose, polyvinyl alcohol, trehalose, polyethylene glycol, Arabic gum, polyvinylpyrrolidone, Fe-EDTA, and tapioca floor [133]. Sucrose when added to liquid inoculum, improves the survival of microbes, mostly in the phosphate solubilizing bacteria and rhizobia [134]. The glycerol contains a significant amount of liquid and slows down the desiccation rate to protect the cell from dehydration [135]. A study reported the addition of glycerol for the preparation of liquid inoculum where Pseudomonas fluorescens was used as a strain and maintains the viability for 6 months, during the storage time [134]. Polyvinylpyrrolidone is also used as a binding material in several formulations, especially in Bradyrhizobium japonicum, a protectant from dehydration, and inhibits seed exudates that are harmful to rhizobia [133]. Carboxymethyl cellulose is a readily available additive and is used commonly. Owing to its semisynthetic polymer nature, it exhibits steady batch quality as compared to other additives. Additionally, it is used in “1/5; w/v,” a very low concentration that makes it economical to use [136]. Another commonly used additive is Arabic gum: a complex mixture of polysaccharides and glycoproteins. Due to its extraction from acacia, it is also called acacia gum. Various studies reported its use in many biofertilizers formulations as an adhesive agent especially rhizobia [137]. It improves the survival of bacteria and protects them against dehydration when used within the liquid formulations [138]. The nature of additives and their concentrations are major factors that affect the inoculum. So, the amount of

Biofertilizer Formulations  239 additives and type of them is highly significant to consider while preparing the inoculum.

7.3.4 Cell Immobilization Scientists have been working on developing the novel formulations that undergo maximum fruitful effects and minimum deleterious effects, for decades. Recently progress has been made by developing a new formulation called cell immobilization. Immobilization encompasses various forms of entrapment or attachment of cells into the matrix. These may include adsorption on surfaces, cross-linking of the cells, flocculation, covalent bonding with the carrier material, and encapsulation of the cells into polymer gel [139]. Encapsulation is the most promising formulation technique that constructs useful carriers for microorganisms, with considerable advantages over other carrier materials. Encapsulation provides a protective nutritive environment to the living cells against environmental stresses such as organic solvents, poison, temperature, and various mechanical stresses along with predators. Such formulations when provided to the soil, microbes living in the soil degrade the capsule slowly and the targeted cells released into the soil in large quantities, usually at seedling emergence time or seed germination time [91]. The bacteria, fungi, and small hyphal segments can be encapsulated that represent the technology a very useful approach for preparing single or mixed strain formulations like rhizobia-­ AMF–based inoculums. The most common type of materials used for encapsulation is the polymers that may be from various sources.

7.3.4.1 Polymer Entrapped Formulations The advancement has been made within the inoculant technology that resulted in polymer entrapped formulations. The cells after the process of mass multiplication are mixed with a certain type of polymer and treated with chemical solidification. It resulted in uniform beads that contain living cells inside of it. The beads are fizzed in the polymer matrix that causes further growth and then dried. The beads are commonly degraded by microorganisms living in the soil when applied to the it. The polymer materials that are used commonly may be natural such as polysaccharides and protein material, or synthetic like polyacrylamide and polyurethane, or homopolymers, heteropolymers, or copolymers. A study has estimated that there are about 1,350 possible combinations of material which can be as an entrapment polymer material, selected depending upon the chemical composition, molecular weights, and their relative ability of interaction

240  Biofertilizers with other components used [140]. Among the most commonly used polymer materials are alginate beads and polyacrylamide, but due to more handling precautions of polyacrylamide, alginate is the preferred one [141].

7.3.4.1.1 Alginate

Alginate is a naturally available polymer, a material of choice for polymer entrapped formulations, and is currently under application. It is obtained in sustainable quantities from marine macroalgae and several bacteria [142, 143]. Alginate formulations enhance the survival of microbial inoculants and support a high population rate at elevated temperature conditions of 40°C [144]. Several alginate-based inoculants are used for various organisms and their study has been conducted for agricultural purposes, results proved the superiority of polymer entrapped formulations over all other formulation such as polymeric formulations of AMF, ectomycorrhizae, and many P solubilizing bacteria [91, 99, 140]. PGPMs can survive within alginate polymeric material for a very long time. The beads are superior to other polymer materials due to less activity of water as targeted microbes will remain on low metabolic activity and then released into the soil when there is the availability of moisture, which commonly corresponds to the seed germination. The field inoculation of alginate-based formulations shows the high survival rate of microorganisms and a comparable population with other carrier-based formulations. A study reported highly efficient results for root colonization through targeted beneficial cells that were released through polymeric material as compared to direct inoculation for the wheat crop. Alginate-based formulations undergo various advantages as well as major constraints some of whom are covered in Table 7.6. • • • • • • •

simpler to be used; uniform and also biodegradable; non-toxic in nature; can hold a large bacterial population; slow release of a large number of bacteria; do not cause any ecological pollution; biological characteristics are dependent on chemical features. So, it is easily controlled; • the beads in a small volume can be stored without any effect on bacterial population; • the targeted microbes are released through the beads that inoculate plants efficiently.

Biofertilizer Formulations  241 Table 7.6  Advantages and constraints of alginate-based materials. Advantages

Constrains

◦◦ usually stored at climatic conditions for a long time [91]

◦◦ polymeric materials are expensive than other solid carrier materials like peat [91]

◦◦ its production and handling is quite easy [91]

◦◦ the industrial process demands more handling [21]

◦◦ nontoxic nature [21]

◦◦ more labor-intensive [146]

◦◦ a consistently good quality batch is produced [146]

◦◦ sometimes, due to low oxygen transfer survival of inoculum becomes limited

◦◦ provides a niche that is most suitable for bacteria [91]

◦◦ strain survival is effective in low water activity. When an activity is increased, strain survival decreases

◦◦ according to different bacterial needs, its manipulation is an easy task [146]

◦◦ the survival rate fluctuates with the type of solute in culturing of strain

◦◦ if inoculant have short survival rate then it can be amended easily by proving different nutrients [91]

◦◦ compounds with low molecular weights have a negative on strain survival

◦◦ it provides a temporary shield against unfavorable conditions [146] ◦◦ microbes are released continuously to colonize the plant roots

• Inoculant Preparation Before the preparation of inoculum, microorganisms are cultured within the nutrient broths placed in the rotatory shaker where they attain a significant growth phase. Bacteria are entrapped within the beads in laminar flow hood when mixing to the sterile alginate. The culture along with 2% powder of sodium alginate is aseptically mixed as well as stirred for about 1 h that results in complete mixing of all the added ingredients. The mixture is filled into a sterile syringe and added dropwise into 0.1 M CaCl2 solution at room temperature that is also sterilized. The beads

242  Biofertilizers entrapment depends upon the syringe used for experiment. The beads are kept within a solution for 1 to 3 h at room temperature that ultimately forms solid beads. After the formation of the beads, the CaCl2 solution is dropped off and beads are washed. Then, they are incubated within freshly prepared nutrient broth for an expended time of almost 24–48 h within rotatory shaker which multiply bacteria in the beads. Then, beads formed are finally washed, collected and then dried. The formed beads are in two sizes: microbeads (20–200 µm) and macro-beads (1–4 mm) [94]. • Macro-Alginate Beads The macro-beads are most acceptable to encapsulate several microbes such as PGPR and mycorrhizae. However, it exhibits some challenges: firstly, during sowing, additional treatment is required even if it is planted by using a seeding machine. Secondly, those targeted bacteria that are released through the capsule needs to move toward the plant through the soil. Agricultural practices showed that when seeds are mixed up with alginate beads and sown together, they may fall apart from the seeds at a distance of few centimeters. After dispersal, the bacteria started to release and migrate from beads to the soil, where a tough competition may occur between inoculated strain and native microflora. Sometimes, there is a need for continuous water film that mobilizes the inoculant strain. However, in the absence of that film, it is considered a limiting factor [94]. • Micro-Alginate Beads Although macro-alginate bveads are more efficient to use as a carrier material for microbial strains, where micro-alginate beads can also be used for this purpose. However, they form a “bead powder-type” formulation. Seed handling facilities make the seeds coated with this bead powder formulation. Subsequently, they result in constantly homogenous spreading of the strain cells near the target place. Small-sized seed could enhance the efficacy of the corresponding application [94]. Moreover, they magnify the strain’s movement through the soil and off-site drift is ultimately reduced [139]. The micro-alginate beads are produced in a quite simple way: a bacterial culture suspended within a nutrient-rich medium is gently mixed up with an alginate solution. The prepared mixture is sprayed into CaCl2 stirred solution with the help of thin nozzle with very low pressure producing mist like extrudes and finally form alginate beads of very small diameter that immediately solidified into 50- to 200-µm diameter range beads. The microbeads entrap nearly 108–1010 CFU g−1 bacterial population that is comparable to macro-alginate beads [145].

Biofertilizer Formulations  243

7.3.4.2 Advantages and Constrains Relative to the conventional formulations, they are highly significant in several prospects like an effective carriage and good resistance against environmental stress and handling. Though it is a relative success, shown by many investigations on a laboratory level, still no commercial product is available due to technical handling and very high production cost. There is still a need to minimize the cost of labor that can be adopted by planters and manufacturers. The economic viability of the chemical industry has been shown that alginate is a costly material. But the massive production of alginate-based formulations in East countries has provided a good opportunity to be used as an inoculant in agricultural productions. However, many attempts have been made that amend alginate with some other cheap material that can act as carrier material. Some materials like bentonite clays, rock phosphate, gypsum, talc, cement, granite powder, rock phosphate, and lignite have been tried in a combination with alginate which could result in comparatively low cost [147]. A combination of skim milk and clay resulted in significantly increased survival of bacteria than single alginate material. Alginate and perlite have been reported to entrap Rhizobium [103]. Furthermore, a byproduct of the starch industry called pero-dextrin was also used. It was a combination of pero-dextrin, Arabic gum, gelatin, and starch granules in 20%, 5%, 20%, and 10%, respectively, impregnated with diazotroph cells. It improves the survival rate and nitrogenous activity [148].

7.3.5 Fluid Bed-Dried Formulation Fluid bed dryer formulation is a novel approach toward the productions of such formulations that helps in sustainable agriculture. Here, a fluid bed drier provides a fluidized condition such that material is suspended against the gravity where an upward air stream flows. Electrical heaters provide heat for drying purposes. This hot air is responsible for the expansion of a bed of material that occurs at terminal velocity causing some sort of turbulence within a product, the process is called fluidization. The phenomenon produces a group of solid particles which results in uniform drying and continuous heat transfer. The quest for appropriate drying technologies for bio inoculants has been led to the idea of using a fluid bed dryer which mainly finds its applications in those industries where processed food is prepared. This dryer has been used for making coffee powders instantly and for several drying operations [131]. In this method,

244  Biofertilizers granulation is commonly done in wet conditions as a closed operation. It is highly acceptable to use because many constituents can be granulated than mixing and drying is done within the same vessel. Moreover, reduced material handling and less process time have made it a better candidate to be used in inoculant formulations. Also, the formulation undergoes the following features: 1. 2. 3. 4. 5.

the cell decline is very limited; no contamination occurs during the process; many ingredients are mixed and dried; drying process demands ambient temperature conditions; while the drying temperature can be set according to the manufacturer’s requirement.

Carrier-based inoculants often have a short shelf life and high contamination which are potential drawbacks in biofertilizer formulations. Also, the moisture content is a significant problem that reduces the efficacy of the product. Improved methods of biofertilizer production introduced the fluid bed dryer, which is highly efficient for drying at 37°C–38°C [149]. Even temperature can be adjusted for sensitive microorganisms. The drying process kept a specific level of water content that minimizes the growth of contamination. Although fluid bed drying is much capable of producing efficient formulation still it is not in working for producing bio inoculants. Several studies have been reported their ability for containing nitrogen fixers, plant growth-promoting microbes, phosphate solubilizers, etc., but due to its applications in food survival, it is not being used for inoculant production. There is a need for further research to fulfill its protocols to be used in the biofertilizer industry.

7.3.6 Mycorrhizal Formulations The AMF are a proven biofertilizer that mobilizes phosphorus present in the soil toward plant roots providing a nutritive environment to the targeted plant. Due to a wide range of hosts, it finds significant utility in the biofertilizer industry. Large-scale production of the mycorrhizal formulation is difficult due to biotrophic nature. This formulation is produced using a pot culture where an association with a host plant occurred. Different type of mycorrhizae-based formulations is presently used for various purposes. Production of mycorrhizal spores is used on a laboratory

Biofertilizer Formulations  245

Infected root inoculum

Mixed bacterial inoculants

Inoculant techniques

Soil based inoculum

Genetically transformed roots of Agrobacterium rhizogenes

Soil based inoculum

Figure 7.7  Mycorrhizae-based formulations can be prepared by the above-mentioned techniques.

scale for in vitro purposes but it is not applicable at large scale [150]. The arbuscular mycorrhizal fungi are potentially applicable on a range of plants and produced on a large scale due to some unique features: it is produced under axenic culture, formulation consists of high shelf life, easy handling, strain is developed that is superior to indigenous fungi, and its economic production with high-quality inoculant formulations [151]. Figure 7.7 shows several techniques used for inoculant production such as infected roots that contain spores and mycelium which results in a good inoculant technique. Methods like hydroponics and aeroponics make it possible. However, it is not feasible economically. While genetically transformed roots of Agrobacterium rhizogenes provide a site for culturing mycorrhiza. The technique is being used for mass multiplication. Soil-based inoculum is the simplest technique where inoculant is produced by pot culture technique, and AMF is multiplied in soil. On-farm inoculant production can be done by enrichment of soil through growing of mycorrhizae with the host. Peat-based inoculants are also called nutrient film technique in which a pre-infected host plant with fungi is placing in a try where nutrients flow in an inclined way, pH is also adjusted. Peat is incorporated in blocks. Also, mixed bacterial inoculants with AMF are very beneficial to use in inoculant technology where co-inoculation results in beneficial effects.

246  Biofertilizers

7.4 Stickers Sticking agents are commonly assimilated with that of peat-based materials which enhance the ability of the formulation to get maximum coverage over the seed [22]. The adhesive materials by plants are particularly polysaccharides like carboxymethylcellulose or gum, caseinate salts, and polyalcohol derivatives. They must be nontoxic, easily dispersible, exhibit better adhesion, and survival of microbes on seeds. In the case of rhizobia, sticking agents have been selected to maintain the viability of bacteria but the mechanism by which viability is increased is not clear yet. The only drawback of their use is the long-term interaction of bacteria with seeds due to improved adhesion [93].

7.5 Additives Inoculant formulation also contains some additive material other than stickers, microbial strain, and carrier material. They include micronutrients, macronutrients, hormones, carbon or some other mineral sources, and sometimes fungicides. The main objective of additives is to provide a nutritive and protective environment. Also, they improve the quality of inoculant as seed adhesion is improved, the product is stabilized, inactivation of toxins, an enhanced strain survival under extreme environmental conditions, and even storage. The additives and strains have a strong connection in terms of cell survival. Glycerol like additives improves cell survival by holding a significant concentration of moisture that shields the cells from dehydration. To improve the performance of strain, there is a need for some amendments. The chemical nature of additive must prevent rapid degradation. The materials like xanthan, clay, sodium alginate, and skim milk have been studied and reported with variable results.

7.6 Packaging The nature of packaging material is highly important in inoculant quality. It must inhibit the moisture exchange but allow some oxygen passage from inside to outside. A sterilized product must be chosen while packing the material. Few materials are more suitable for autoclave but they are radiation sensitive and may damage by radiation.

Biofertilizer Formulations  247 However, the complete process of biofertilizer formulation can be summarized in a flowchart which describes different steps involved in the process. Mainly, it exhibits the choice of bacteria for inoculant formulation, the scale-up production of beneficial strain, choice and sterilization of carrier material, and the use of various materials like stickers and other additives. The packaging material selection is also a crucial step involved in the process. After inoculant formulation, it is transported to the market and finally it is used by farmers for better crop yield.

7.7 Conclusion Biofertilizers are an essential part of the sustainable cultivation that has been adopted globally to comply with the growing population so that it can cope with the climate changes, food shortage, and degradation of the land available for food production. In this chapter, discussion was underlined about the complete formulation strategy especially the type of biofertilizer formulations. It discussed reported microbes used in formulation depending upon the targeted soil and mechanism of action. Several formulations such as powder formulations, granular formulations, liquid formulations, and cell encapsulation were discussed along with their benefits and limitations. Also, some additives and sticking materials were added to improve the efficacy of biofertilizer. Despite brilliant efforts made to improve the efficiency in inoculant production, the variation is still lagging behind the real potential. Efforts have been made to get maximum possible microbial count, low contamination level, and the longer shelf life. In addition, more formulation strategies to get better biofertilizers with higher yields are being investigated. The future challenge for biofertilizers is to produce efficient microbial inoculants, with higher microbial count, better shelf life, and resistance toward biotic and abiotic stress, economic, easy-to-handle, and more effective toward sustainable agriculture.

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Biofertilizer Formulations  253 91. Bashan, Y.J.B.a., Inoculants of plant growth-promoting bacteria for use in agriculture. 16(4): p. 729–770, 1998. 92. Sivasakthivelan, P. and P.J.I.J.o.M.R. Saranraj, Azospirillum and its formulations: A Review. 4(3): p. 275–287, 2013. 93. Deaker, R., et al., Legume seed inoculation technology—a review. 36(8): p. 1275–1288, 2004. 94. John, R.P., et al., Bio-encapsulation of microbial cells for targeted agricultural delivery. 31(3): p. 211–226, 2011. 95. Egamberdiyeva, D.J.A.S.E., The growth and nutrient uptake of maize inoculated with plant growth promoting bacteria affected by different soil types. 36: p. 184–189, 2007. 96. Rekha, P., et al., Effect of free and encapsulated Pseudomonas putida CC-FR2-4 and Bacillus subtilis CC-pg104 on plant growth under gnotobiotic conditions. 98(2): p. 447–451, 2007. 97. Van Dyke, M., J.J.S.B. Prosser, and Biochemistry, Enhanced survival of Pseudomonas fluorescens in soil following establishment of inoculum in a sterile soil carrier. 32(10): p. 1377–1382, 2000. 98. Egamberdieva, D., et al., Bacteria able to control foot and root rot and to promote growth of cucumber in salinated soils. 47(2): p. 197–205, 2011. 99. Fages, J.J.A.m. and biotechnology, An optimized process for manufacturing anAzospirillum inoculant for crops. 32(4): p. 473–478, 1990. 100. Mishra, B. and S.J.J.A.D.R. Dadhich, Methodology of nitrogen biofertilizer production. 1(1): p. 3–6, 2010. 101. Bazilah, A., et al., Influence of carrier materials and storage temperature on survivability of Rhizobial inoculants. 10: p. 331–337, 2011. 102. Muresu, R., L. Sulas, and S.J.R.d.A. Caredda, Legume-Rhizobium symbiosis. Characteristics and prospects of inoculation. 2003. 103. Hegde, S., G.J.P. Brahmaprakash, and Soil, A dry granular inoculant of Rhizobium for soil application. 144(2): p. 309–311, 1992. 104. Smith, R.J.C.j.o.m., Legume inoculant formulation and application. 38(6): p. 485–492, 1992. 105. Herridge, D., Inoculation technology for legumes, in Nitrogen-fixing leguminous symbioses. Springer. p. 77–115, 2008. 106. Okon, Y. and R.J.C.r.i.b. Baker, Microbial inoculants as crop-yield enhancers. 6(1): p. 61–85, 1987. 107. Hungria, M., et al., Inoculant preparation, production and application, in Nitrogen fixation in agriculture, forestry, ecology, and the environment. Springer. p. 223–253, 2005. 108. Graham-Weiss, L., M.L. Bennett, and A.S.J.A.E.M. Paau, Production of bacterial inoculants by direct fermentation on nutrient-supplemented vermiculite. 53(9): p. 2138–2141, 1987. 109. Tittabutr, P., et al., Growth, survival and field performance of bradyrhizobial liquid inoculant formulations with polymeric additives. 33(1): p. 69–77, 2007.

254  Biofertilizers 110. Van Elsas, J.D., C.J.B. Heijnen, and F.o. Soils, Methods for the introduction of bacteria into soil: a review. 10(2): p. 127–133, 1990. 111. Chao, W.-L. and M.J.A.E.M. Alexander, Mineral soils as carriers for Rhizobium inoculants. 47(1): p. 94–97, 1984. 112. Smith, R., Inoculant formulations and applications to meet changing needs, in Nitrogen fixation: fundamentals and applications. Springer. p. 653–657, 1995. 113. Meyer, S.L., et al., Application of Burkholderia cepacia and Trichoderma virens, alone and in combinations, against Meloidogyne incognita on bell pepper. 2001. 114. Nandakumar, R., et al., A new bio-formulation containing plant growth promoting rhizobacterial mixture for the management of sheath blight and enhanced grain yield in rice. 46(4): p. 493–510, 2001. 115. Shanmugam, V., et al., Biocontrol of vascular wilt and corm rot of gladiolus caused by Fusarium oxysporum f. sp. gladioli using plant growth promoting rhizobacterial mixture. 30(7): p. 807–813, 2011. 116. Lehmann, J. and S. Joseph, Biochar for environmental management. 2009: Earthscan London. 117. Lehmann, J.J.F.i.E. and t. Environment, Bio‐energy in the black. 5(7): p. 381–387, 2007. 118. Grossman, J.M., et al., Amazonian anthrosols support similar microbial communities that differ distinctly from those extant in adjacent, unmodified soils of the same mineralogy. 60(1): p. 192–205, 2010. 119. Hale, L., et al., Evaluation of pinewood biochar as a carrier of bacterial strain Enterobacter cloacae UW5 for soil inoculation. 84: p. 192–199, 2014. 120. Lehmann, J., et al., Biochar effects on soil biota–a review. 43(9): p. 1812–1836, 2011. 121. Glaser, B.J.P.T.o.t.R.S.B.B.S., Prehistorically modified soils of central Amazonia: a model for sustainable agriculture in the twenty-first century. 362(1478): p. 187–196, 2007. 122. Saranya, K., et al., Potential for biochar as an alternate carrier to lignite for the preparation of biofertilizers in India. 4(2): p. 167–172, 2011. 123. Hemphill, D. and H. DD JR, Anticrustant effects on soil mechanical resistance and seedling emergence. 1982. 124. Lima, J.d.A., et al., Effects of organic matter and vermiculite on garlic yields [Allium sativum]. 1984. 125. Walter, J.F. and A.S.J.S.M.E. Paau, Microbial inoculant production and formulation. p. 579–594, 1993. 126. Atieno, M., et al., Efficiency of different formulations of Bradyrhizobium japonicum and effect of co-inoculation of Bacillus subtilis with two different strains of Bradyrhizobium japonicum. 28(7): p. 2541–2550, 2012. 127. Diane Knight, J.J.C.j.o.p.s., Evaluation of Rhizobium inoculant formulations for alfalfa yield and N fixation. 87(2): p. 267–272, 2007. 128. Clayton, G., et al., Inoculant formulation and fertilizer nitrogen effects on field pea: Nodulation, N2 fixation and nitrogen partitioning. 84(1): p. 79–88, 2004.

Biofertilizer Formulations  255 129. Malusá, E., L. Sas-Paszt, and J.J.T.s.w.j. Ciesielska, Technologies for beneficial microorganisms inocula used as biofertilizers. 2012. 2012. 130. Bashan, Y., et al., Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013). 378(1–2): p. 1–33, 2014. 131. Brahmaprakash, G. and P.K.J.J.o.t.I.I.o.S. Sahu, Biofertilizers for sustainability. 92(1): p. 37–62, 2012. 132. Bashan, Y., et al., Survival of Azospirillum brasilense in the bulk soil and rhizosphere of 23 soil types. 61(5): p. 1938–1945, 1995. 133. Singleton, P., et al., Development and evaluation of liquid inoculants. p. 52–66, 2002. 134. Taurian, T., et al., Phosphate-solubilizing peanut associated bacteria: screening for plant growth-promoting activities. 329(1–2): p. 421–431, 2010. 135. Manikandan, R., et al., Standardization of liquid formulation of Pseudomonas fluorescens Pf1 for its efficacy against Fusarium wilt of tomato. 54(2): p. 83–89, 2010. 136. Jha, C.K. and M.J.J.o.p.g.r. Saraf, Evaluation of Multispecies Plant-GrowthPromoting Consortia for the Growth Promotion of Jatropha curcas L. 31(4): p. 588–598, 2012. 137. Rose, M.T., et al., The survival of plant growth promoting microorganisms in peat inoculant as measured by selective plate counting and enzyme-linked immunoassay. 27(7): p. 1649–1659, 2011. 138. Wani, P.A., M.S. Khan, and A.J.C. Zaidi, Effect of metal tolerant plant growth promoting Bradyrhizobium sp.(vigna) on growth, symbiosis, seed yield and metal uptake by greengram plants. 70(1): p. 36–45, 2007. 139. Cassidy, M., H. Lee, and J.J.J.o.I.M. Trevors, Environmental applications of immobilized microbial cells: a review. 16(2): p. 79–101, 1996. 140. Vassilev, N., et al., Polymer-based preparation of soil inoculants: applications to arbuscular mycorrhizal fungi. 4(4): p. 235–243, 2005. 141. Date, R.A.J.A.J.o.E.A., Advances in inoculant technology: a brief review. 2001. 41(3): p. 321–142. Yabur, R., Y. Bashan, and G.J.J.o.A.P. HernándezCarmona, Alginate from the macroalgae Sargassum sinicola as a novel source for microbial immobilization material in wastewater treatment and plant growth promotion. 19(1): p. 43–53, 2007. 143. Smidsrød, O. and G.J.T.i.b. Skja, Alginate as immobilization matrix for cells. 8: p. 71–78, 1990. 144. Viveganandan, G. and K.J.M.r. Jauhri, Growth and survival of phosphatesolubilizing bacteria in calcium alginate. 155(3): p. 205–207, 2000. 145. Bashan, Y., et al., Alginate microbeads as inoculant carriers for plant growth-promoting bacteria. 35(5): p. 359–368, 2002. 146. Bashan, Y., L.E.J.A.M. González, and Biotechnology, Long-term survival of the plant-growth-promoting bacteria Azospirillum brasilense and Pseudomonas fluorescens in dry alginate inoculant. 51(2): p. 262–266, 1999.

256  Biofertilizers 147. Lewis, J. and G.J.P.P. Papavizas, Characteristics of alginate pellets formulated with Trichoderma and Gliocladium and their effect on the proliferation of the fungi in soil. 34(4): p. 571–577, 1985. 148. Ali, S.M., et al., Production of biofertilizers using baker’s yeast effluent and their application to wheat and barley grown in north Sinai deserts: (Produktion von Biodüngern unter Verwendung von Backhefeabwasser und ihre Anwendung zu Weizen-und Gerstenanbau im Norden der Sinai-Wüste). 51(6): p. 589–604, 2005. 149. Sahu, P., G. Lavanya, and G.J.J.S.B.E. Brahmaprakash, Fluid bed dried microbial inoculants formulation with improved survival and reduced contamination level. 33(1and2): p. 81–94, 2013. 150. Bagyaraj, D., V. Mehrotra, and C.J.B.o.B. Suresh, 23. Vesicular Arbuscular Mycorrhizal Biofertilizer for Tropical Forest Plants. p. 299, 2002. 151. Hua, S. Prospects for axenic growth and feasibility of genetic modification of vesicular-arbuscular mycorrhizal (VAM) fungi. in Innovation and Hierarchical integration. Proceedings of the Eighth North American conference on Mycorrhiza. 1990.

8 Scoping the Use of Transgenic Microorganisms as Potential Biofertilizers for Sustainable Agriculture and Environmental Safety Vasavi Rama Karri* and Nirmala Nalluri Department of Biotechnology, GITAM Institute of Technology, G.I.T, GITAM (Deemed to be University), Visakhapatnam, India

Abstract

Biofertilizers are the substances prepared with different environmental friendly microorganisms to improve the nutrient uptake of plants and fertility of soil. Presently, inorganic-based chemical fertilizers are being used as one of the strategy to improve soil fertility. But, continuous practice of this type of soil fertility management through chemical fertilizers creates a major threat to human health and environment. In this scenario, desirable microorganisms can be exploited as biofertilizers to enhance crop yield for sustainable farming. Biofertilizers application is one of the essential constituents of integrated nutrient management, as they are cost-effective and renewable plant nutrients source to substitute the chemical fertilizers. Nowadays, various microbes are being used as biofertilizers depending upon their competence to access different minerals and nutrients from soil and atmosphere. Different kinds of beneficial microorganisms capable of fixing atmospheric nitrogen and solubilizing potassium and phosphorus are included as major constituents in organic biofertilizers. Modern biotechnology promotes the development of novel strains of genetically modified or transgenic microorganisms by exploiting the recent techniques of genetic engineering to produce biofertilizers. Employing the utilization of genetically altered microbes as biofertilizers would be a scope for versatile prospects to develop novel eco-friendly biofertilizers for sustainable agriculture. Keywords:  Microorganisms, nutrients, biofertilizers, inorganic fertilizers, genetically modified organisms, PGPR, sustainable agriculture *Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (257–292) © 2021 Scrivener Publishing LLC

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258  Biofertilizers

8.1 Introduction Feeding the increasing population of world which is expected to reach 9 billion by 2050 requires adoption of powerful and sustainable farming practices to conserve the natural resources along with simultaneous hike in growth and yield are the few important challenges during agricultural practices [1]. In order to combat the needs of continuously growing population, agriculture practices must be comprehensive and inhabitable in the future. But, it is well clear that crop yield cannot be enhanced unless the nutrients exhausted from the soil during crop production are replenished. Majority of the agricultural soils due to scarcity of adequate quantity of necessary nutrients cannot support optimal level of plant growth. To counteract this issue and to achieve more production, farmers are frequently relying upon chemical based fertilizers [2]. Utilization of these inorganic-based fertilizers during the last six decades increased the yield of grains in majority of countries, but their long-term continuous application results in toxicity problems in plants due to environmental pollution which deteriorates the human health [3, 4]. Although, these fertilizers encouraged the growth of plants, they are not appropriate to amend the soil properties. It is perspicuous that extensive application of inorganic chemical–based nitrogenous, potassic, and phosphorous fertilizers has adverse side effects on climatic conditions of an environment [5] and led to a decline in crop yield and soil fertility in the intensive cropping system [6]. Furthermore, lowincome–gaining farmers of developing countries cannot invest more money to acquire costly chemical fertilizers for agricultural practice. So, one of the tough challenges for the future coming humanity is to generate sufficient food material in a sustainable way, which, in turn, paved the approach for consolidative management of plant nutrients. Therefore, in this scenario, to boost the crop yield with low environmental risk, novel cultivation systems that include biological fertilizer [7] as a source of nutrients are needed. Microorganisms living in soil play a crucial role in cycling of elements in biogeochemical cycles on our planet [8]. Plants as producers generate biomass through photosynthesis and the products of photosynthesis will be liberated partly into the soil through exudates of roots or by degradation of plant debris. Due to this continuous process, organic matter accumulates in the soil, which can be utilized as a substrate by various heterotrophs. Since the period of evolution, several microbes were recognized to promote growth of plants and these plant/microbe interrelations were identified to accomplish mutual gain for both the collaborating organisms (Figure 8.1). These microorganisms which are involved in symbiosis mostly inhabit in

Transgenic Microorganisms as Biofertilizers  259

Nitrogen Fixation

Antibiotics

Siderophores

Systemic resistance

Indirect Mechanisms

Direct Mechanisms

Increased uptake of minerals

Secretion of Plant growth hormones

Figure 8.1  Plant growth stimulating mechanisms regulated by microorganisms.

the rhizosphere, i.e., the region of soil which is under the direct clout of root secretions [9]. So, practically this group of organisms might play a leading role in plant growth improvement and ensure ample food production in sustainable process to abate the necessity of chemical fertilizers, whose manufacture relies on non-renewable sources of energy. Considering all of these advantages into account, in an attempt to protect the environment substitution of inorganic chemical fertilizers with biofertilizers is the best strategy to enhance the sustainable yield of crops without any ecological problems. A biofertilizer is a preparation consisting of live or latent cells of competent microbial strains obtained from plants rhizosphere capable of fixing atmospheric nitrogen, solubilizing phosphates and hydrolyzing cellulose applied to the soil in the areas of composting with the aim of boosting the number of microbes to enhance the nutrients availability to plants for easy assimilation [10]. It was demonstrated that microbes living in the biofertilizers accelerate proper supply of required nutrients by eliciting various mechanisms which promote plant growth and assure sustainable yield [11]. Sometimes, application of biofertilizers along with natural manure endeavors a small capital investment in environmentally beneficial way to enhance farm productivity [12]. In this way, globally organic-based farming has commenced as main priority area in the aspect of the expanding demand for safe and healthy food in the view of abiding sustainability with a concern toward increasing environmental pollution due to more use of agrochemicals.

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8.2 Role of Nitrogen in Plant Growth and Development Widely, plants constitute carbon, hydrogen, and oxygen as major constituents in their body. Apart from these, nitrogen (N) is one of the important elements needed by plants in considerable quantity [13] which regulates the accumulation of organic matter [14]. Nitrogen (N) is ample in earth’s atmosphere and highly inert [15] in nature. It occurs in proteins, amino acids, and several other organic compounds formed during the process of nitrogen fixation [16]. Although it is plenty in nature, not all organisms can access this except few organisms and plants which are involved in either symbiotic or non-symbiotic relationships. Due to this condition, the productivity of plants will be declined. It was studied that free living active microorganisms involved in nitrogen fixation are important to promote growth and development of plants [17]. Biological nitrogen fixation is an indispensable function of the microbes [18] and about 70% or 175 million metric tons of atmospheric nitrogen is fixed by means of biological nitrogen fixation process, and rest of the portion is completed by either auto or heterotrophs [19]. It was predicted that nodulated legumes play a major role by reverting 65% (139 × 109 kg out of 386 × 1,016 kg) of the total nitrogen by microbial process [20]. The conversion of nitrogen in gaseous state (N) to the form (NO or NH) utilized by other organisms and plants is interceded by various plant-microbial interactions like (i) bacteria involved in symbiotic relation with plants, (ii) symbiotic interaction of blue-green algae or cyanobacteria and plants or fungi (lichens), and (iii) free living auto or heterotrophic bacteria which commonly associate with detritus or soil.

8.2.1 Microorganisms Involved in Nitrogen Fixation Azospirillum is a familiar gram negative nitrogen-fixing bacteria [21] that extensively grows in the subtropical, tropical, and temperate regions of soil and establish close association with different agricultural and of wild plants roots [22, 23]. It benefits plants directly by increasing the development of root and shoot with simultaneous raise in the rate of mineral and water uptake by the roots [24]. Next set of organisms important in nitrogen cycle globally is cyanobacteria [25] and is potent fixers of nitrogen in marine and fresh water ecosystems [19, 26]. Nowadays, cyanobacteria are preferred to use as biofertilizer in modern agricultural practices [25] because they survive in severe environmental conditions as a result of their peculiar modifications like capacity of nitrogen fixation and tolerance to dehydration.

Transgenic Microorganisms as Biofertilizers  261 Furthermore, atmospheric nitrogen is also fixed by another family of a bacterium called Gluconacetobacter diazotrophicus which is an acetic acid bacterium initially identified from the plants of sugar cane crop. This family has three genera of nitrogen fixers constituting seven species, namely, Acetobacter nitrogenifigens, Acetobacter peroxydans, Gluconacetobacter johannae, Gluconacetobacter kombuchae, Gluconacetobacter diazotrophicus, Gluconacetobacter azotocaptans, and Swaminathania salitolerans [27]. A specific kind of bacteria referred as “rhizobia” [28] inhabits more in the rhizosphere where they make nitrogen available to plants by establishing symbiotic relation with leguminous plants [24].

8.3 Importance of Phosphorus Next to nitrogen, phosphorus (P) is one among the primary growth limiting macronutrients needed by the plants for proper growth, especially in the areas of tropics, where the phosphorus availability is poor in the soils [29]. Out of different forms of phosphate available, plant cells mostly absorb H2PO4− or HPO24− forms based on pH of the soil [30–34]. It constitutes around 0.2% to 0.8% of plants dry weight [35] and is reported to be crucial for their development and growth starting from the molecular level to various biochemical and physiological activities such as photosynthesis [35], root growth and development, stalks and stems strengthening, seeds and flowers formation, crop quality and maturity, production of energy, growth and division of cells, fixation of atmospheric N in legumes, development of disease resistance in plants [30–32, 35, 36], conversion of sugar into starch, and transfer of genetic traits [31, 37]. Sufficient quantity of phosphous aids in earlier maturation and proper seed formation in cereals and leguminous crops [35]. This element is also needed in sufficient quantity during the early developmental stages of the plant for primordia formation in reproductive parts of plants [31]. Generally, the values of phosphorus in soil test are much greater, but large part (95% to 99%) of it exists in insoluble phosphate forms like aluminium, calcium, and iron phosphates [32, 35, 37–39]. Due to this, soluble form of phosphorus is very less, ranging from ppb in less fertile soils to 1 mg/ml concentration in the densely fertilized soil [32, 33, 35, 36]. Consequently, phosphorus deficiency markedly decreases the crop growth and yield [32]. During the application of chemical fertilizer to acidic and calcareous soils (containing active Fe3+, Al3, and Ca2+ ions) majority of phosphorus (75%– 90%) will be locked in the soil as metalcation complex precipitate and exhibit longstanding effects like depletion of soil fertility, eutrophication,

262  Biofertilizers and carbon foot print in the environment [35, 40, 41]. So, in the solutions of agricultural soils the levels of this element are diminished and inadequate to satisfy the necessity of crop plants.

8.3.1 Microbes Involved in Phosphate Solubilization Normally, soil is a universally supporting indispensable media for growth of microbes and the microorganisms inhabit in soil have crucial role in dynamics of soil phosphate and regulate the subsequent availability of phosphorus to plants [42]. With phosphate solubilizing efficiency microbial systems can balance required phosphate needs of crop plants [35]. Certain species of rhizobia like R. meliloti, R. leguminosarum, and M. mediterraneum and species of Bradyrhizobium and B. japonicum were reported to solubilize phosphorus [43, 44] by release of small molecular weight organic acids which act on the inorganic form of phosphorus. The activity of phosphate solubilization by few species of rhizobia has been reported in barley and chick pea [45]. A large number of other bacteria and fungi or molds isolated from soil are also reported to solubilize organic and inorganic phosphates present in the soil [46–48]. Microorganisms belonging to a group called as PSM (phosphate solubilizing microorganisms) are efficient in hydrolyzing inorganic and organic form of phosphate compounds from insoluble compounds. These PSM were reported to be active metabolically in plants rhizosphere from where they were identified [32, 36, 49]. Different strains of organisms from genera of bacteria (Rhizobium, Bacillus, and Pseudomonas), genera of fungi (Aspergillus and Penicillium), arbuscular mycorrhizal (AM), and actinomycetes are conspicuous in the group of PSM. Among the entire population of microbes exist in soil, bacteria and fungi hydrolyzing phosphorus constitute 1%–50% and 0.1%–0.5%, respectively [32, 36, 50]. In addition to above specified species, following species such as symbiotically nitrogen fixing rhizobia [32, 33, 36] and nematofungus Arthrobotrys oligospora [32, 36, 51, 52] were also noticed to have phosphate solubilization activity. PSMs adopt various stratagies like reducing the soil pH, mineralization and chelation for solubilization of phosphorus.

8.3.2 Reducing the pH of Soil During this process, microbes lower the soil pH by the release of various organic acids like lactic, citric, 2-ketogluconic, gluconic, acetic, oxalic, malic, glyconic, fumaric, tartaric, succinic, glutaric, malonic, butyric, propionic, adipic, and glyoxalicacids [30–33, 38, 49, 53–55].

Transgenic Microorganisms as Biofertilizers  263 Out of these, gluconic acid (by Erwinia herbicola, Pseudomonas sp., and Burkholderia cepacia species of bacteria) and 2-ketogluconic acid (by Rhizobium leguminosarum, Rhizobium meliloti, and Bacillus firmus) are majorly produced organic acids in microbial phosphate solubilization process [31–33].

8.3.3 Mineralization Through this process, organic phosphate is converted into usable form by microbes from animal debris which is a major source of organic phosphate [36]. Organic phosphate is transformed into utilizable form by PSMs through the process of mineralization, and it occurs in soil at the expense of plant and animal remains, which contain a large amount of organic phosphorus compounds such as nucleic acids, phospholipids, sugar phosphates, phytic acid, polyphosphates, and phosphonates [36]. By enzymatic action of acid or alkaline phosphatases (phytases), PSMs hydrolize organic phosphate into inorganic phosphate that will be immobilized by the plants present in the soil [29–31, 36, 49, 56–58]. Following fungi are few examples observed to release phytases in mineralization process: Aspergillus fumigates, Aspergillus candidus, Aspergillus niger, Aspergillus rugulosus, Aspergillus parasiticus, Aspergillus terreus, Penicillium simplicissimum, Penicillium rubrum, Pseudeurotium zonatum, Trichoderma viride, and Trichoderma harzianum [56, 57]. In addition to the above, other microbes like Streptomyces spp. and soil Bacillus also hydrolyze complex form of organic phosphate into simple inorganic by secreting various extracellular enzymes such as phospholipases, phosphodiesterases, phytases, and phosphoesterases [32]. Based on previous studies, it was noticed that application of PSM mixed cultures (Pseudomonas, Streptomyces, and Bacillus) is an effective strategy for mineralization process [36].

8.3.4 Chelation Carboxyl and hydroxyl groups of inorganic and organic acids produced by PSMs chelate the cations associated with phosphate group and change insoluble form of phosphate into soluble form [36, 38].

8.3.5 Promotion of Plant Growth by PSMs Phosphate solubilizing group of microorganisms exerted the ability to bring back the fertility of debased and unproductive soils for agricultural practice [40]. It was observed that application of this particular group

264  Biofertilizers of microorganisms to soil facilitate easy absorption of phosphorus from broad area by producing a protracted network around roots and augment hydrolyzation of fixed as well as applied soil phosphates and ensure better yield of crops [37, 49]. Because of this nature, these organisms displayed significant outcomes when used either singly or with other microbes in rhizosphere in conventional method of agricultural practice [32, 59]. A clear relation was noticed between PSMs inoculation and other parameters in plants like biomass production, height, and content of phosphorus [29]. It was observed that, when soil was inoculated with bacteria like Achromobacter, Agrobacterium, Bacillus, Erwinia, Flavobacterium, Micrococcus, Pseudomonas, and Rhizobium capable of solubilizing phosphate, an increased yield of crops was recorded [31, 33]. Phosphate solubilizing microbes (PSMs) ensure better plant growth by producing plant growth regulators like cytokinins, auxins, and gibberellins and rise the availability of various other trace elements like siderophore [29, 32, 33, 54, 60–62]. Moreover, they also help in improving nitrogen fixation efficiency by bioinoculation methods [63]. Afzal (2008) [64] mentioned that in the case of Pseudomonas sp. (54RB) and Rhizobium leguminosarum, production of phytohormones is coupled to phosphate solubilization.

8.3.6 Approach of Using PSMs as Biofertilizer and the Future Perspective Since many years, utilization of microbes solubilizing phosphate (PSMs) as biofertilizers for enhancement of agriculture has been an object of an exercise. Competence to avail phosphorus in agriculre soil can be made better by inoculating PSMs, and there were evidences of their benefaction in mineral and inorganic phosphate hydolyzation [56, 57, 65, 66]. These organisms were reported to improve the uptake of phosphorus and enhance the yield of crops [61, 67]. In this context, PSMs have increased the yield of wheat and sugar cane upto 43% with Bacillus and upto 30% (wheat) and 12.6% (sugar cane) with Azotobacter inoculations [32, 37, 68]. Commercially inoculum of PSMs is prepared using Penicillium bilaii strain and is avialble on large scale as JumpStart [31]. Likewise, other organisms like Bacillus circulans, Bacillus subtilus, Bacillus megaterium, and Pseudomonas striata were also demonstrated to use as potential biofertilizers [31]. So, PSMs play a key role in substituting the inorganic phosphate fertilizers and enhance the production by combating the needs of phosphate through sustainable method of agricultural practices. Hence, it is beneficial to investigate persuasive biofertilizers with multifarious growth exhilarating characteristics.

Transgenic Microorganisms as Biofertilizers  265

8.4 Significance of Potassium (K) In addition to nitrogen and phosphorus, potassium is one of the essential nutrient which is important for plant growth, development, and metabolism. Furthermore, this element has a crucial role in enhancing the resistance of plants to biotic as well as abiotic stresses and is also needed for activation of enzymes regulating several plant processes like photosynthesis, sugar degradation, starch synthesis, nitrate reduction, and other metabolic activities [69–74]. Eventhough, potassium content is high in the soil, about 1%–2% of it is only accessable to plants [75] and remaining part of it is not available due to its association with other minerals. In soil, this element exists in various forms such as exchangeable K, non-exchangeable K, solution K, and mineral K. Based on the type of soil, majority (90%–98%) of this element exists as mineral K and is unacccessible for uptake by plants [75] and around 1%–10% of it occurs as non-exchangeable K that is captured in between certain types of earths mineral sheets or layers [76]. Next form of K is solution K which is present around 2 to 5 mg L−1 concentrations in normal agricultural soils, is easily absorbed by microbes and plants, and is frequently exposed to leaching [75]. As a result of launching new varieties for high yield along with continuous expansion of agriculture, potassium levels in soil are getting depleted rapidly. Considerable amount of potassium occurs in soil as fixed form (unavailable to plants) due to unbalanced use of fertilizer to achieve great yield that finally makes the soil deficient in potassium that has been described in majority of crops [77, 78]. Due to rising cost of potassium fertilizers (the price of potash $ 470 per ton since 2011) [77] along with their negative impact on environment, it is imperative to identify an alternative and innate potassium source to retain required levels of K for plant development.

8.4.1 Microorganisms Involved in Potassium Hydrolyzation It has been demonstrated that community of microbes present in soil is capable of regulating the fertility of soil through various processes like mineralization, decomposition, and deposition of nutrients [79]. It was evident that few useful microbes like broad range of fungi, saprophytic bacteria, and actinomycetes which inhabit in soil can hydrolyze insoluble form of potassium by different mechanisms [77, 80–85]. In these microorganisms, potassium solubilizing bacteria (KSB) draw the diligence of agricultural scientists as potent soil inoculum in boosting the growth and yield of crop plants. These KSB are efficient in liberating

266  Biofertilizers the potassium from insoluble inorganic pools of potassium present in soil by solubilization [77, 79, 85–91]. These organisms live in different soils like non-rhizosphere soil, paddy soil, rhizosphere soil [81], and saline soils [82] and are significantly more in soils rhizosphere compared to non-rhizosphere [83]. Potassium mobilization or solubilization is accomplished by good number of bacteria like B. edaphicus, B. mucilaginosus, B. circulans, Burkholderia, Acidithiobacillus ferrooxidans, Pseudomonas, and Paenibacillus spp. Out of various soil bacteria, B. circulanscan, B. edaphicus, and B. mucilaginosus were reported as potent solubilizers of K [77, 84, 85].

8.4.2 Effect of KSB on Plant Growth and Yield Previously, it was reported that KSB had an enhancing growth effect on various plants [78, 80, 81]. Seedlings and seeds of various plants inoculated with potassium solubilizing bacteria (KSB) exihibited considerable improvement in percentage of germination, vigor of seedling, growth and yield of plants, and potassium (K) uptake under field and greenhouse conditions [77, 84, 92–97]. It was observed that in tomato plants inoculated with B. mucilaginosus strain RCBC13 (silicate dissolving bacteria) biomass and uptake of potassium and phosphorus were increased by more than 125% and 150% compared to control plants [98]. Similarly, in WH711 variety of wheat plants, 51.46% gain in root dry weight and 44.28% shoot dry weight were reported when treated with HWP47 strain of K-solubilizing bacteria [99]. In addition to these KSB also displayed good results in development and yield of various crops such as tomato [94], Okra [100], black pepper [101], potato [102], peanut [103], pepper and cucumber [104], rape, and cotton [105]. The above investigations demonstrate that KSB can be employed as biofertilizers to improve agricultural yields through enviromnmentally safe sustainable method of farming by decreasing the usage of chemical fertilizers [88, 100, 106]. Hence, isolation of competent strains of bacteria efficient in potassium solubilization can promptly preserve present resources and avert environmental hazrds due to excessive use of potassium fertilizers.

8.4.3 Abilities and Objections of K Solubilizing Bacteria To enrich plant nutrition, efforts have been put together to avail bacteria hydrolyzing potassium from diverse mineral sources [85]. Eventhough, KSB is another possible capable technology to transform potassium (K) from insoluble form into soluble form, and because of various aspects

Transgenic Microorganisms as Biofertilizers  267 related to their utilization to practice agriculture, it is limited; for example, (i) farmers are not aware of biofertilizers application, (ii) gradual effect of potassium biofertilizer on crop yield, (iii) less concern of researchers toward biofertilizers improvment, (iv) KSB culture collection banks were not ready due to unavailability of identified strains, and (v) inadequate information regarding the formulation of the product. These are few constraints which need to be solved in future to boost the importance of KSB as biofertilizers. So, considerable number of fungi or molds along with certain bacteria are noted to work as nitrogen fixers and potassium and phosphate solubilizers, which can be formulated with other type of microorganisms in various proportions to prepare biofertilizers. These biofertilizers as substantial segments of organic farming perform an imperative role in retaining the sustainability and fertility of soil for long duration and minimize the degradation of land by simultaneously increasing the crop productivity [11, 107, 108].

8.5 Biofertilizers Used in Agriculture Different types of microorganisms used as biofertilizers in agricultural soils are as follows: a) Nitrogen fixing biofertilizers (NFB): examples include Rhizobium spp., Azospirillum spp., and cyanobacteria; b) Phosphate solubilizing biofertilizer (PSB): examples include Bacillus Spp., Pseudomonas Spp. and Aspergillus Spp., c); Phosphate mobilizing biofertilizers (PMB): examples are Mycorrhiza; d) Plant growth promoting biofertilizer [36–38] (PGPB): examples include Pseudomonas Spp.; e) Potassium solubilizing biofertilizer (KSB): examples include Bacillus Spp. and Aspergillus niger; f) Potassium mobilizing biofertilizer (KMB): examples include Bacillus Spp.; and g) Sulfur oxidizing biofertilizer: examples include Thiobacillus Spp. (Figure 8.2). Biofertilizers

Nitrogen fixing Azolla Microorganisms

Bacteria

Phosphate solubilizing Microorganisms

Potassium solubilizing Microorganisms

Blue green algae

Figure 8.2  Microbes utilized as biofertilizers.

Mycorrhizae (VAM)

Microorganisms releasing plant growth regulating harmones and enzymes

268  Biofertilizers

8.5.1 Mycorrhiza Out of various soil organisms, Arbuscular Mycorrhizae (AM) fungi have proven to be a crucial constitutent of viable plant-soil coordination systems [109] and colonize in plants roots. They can escalate growth and progress of plants by promoting absorption of various elements like nitrogen [110], phosphorus [111], and micronutrients [112], by enhancing photosynthesis and fruit yield, by exhilarating substances influencing the growth, by maintaining osmotic regulation in salt and drought conditions, and also aid in improving pest tolerance by acting as antagnonists aginst disease causing pathogens of plants [113, 114]. Previous studies report that these type of mycorrhiza are not present in substrates without soil [115–117]. Mycorrhizal application minimizes the fertilizer use by providing easy access of nutrients and essential elements needed for plant growth in inoculated (with mycorrhiza) soils compared to non-inoculated soils [118–121].

8.5.2 Plant Growth-Promoting Rhizobacteria (PGPR) They can increase the growth competence of plants by enhancing the development and yield of plants along with seed emergency. Few PGPR elicit systemic resistance to bacteria, fungi, and viruses, and in certain cases of nematodes [122–124]. Mostly, Bacillus or Pseudomonas species are used as PGPR strains [122, 125].

8.6 Role of Biotechnology in Agricultural Sector Biotechnology has iniated to reform agriculture with methods to cultivate different new varieties of crops with better yield and enriched nutritional content and competence to survive under adverse conditions with minimum requirement of pesticides and fertilizers. GMOs or genetically modified organisms are described as living organisms, whose genome has been modified or transformed to develop transgenic organisms with desired characteristics by genetic recombination [126]. Modern techniques in science especially genetic engineering enable accomplishing of best characters in organisms by incorporation of specific gene of interest (DNA) resulting in development of new crops with novel traits like pathogen, pest, and herbicide resistance and in invention of potent microbial strains (to promote the growth of plants and can defend them from unfavorable conditions). Due to restraints on use of inorganic-based chemical fertilizers and pesticides because of their environmental complications, at present,

Transgenic Microorganisms as Biofertilizers  269 Table 8.1  Comparing the effects of organic and inorganic fertilizers on plant growth. Biofertilizer/organic-based fertilizers

Chemical fertilizer/inorganicbased fertilizers

1.

In addition to macronutrients, biofertilizers also supplies growth supporting substances like hormones, vitamins, and amino acids required to enhance yield

Provides only macronutrients necessary for growth

2.

Facilitates nutrient supply during the entire period of plant life and supports growth

Frequent treatments are needed

3.

Encourages and amplifies the ability of microbes in root colonization

Intimical effects on microbes inhabiting rhizosphere

4.

Helps in recovery of soil properties and improves the absorbtion of nutrients.

Results in disproportion of nutrients and soil degradation

5.

Inexpensive and environmentally amiable

Costly and environmentally abominable

S. no

biotechnology is offering advanced products in the form of biofertilizers for welfare of society (Table 8.1) where there is no other alternate stratagies [127]. Further, different strains of microbes which are capable of reducing the onset of disease and stimulating growth of plants appear to limit the application of fungicides, pesticides, and fertilizers and open up new avenues to prevent major loss of crop yield that cannot be handled even today by available agrochemicals. So, it is visualized that biotechnology can act as a reliable source to achieve sustainable food production in an environmental friendly manner.

8.6.1 Development of Potent Microbial Strains Through Genetic Engineering Approach to Produce Efficient Biofertilizers Biofertilizer technology has considerably developed with in the market. The characteristics of diversified processes explored regarding the functioning

270  Biofertilizers of PGPR along with feasibility of altering the genome of specific microbial strain as an appropriate strategy to promote growth, deveopment, and yield of plants recommend employement of genetically altered transgenic microorganisms as biofertilizers to exploit numerous and disparate prospects of execution in the coming future. Majority of the inorganic fertilizers used today are manufactured industrially through Haber-Bosch process [128, 129] that is budgetary to developed countries which can support the cost of that process, but it is proscribed in poor countries where such expenses are not bearable. In addition, during this process, fossil fuels are burnt to produce ammonia from nitrogen gas (molecular nitrogen) which consumes around 5% of the total global natural gas produced. So, use of in situ mode of functioning transgenic diazotrophs developed through genetic engineering by gene modification could alleviate problems of environmental pollution and reduce shipment costs compared to fertilizers derived from Haber-Bosch process. Further, they enhance the assimilation of nutrients and facilitate their release promptly during the plant development that may address present agricultural leftovers discharge-related problems [130]. The underlying processes crucial for microbial induced growth and development of plants are at starting level and need to be deciphered at various aspects of molecular level. This information was accustomed for improvement of microbial strains through genetic engineering by modifying their genetic material. There are various such examples, viz., transforming PGPB strains with1-aminocyclopropane-1-carboxylate (ACC) deaminase gene to reduce the levels of ethylene in plants [131], developing microbial strains (Azospirillum) secreting higher levels of IAA [132], strains of microbes changed genetically to produce fixed form of ammonium [133], where substantial advancement to exploit the potential of microorganisms as plant growth inducers can be accomplished. Environmental safety evaluation regarding the administration of corresponding strains necessitates a study perception regarding the mechanisms inducing the plant growth. For example, in rhizospheric bacteria, lateral gene transfer of ACC deaminase genes has been recommended [134].

8.6.2 Genetically Altered Transgenic Azotobacter vinelandii as an Effective Diazotrophs Biofertilizer Genetically engineered bacteria of Azotobacter vinelandii (A. vinelandii) secrete considerable quantity of nitrogen containing substances like urea or ammonia compared to their wild types. This kind of end products generated during microbial metabolism could be utilized adequately as

Transgenic Microorganisms as Biofertilizers  271 biofertilizers. Dissimilar to majority of bacteria producing nitrogen which work only during anaerobic conditions, A. vinelandii functions in aerobic conditions which makes it as an excellent biological or organic forge that bolsters growth of routine crops in agriculture or algal production [135].

8.6.3 Phytostimuators and Biofertilizers Few examples of different genera of bacteria used as biofertilizers for stimulating growth and development of plants include Bradyrhizobium, Sinorhizobium, and Rhizobium [136–138]. They associate symbiotically with leguminous plants and supply nitrogen to host in accessible form. Biofertilizers have been developed by genetic modification of microor­ ganisms through IMPACT (Interactions between Microbial inoculants and resident Populations in the rhizosphere of Agronomically important Crops in Typical soils) program which are potent in developing symbiotic asssosiation with host plants and decrease the necessity of fertilizers application. Azospirillum (bacterea) can be considered as one of the phytostimulator that induces growth of plants by releasing growth stimulating substances which increase growth of plant roots and facilitates enhanced uptake of nitrogen and water. Strains have been developed by genetic modification that are able to produce significant levels of growth stimulating factors with an aspect to enhance the yield of crops and protect environment from hazardous chemical fertilizers. In IMPACT program, impact of genetically modified phytostimulators and biofertilizers on naturally existing population of microbes was determined in addition to testing their efficiency in hiking agricultural yields [139]. Other than carbon dioxide (acquired from atmosphere), plants acquire majority of nutrients from soil, which needs to be ameliorated with nutrients obtained from renewable sources according to present policy of sustainable agriculture practice. The excellent illustration to demonstrate this concept is mechanism of biological nitrogen fixation in plants of leguminaceae by which enormous form of nitrogen (nitrate or ammonia) discharged into surface water bodies is evaded. Bacteria capable of fixing nitrogen could be explored as self-proliferating source of nitrogen for plants, where desperately all plants (like rice, maize, and wheat) cannot commence symbiotic relation with nitrogen fixing bacteria even though they exist in huge number on their roots. Until advanced methods are evolved to prompt symbiotic relation between the microorganisms involved in nitrogen fixation and the crops of agronomical importance, agricultural yield relies majorly on inorganic source of fertilizers. To supplement nitrogen content, fertilizers are added

272  Biofertilizers to soil as nitrate which is highly movable in nature, due to this reason plenty of nitrogen is applied to soil than the necessary concentration to support ideal plant development. Currently, in paddy fields to obtain better yield around 450 kg N/ha is used, out of which only 200–250 kg N/ha is being utilized for plant growth. So, more than half of the nitrogen furnished is seeped (budgetary loss) into environment that has severe implications on atmosphere (environmental adulteration) [140]. Distinct systems to enhance fertilizer intake by roots have been improved, that comprise alternative fertilizer formulations like controlled and slow release fertilizers and employment of PGPR (Plant Growth Promoting Rhizobacteria). These PGPR act either directly in development of plants or indirectly by functioning as biocontol agents in elimination of devastating deleterious pathogens and microorganisms [141].

8.6.4 Azospirillum The well-studied case to illustrate direct action of PGPR in stimulating the growth of plants is phytostimulation. Species of Azospirillum bacteria colonize in the roots of plants and deliver plant growth enhancing factors (cytokinins, auxins, etc.) to promote growth and development of plants (Figure 8.3). This ensures improved absorption of nutrients and water and helps in greater yield of crops. Moreover, better intake of nitrogen reduces its remnants in soil and substantially minimizes the possibility of groundwater pollution (Figure 8.4).

Azospirillum: Free living rhizospheric N2 fixing bacteria, PGPR (Genus)

–

–

+

+

Figure 8.3  Enhanced root development with the application of Azospirillum inoculum (+ Inoculated − Non-inoculated). (Source: Harnessing the potential of genetically modified microorganisms and plants, European commission community research).

Transgenic Microorganisms as Biofertilizers  273 Amount of remaining nitric N at harvesting period in soil (0-30 cm) 120 100

Kg N/ha

80 Control

60

Azogreen 40 20 0 0N

50 N

100 N 150 N N level

200 N

250 N

Figure 8.4  Decreased levels of nitrogen content in soil planted with Azospirillum. (Source: Results of Azogreen-m field experiment 1997).

Pseudomonas fluorescens strain F113 (biological control agent) and strains of Azospirillum with genes for non­antibiotic resistance as markers (genes which enable the identification of bacteria in the soil) were developed by genetic modification in IMPACT program to evaluate their performance on yield and production of sorghum grains. Several trails were conducted in fields by research and industrial associates on commercial scale in translational scheme of IMPACT group. The ability of colonization, endurance, and continuity of genetically altered bacteria on sorghum roots and their impact on enhancing the yield of sorghum grains and endemic microflora was determined [139]. Correspondingly, sorghum was grown in three separate plots emended with various concentrations of nitrogen to check the influence of microbial inoculants. The two strains of Azospirillum, namely, Azospirillum brasilense Sp6 secreting ordinary levels of plant growth regulating factor IAAs and Azospirillum brasilense Sp6 IAA++ producing higher levels of IAA were under examination as per regulations mentioned in EU Directive 90/220. It was observed that inoculants of Azospirillum elevated development of sorghum roots and raised grain production that indicates similar yield of grains can be acquired by the use of Azospirillum inoculum along with seeds by decreasing the application of nitrogen fertilizer [139].

274  Biofertilizers 9.0

CFU/g Soil dry weight (Log)

8.0 7.0 6.0 5.0

Untreated control

4.0

A. brasiensi Sp6

3.0

A. brasiensi Sp6 IAA ++

2.0 1.0 0.0

Total Aerobic Micro bacterial fungi population

Strepto Fluorescent Aerobic spore mycetes Pseudomonads forming bacteria

Figure 8.5  Effect of genetically modified Azospirillum brasilense strains on native microbial population. (Source: Harnessing the potential of genetically modified microorganisms and plants, European commission community research).

Analysis showed that as long as the genetically modified microbial strains are satisfactorly colonizing the roots, the density of these cells was maximum on the roots after 15–20 days of sowing, after this stage, Azospirillum colonization was continuously decreased and, finally at harvesting period density of cells, was particularly low (9 × 103 cells/g soil dry weight) [139]. Further, seed inoculation with these genetically modified transgenic strains did not disturb the population of native microbes specific to that area (Figure 8.5).

8.6.5 Generation of Genetically Modified Transgenic Azospirillum Strains With Enhanced Levels of Phytoharmone Secretion To develop a process to stimulate the induction of plant roots and to improve nitrogen uptake using Azospirillum strains, a proper knowledge about regulating mechnanisms and controlled conditions that elicite these bacteria to produce phytoharmones is needed. Additionally, a clear understanding of plant-bacterial interaction is also important. The three prime functions in IMPACT to address the problem are follows: 1) To acquire information about biochemistry and genetics related to IAA (Indole­ -3­ -Acetic Acid) synthesis, i.e., the phyto­harmone produced by Azospirillum bacteria.

Transgenic Microorganisms as Biofertilizers  275 2)  To generate genetically modified stains of Azospirillum producing accepted IAA levels (IAA-­over producers, IAA­ attenuated, and IAA-minus). 3)  To check the impact of these genetically altered bacteria on various growth parameters of plants (uptake of nutrients and nitrogen and promotion of growth) and on the habitat or environment (synergy with inhabitant microbial flora, survivance and transmission) under the field circumstances. These efforts have been exercised in research program that implicates engineering of bacteria, assessing of various physiological process followed by final screening under field conditions. The process of IAA biosynthesis by strains of Azospirillum appears to be complicated and interceded by three pathways atleast. Enhanced production of IAA by Azospirillum was accomplished by engineering the gene ipdC which is responsible for the expression of an important enzyme in the indole-­3­-pyruvic pathway (major biosynthetic pathway). These strains were designed with certain genes as markers to enable their identification in the soil during field trails. The two genes selected to use as marker genes are gfp and lue; where the gene gfp is coding for green fluorescent protein which makes the cell to fluoresce and expression of lue gene causes bioluminescence that makes cells of bacteria to glow [139]. Currently, strains of Azospirillum are available with basic features. But, prior to their release for field trails, comprehensive and accurate experimental analysis under controlled conditions is mandatory. In IMPACT, nowadays, studies are concentrating on effect of Azospirillum strains developed by genetic modification on native population of microbes, rate of nitrogen uptake from soil by plants, and growth rate of plants. These experiments are carried out in glass house and growth cabinets to obtain essential knowledge about behavior of GM strains under the field conditions [139]. IMPACT consortium transational partnership aids to conduct studies with various categories of crops in different soil types under diversified conditions of climate.

8.6.6 Development of Rhizobium Strains With Increased Competitiveness by Genetic Modification The yield of leguminous crops like clover, peas, and beans can be enhanced by using more competent nitrogen fixing bacteria for inoculating the seeds. However, inoculation of legumes is usually unproductive due to presence of indigenous microorganisms inferior in nitrogen fixing capacity that can

276  Biofertilizers challenge with strains imported or introduced for iniation of nodules. The capacity to preeminent nodulation is described as competitiveness which is very crucial for promising application of rhizobial bacteria as inoculants [142]. Hence, it is advisable that the strain used as inoculant should be altered to assure that it should occupy ample number of root nodules to support increased level of nitrogen fixation for host plant. It has been determined that nodulation competitiveness of various Sinorhizobium meliloti bacterial strains obtained from different geographical areas can be increased by genetic modification. This genetic modification includes nifA gene expression which is important in regulating all the genes (nif genes) responsible for the nitrogen fixation process. It was noticed that in alfalfa roots (Table 8.2) compared to wild type, genetically manipulated strains of S. meliloti occupied majority of root nodules in mixed inoculation experiments. The genetic basis for this advancement is not interpreted clearly, but it has been hypothecated that gene nifA controls expression of other genes apart from nif genes. It was postulated that altered expression of those genes will support and favors the formation of root nodules during the development [139]. In addition to gene expression, other aspects important in nodulation competitiveness is competence of rhizobium bacterial strains to the ability of rhizobium strains to recognize the plant root effectively. It has been speculated that employement of microbial inoculants that are captivated Table 8.2  Potentiality of transgenic rhizobium strains in nodule occupancy in comparison with wild-type strains. (Source: Harnessing the potential of genetically modified microorganisms and plants, European commission community research). S. no.

Rhizobial strains

Nodule occupancy in coinoculation (%)

1

2011

5

2

2011-GM

95

3

L5.30

22

4

L5.30-GM

78

5

GR0-13

7

6

GR0-13-GM

93

7

Rm41

13

8

Rm41-GM

87

Transgenic Microorganisms as Biofertilizers  277 particularly to desired plant roots could permit more productive inoculation; hence, demand reduced dosage of bacterial strains as an inoculums [139]. The function of bacterial movement approaching roots on the bacterial strains competitive ability has been assessed using strains of Rhizobium leguminosarum which have been genetically engineered for a reporter gene, ß-glucuronidase (gusA gene) expression that enables their identification easy in root nodules. Based on the studies, it was stated that induction percentage of root nodules was higher in gusA-labeled bacterial strains compared to flagella deficient non motile bacterial strains. When compared to flagella-deficient non-motile strain (a flagellum is a whiplike structure which is responsible for propelling the bacterium through water) gusA-labeled strain produced high percentage of nodules [139]. In this manner, it was demonstrated that functional flagella are needed for adequate competition in nodule formation. The initial action of Rhizobium toward roots of host plant is detection of substances secreted by plants roots. These substances are identified by Methyl-accepting chemotactic (Mcp) proteins which are present in the Rhizobium bacterium cell wall and induce bacteria to move toward host roots (Figure 8.6). Genes like Mcp were discovered in R. leguminosarum

Plant

Root Exudates

MCP

Figure 8.6  Movement of rhizobium nearer to the plant roots. MCP (Methyl accepting Chemotaxins proteins) recognize the exudates of distinct plant roots.

278  Biofertilizers bacteria and recently the compound (secreted by the plant) perceived by Mcp proteinis being investigated [139]. This will contribute valid knowledge regarding the process of root affinity permitting the growth of Rhizobial bacterial strains with improved nodulation competitiveness and accelerated host specificity.

8.6.7 Effect of GM Rhizobial strains on Arbuscular Mycorrhizal (AM) Fungi

Myconhizal Entry Points (No.)

As discussed earlier, AM fungi are crucial group of fungi that develop symbiotic mode of association with plants. In the project of IMPACT, an important part is to investigate whether modified Rhizobial strains aimed for enhancing the competitiveness of producing more colonization and nodule formation of host plant root has an impact on the capacity of mycorrhizal fungi to infect roots of plants to etablish advantageous symbiotic association. In a set of experiments conducted in glass house and growth room, it has been entrenched that strain Sinorhizobium meliloti developed by genetic modification with enhanced nodulation potential did not impede with any perspective related to mycorrhiza formation in the characteristic AM fungi (Glomusmosseae). Certainly, when compared to wild isolates, the genetically manipulated strains of S. meliloti raised the AM colonization units number and the nutrient attaining capacity (Figures 8.7 and 8.8). Formation of symbiotic relation also stimulated changes in morphology of roots, especially in 700 600 500 400

Wild type

300

GM derivative

200 100 0

2 4 6 8 10 Time After Inoculation (Weeks)

Figure 8.7  Comparing the impact of wild-type and its GM Sinorhizobium meliloti strains in alfafa plants associated with Arbuscular mycorrhizal fungi (Glomusmosseae). (Source: Harnessing the potential of genetically modified microorganisms and plants, European commission community research).

Nitrogen Content (mg)

25 20 15 10 5 0

2

4

6

8

10

Phosphorus Concentration (mg)

Transgenic Microorganisms as Biofertilizers  279 1.4 1.2 1 0.8 0.6

Wild type

0.4

GM derivative

0.2 0

2

4

6

8

10

Time After Inoculation (Weeks)

Figure 8.8  Comparing the effects of Sinorhizobium meliloti wild-type and GM strains on nitrogen and phosphorous assimilation in alfafa plants associated with Arbuscular mycorrhizal fungi (Glomusmosseae). (Source: Harnessing the potential of genetically modified microorganisms and plants, European commission community research).

the plants inoculated with GM S. meliloti strain, the extent of branching and number of developed lateral roots was greater [139].

8.6.8 Release of Genetically Manipulated Rhizobium for Field Trails Genetically modified strains of Rhizobium leguminosarum bv. viciae, with HgCb resistance (mer genes) and lacZ genes were produced in IMPACT association and their influence and consequence under the field circumstances was evaluated. Three GM R. leguminosarum bv. viciae 1003 (wildtype) derivatives were used for trails in field. They are strain 1110 which consists of pDG3 plasmid containing genes imparting HgCb (mer genes) resistance and lacZ gene whose function is under the lacl-lacO system control, next strain 1111 which contains pDG4 plasmid where the gene lacZ is over expressed at elevated levels, followed by another strain 1112 carrying a set of mer genes and a controlled lacZ gene incorporated into part of the chromosome. These strains produced by genetic modification were detected using lacZ/mer reporter system and microbial populations were screened by using the most probable number (MPN) method. Further, the activity of microbes in soil was estimated by checking CO2 levels whose concentrations provide evidence for metabolic activity in soil and emissions of N2O is to ascertain the transformation of nitrogen [139]. The existence of inoculant microbial strains in pea plants rhizosphere was identified after ten days of sowing (Table 8.3). It was noticed that majority of the tested strains colonized to the same extent in the rhizosphere. When these bacterial stains were studied for stability assays revertants were not

280  Biofertilizers Table 8.3  Population density of genetically modified and control R. leguminosarum bv. Viciae strains in soil rhizosphere during the development of pea plants after 10 days of sowing. (Source: Harnessing the potential of genetically modified microorganisms and plants, European commission community research). S. no.

Strain

Rhizobium leguminosarum bv. viciae Total

Genetically modified

Revertants

1

1003

1.9 × 104

–

–

2

1110

3.4 × 105

3.4 × 105

< 102

3

1111

1.3 × 105

9.9 × 104

2.6 × 104

4

1112

2.9 × 104

2.9 × 104

< 102

found from 1110 and 1112 (both the strains are designed to have regulated or controlled gene expression) by the plate count method, whereas a significant instability was detected in strain 1111, due to the constitutive lacZ gene expression. In the case of uninoculated plants, emission of CO2 in nonrhizosphere soil appears to be remarkably smaller than in rhizosphere soil. Nonrhizosphere soil had reduced rate of respiration compared to plants inoculated in rhizosphere part of soil [139]. Furthermore, levels of CO2 produced are similar in soil inoculated with non GM and GM strains. These investigations imply that even though the plant existence had aappreciable effect on mineralization of carbon in the soil, the influence of genetically modified strains of Rhizobium is insignificant with respect to wild-type strain. About N2O production, soil with no plants had a method of N2O emission considerably dissimilar compared to soil with plants. However, production of N2O was not substantially distinguishable among uninoculated and inoculated plants or in between nonmodified or GM strains. These resuls agree with those of CO2 production and recommend that the influence of the plant on activity of microorganisms is remarkably higher than the effect of genetically altered microbial inoculants when compared to wild-type strains [139].

8.7 Conclusion Biofertilizers prepared from the organic matter are important constituents for maintaining prolonged soil fertility by facilitating efficient nitrogen

Transgenic Microorganisms as Biofertilizers  281 fixation and phosphate solubilization and play a crucial part in increasing the availability of various vital nutrients present in soil to the plants. To attain better yields, farmers routinely apply inorganic fertilizers, which are not safe and accountable for air, water, and soil pollution and ultimately causes various human disorders. In this situation, they can be a reliable choice for farmers to enhance the yield per unit area. Recently developed technologies have accelerated the understanding of gene modifications, proteomics, and profiling of metabolites to exploit the microbe mediated strategies for promoting the plant growth. The ultimate objective to ameliorate the plant microbe associations would be microbial biofertilizers integration, advanced microbiomes, soil improvement, and suitable microbe-amended crops for various types of soil. The existing newly emerged transgenic techniques promotes the generation of potent genetically modified microbial strains to develop efficient biofertilizers. Certainly, this is a prime area that entitles the main research work, since it retains the assurance to enhance the crop production and addresses food security in an eco-friendly and sustainable approach.

Acknowledgements The authors acknowledge the support of Department of Biotechnology, GITAM Institute of Technology, GITAM (Deemed to be University), Visakhapatnam in successful completion of this study.

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Transgenic Microorganisms as Biofertilizers  289 104. Sheng, X., Growth promotion and increased potassium uptake of cotton and rape by a potassium releasing strain of Bacillus edaphicus. Soil Biol. Biochem., 37, 1918–1922, 2005. 105. Archana, D.S., Nandish, M.S., Savalagi, V.P., Alagawadi, A.R., Screening of potassium solubilizing bacteria (KSB) for plant growth promotional activity. BIOINFOLET-A Quarterly J. Life Sci., 9, 627–630, 2012. 106. Hari, M., Seshadri, S., Perumal, k., Bio-fertilizer (phosphobacteria), Shri Amm Murugappa Chettair Research centre, Taranmani. Science., 5(6), 25–40, 2010. 107. Khosro, M., Yousef, S., Bacterial bio-fertilizers for sustainable crop production: A review. APRN Journal of Agricultural and Biological Science., 7(5), 237–308, 2012. 108. Schreiner, R.P., Mihara, K.L., Mc Daniel, H., Benthlenfalvay, G.J., Mycorrhizal fungi influence plant and soil functions and interactions. Plant Soil., 188, 199–209, 2003. 109. Barea, J.M., Azcon-Aguilar, C., Azcon, R., The role of VA mycorrhizae in improving plant N acquisition from soil as assessed with 15N. In: Stable isotopes in plant nutrition. IAEA, Vienna., 209–216, 1991. 110. Bolan, N.S., A critical review on the role of mycorrhizal fungi in the uptake of phosphorus by plants. Plant Soil., 134, 189–207, 1991. 111. Burkert, B., Robson, A., 65Zn uptake in subterranean clover (Trifolium subterraneum L.) by 3 vesicular arbuscular mycorrhizal fungi in a root-free sandy soil. Soil Biol. Biochem., 26, 1117–1124, 1994. 112. Al-Karaki, G.N., Nursery inoculation of tomato with arbuscular mycorrhizal fungi and subsequent performance under irrigation with saline water. Sci. Hortic., 109, 1–7, 2006. 113. Duponnois, R., Colombet, A., Hien, V., Thioulouse, J., The mycorrhizal fungus Glomus intraradices and rock phosphate amendment influence plant growth and microbial activity in the rhizosphere of Acacia holosericea. Soil Biol Biochem., 37, 1460–1468, 2005. 114. Ikiz, O., The effects of mycorrhiza on soilless pepper cultivation. Ph.D thesis. Code number: 730. Department of Horticulture, Institute of Natural and Applied Sciences, University of Cukurova, Adana-Turkey, 2003. 115. Rehber, Y., The effectiveness of vesicular arbuscular mycorrhizas in soilless culture of muskmelon. MSc thesis. Code number: 2365. Department of Horticulture, Institute of Natural and Applied Sciences, University of Cukurova, Adana-Turkey, 2004. 116. Ikiz, O., Abak, K., Dasgan, H.Y. and Ortas, I., Effects of mycorrhizal inoculation on soilless pepper plant growth. Acta Hort., 807, 533–539, 2008. 117. Cozzolino, V., Di Meo, V., Piccolo, A., Impact of arbuscular mycorrhizal fungi applications on maize production and soil P availability. J. Geochem. Explor., 129, 40–44, 2013. 118. Santos, E.A., Ferreira, L.R., Costa, M.D., Silva, M.C.S., Reis, M.R., França, A.C., Occurrence of symbiotic fungi and rhizospheric phosphate

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9 Biofertilizer Utilization in Agricultural Sector Osikemekha Anthony Anani1, Charles Oluwaseun Adetunji2*, Osayomwanbo Osarenotor3 and Inamuddin4 Laboratory for Ecotoxicology and Forensic Biology, Department of Biological Science, Faculty of Science, Edo State University, Uzuairue, Edo State, Nigeria 2 Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Department of Microbiology, Edo State University Uzuairue, Auchi, Edo State, Nigeria 3 Department of Environmental Management and Toxicology, Faculty of Life Sciences, University of Benin, Benin City, Edo State, Nigeria 4 Department of Chemistry, King Abdulaziz University, Jeddah, Saudi Arabia 1

Abstract

It has been discovered that the present agricultural practices heavily depends on the application of synthetic inputs in order to boost increase in agricultural production. The application of these synthetic chemicals has been documented with several adverse effects when applied in excess most especially with health and environmental hazards couple with high level of pesticides residues on agricultural products. Moreover, this has led to increase in the accumulation of these pesticides with negative effects on the soil as well as buildup of heavy metals in the soil. It has been observed that these chemical fertilizers contain acid radicals like sulfuric radicals and hydrochloride, which has led to increase in the level of soil acidity which has a detrimental effect on the health of the plant and the soil. Therefore, there is a need to look for a sustainable, reliable, biocompatible, and eco-friendly fertilizer that could prevent all the highlighted challenges with synthetic fertilizer. The application of microorganism as a biofertilizer has been realized as a sustainable methods that could enhance or led to increase in agricultural productivity. Therefore, this chapter intends to provide a detailed information of the application of biofertilizer as a sustainable biotechnological tool that could led to increase in agricultural production. Detailed information was *Corresponding author: [email protected]; [email protected];   [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (293–308) © 2021 Scrivener Publishing LLC

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294  Biofertilizers provided on the modes of action of these biofertilizer while specific examples of cases where biofertilizer has been utilized for improving increase in agricultural production were also discussed. Recent advances in the application of microbial inoculum as a sustainable technology that could improve plant health were also provided. Keywords:  Biofertilizer, agricultural health and environmental, hazards, biocompatible and eco-friendly

9.1 Introduction In order to meet the growing demand for food globally, it is necessary to increase agricultural productivity. This is more worrisome owing to the fact that insufficient land for crop farming, wherefore farmers are mostly involved in continuous cropping of land. Continuous tilling of farmlands can result in the partial or total loss of soil fertility. To overcome this challenge, farmers resolve to the use of inorganic fertilizer in order to maintain or obtain high crop yield; however, uncontrolled and continuous use of inorganic fertilizer is detrimental to the environment [11]. The nitrogen and phosphorus from fertilizer when washed off from applied soil may result in the enrichment of water bodies as a result of runoff through non-point source pollution. This excess nutrient when present in water bodies can cause eutrophication especially if the water is stagnant [12]. The use of organic manure has been an alternative to inorganic fertilizer usage. However, owing to its bulky nature coupled with the fact that some might contain pathogenic organisms which may be harmful to plants, its use has also been accompanied with some criticism. To overcome these above challenges, biotechnological advances have identified some microbial species capable of enriching the soil through association with their roots or independently. Biofertilizer is a substance containing live or latent cells, applied to plants or soil, which, upon application, colonizes the plant’s rhizosphere. This colonization results in increase nutrient availability to the plant [11]. Biofertilizers use different mechanisms to provide nutrient to the plants such as fixing atmospheric nitrogen and phosphate solubilization and to increase generation of plant growth-promoting compounds in the soil [2, 11, 12].

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9.2 Application of Biofertilizer as Bioaugmentation Agent for Bioremediation of Heavily Polluted Soil The concept of bioaugmentation is not new in environmental biotechnology; it involves the introduction of microbial inoculants into a polluted environment in order to enhance the bioremediation process. It is mostly used in polluted sites when remediation by indigenous microflora is slow or difficult. Several studies have shown that biofertilizer application to polluted soil has yielded promising results. Ma et al. [10] compared the bioremediation potential of biochar and plant growth-promoting bacteria, Bacillus sp. TZ5–amended biochar as biochemical composite material (BCM) for the remediation of cadmium in a pot experiment. Their result showed that the bacteria-amended biochar was able to immobilize cadmium more than ordinary biochar. Their study also showed increase in soil microbial enzyme activity in soil with biochar-bacteria composite. The study therefore concluded that BC-TZ5 composite was a good source of nutrient and successfully remediated the polluted soil. Hindersah et al. [23] isolated Azobacter from mercury contaminated and suggested that bioaugmentation of heavy metal contaminated soil using Azobacter is a renewable biological process, which is cheap, easy, and safe. Girigiri et al. [7] studied the ecorestoration of crude oil contaminated soil using two nitrogen fixing bacteria (Azotobacter sp. and Rhizobium sp.) and phosphate solubilizing bacteria (Pseudomonas and Bacillus) as identified by 16sRNA gene sequence analysis. The two groups of bacteria were used individually and in combination. The bioremediation experiment was monitored in microcosm study for 4 weeks. Their results revealed that total petroleum hydrocarbon reduction efficiency was 97.8% and 94.3% for NFB and PSB, respectively, as against 92.1% and 34.6% recorded for NPK and control, respectively. These authors thereby stated that the removal ability observed with NFB and PSB is evidence that nitrogen and phosphorus are key limiting factors during bioremediation of crude oil. Their observation revealed that hydrocarbon-based contaminated soil with amended microbes such as nitrogen-fixing bacteria could play a crucial role in nitrogen fixation and cometabolism to autochthonous bacteria which help in absorption of essential nitrogen which could facilitate dilapidation of hydrocarbons. It also showed that showed that phosphorus is needed by microbes during bioremediation. Franchi et al. [24] assessed the ability of plant growth-promoting bacteria from heavy metal contaminated soil to remediate soil co-contaminated

296  Biofertilizers with mercury and arsenic. Their results showed that the thirteen indigenous bacteria strain on in vitro characterization showed great potential in augmenting the heavy metal bioremediation process. This was evident in their tolerance to high concentration of the heavy metal coupled with their expression of high plant growth-promoting ability. Bioaugmentation potential of Rhodobacter sphaeroides biofertilizer (RBF) in a petroleum contaminated soil has also been demonstrated by Jiao et al. [25]. These authors reported that after, 120 days using combination of three plants species, wheat, cabbage, and spinach. Their result showed that elimination of TPH from polluted soil in the Rhodobacter sphaeroides biofertilizer bio-augmented rhizosphere soils were found to have the following values for wheat (46.2%), cabbage (65.4%), and spinach (67.5%) rhizosphere, respectively.

9.3 Advantages of Biofertilizer in Comparison With Synthetic Fertilizer The advantages of biofertilizers over chemical/synthetic or conventional fertilizers are based on the role or functions they play in soil functions and plant physiology. Originally, chemical fertilizers make readily available three main nutrients: NPK (nitrogen, phosphorus, and potassium) to plants for development and growth. However, because of their expensive nature and deficiency in providing other required plant nutrients, a more fortified, cheap, and eco-friendly form (biofertilizer), which is rich in plant beneficial microbes and organic matter as well as NPK, makes it more perfect over the conventional type for farming purposes. The overutilization of commercial chemical fertilizers for agricultural activities has increased the impact on the soil functions, thus affecting the eco-balance of nutrients tranportation between the soil components and the plants. To mitigate and combat these impacts farmers and producers need a sustainable alternative. On this vein, Carvajal-Muñoz and García [6] in review, looked at the advantages and disadvantages of utilizing biofertilizers over chemical fertilizers in agriculture. The authors reported that food supply and processing needs to be improved in this current state of population expulsions. That to meet up with this, biofertilizers need to be applied to plants in order to boost the rapid development and growth of agricultural produce. They reported that biofertilizers have many benefits over the widely used fertilizers because they are can be used without any technical assistance, able to give better quality during and after harvest, able to boost the efficiency event at very small cost, more economical

Biofertilizer Utilization in Agriculture  297 compared to the convention types, and possess little or no invasion to the ecosystem. The authors recounted that biofertilizers have come to replace the chemical fertilizers as novel technology that be used to reduce the ecosystem impacts caused by the latter on farmlands. Some of the limitations based on their efficiency of recent decades were highlighted. Ajmal et al. [2] in a review, looked at the utilization of biofertilizers in farming as a substitute for conventional fertilizers. The authors recounted that in the quest for green revolution, the agricultural sector exhibits a major function in the substance of the human population. That biotechnology plays a pivotal role in the production of biopesticides and biofertilizers for a sustainable remedy and as substitute for chemical fertilizers which have propensity to influence human health, water, air, and soil. They also stated biofertilizers have high nutrient values as well as houses microbes like rhizobacteria that functions in the bioavailability of nitrates to soil for plant physiology. In addition, the biofertilizers also enhances the efficacy of convention fertilizers by utilizing a carrier substance on which the microorganism is straddled. Products from these bio-chemical fertilizers can last between 6 months to 2 years on both in the liquid and solid phase which can also be used as feed for aquatic biota like fish. In conclusion, the authors stated that a part from the immense benefits derived from biofertilizer use, there are also some limitations in their utilization, in that, producers might encounter some problems at the course of manufacturing and there might be probable health risk from sudden chances of pollutant like heavy metals during the production process. That if these problems can be by-passed, pesticides and biofertilizers can be solution of the contamination faced by the conventional fertilizers. In order to boost agricultural output and quality, fertilizers especially the chemical ones play a pivotal function in the process. However, the extensive use of them portends great danger to soil function, plant physiology, and human health. To solve this problems, an eco-friendly and sustainable alternative is highly needed. On this ground, Ye et al. [22] tested and evaluated the potential of substituting chemical fertilizers for bioorganic fertilizer in enhancing so the fertility of soil and the yield of tomato quality. A field setting was carried on tomato plant with amalgam of bioorganic fertilizers with Trichoderm sp, using four treatments: CF (chemical fertilizer) as control, TSS (Trichoderma spore suspension), OF (organic fertilizer), and BF (bioorganic fertilizer). The outcome from their study showed that there was accumulation of nitrate, vitamin C, and soluble sugar of percentage 62, 57, and 24 respectively, of the bioorganic fertilizer treatment in the tomato plants in comparison to the untreated. The results from the field and pot trials showed a reduction in the proportion of

298  Biofertilizers bioorganic and chemical fertilizers on tomato yield and were equal to what was obtained in hundred percent chemical fertilizer alone. But, the use of Trichoderma spore suspension and with the combination with organic fertilizer was observed to have decrease the yield (9%–35% and 6%–38%) over the control. Subsequently, there was a rise in the microflora level in the soil as well as the improved soil fruitfulness spontaneously showing a positive direct association specifically in the biofertilizer treated soils. The authors in conclusion stated that the efficiency of the bioorganic fertilizers for upholding a balance tomato production and enhancing their quality was as a consequence of soil and microorganism activities. Therefore, from their findings, the authors suggested Trichoderma bioorganic fertilizer might be utilized in the amalgam with specific conventional chemicals to achieve a maximum beneficial output of crops and for a sustainable future farming tool.

9.4 Specific Examples of a Biofertilizer for Crop Improvement in Agricultural Sector Saeed et al. [16] in a biological experiment, tested and evaluated the potential influence of biofertilizer and chemical fertilizers on the yield and growth of Cucumis sativus under a greenhouse setting. Four duplicates (control = T1, biofertilizer (Azotobacter) = T2, chemical fertilizer = T3, and combination treatment: half chemical and biofertilizer = T4) under RCBD (Randomized Completely Blocks Design) settings were utilized for this study. The outcome from their biological controlled experiment showed that there was a p < 0.05 (significant difference) between the use of chemical fertilizer and the biofertilizer for the yield constituent traits and the yield of the plant using a DMRT (Duncan Multiple Regression Test). The amalgam of chemical fertilizer and biofertilizer had important influence on the growth characters and yield of Cucumis sativus. The outcome from the relationship analysis revealed a string association between total Cucumis sativus weight and Cucumis sativus yield per plant at r = 0.89. A stepwise regression evaluation further showed that the yield of Cucumis sativus showed that the individual Cucumis sativus weight can be validate 50.9% of the yield differences. The findings from their biological controlled experiment showed that biofertilizer increased the yield constituent traits and total yield of Cucumis sativus significantly. In recent years, the concept of organic farming is gaining energy over the convention type consequent of the adverse impact of chemical use on the ecology and health of the soil and biota. Mahdi et al. [13] in a review,

Biofertilizer Utilization in Agriculture  299 looked at the possibility of using biofertilizers in organic farming. The authors stated that organic farming makes the farming system sustainable for both the present and future users based on its synergy with nature. That the soil ecosystem needs to be revived for both biotic and abiotic functioning. They also recounted that biofertilizers show an significant function toward sustaining soil fruitfulness and the maintenance of the nitrogen contents therein, thus assembling the micro- and macronutrients and the conversion of the soil phosphate and make them bioavailable to plants in order to enhance their availability and efficiency. The authors also reported the biofertilizer is a promising tool for a sustainable agriculture over the chemical ones, because of the negative impact it portends to both soil, soil microfauna, and humans at the top food chain level. More so, the association of the mycorrhizal found in the biofertilizer as well as the soil increases the perfect soil function and productivity/area, thus increasing the soil structure, nutrient uptake via increase in the nitrogen, and phosphate level, and alleviating soil and plant toxicity by production of Glomulin a protein component. They pinpointed that a new biofertilizer in form of a liquid has been proven more effective over the convention biofertilizer which is a breakthrough in the biotechnology area of biofertilizers. In conclusion, the authors recommended that this novel biofertilizers should be generally accepted by commercial: biofertilizer producers, extension workers, as well as the farmers in order to forestall a better agricultural fortified system of effective production of desirable yields. One of the greatest threats to food safety and security is overpopulation. Population can impound on land, thereby making it scare for farm activities. Therefore, there is a clarion need to invest on sustainable organic farming in other to feed the ever increasing populace using effective biotechnological tactic. In light of this, Mahanty et al. [14] in a review, looked at the possible method for a sustainable farming using biofertilizers. They stated that the over use of chemical fertilizers in recent times have not only helped in the boosting of crop production but as well impact negatively on the land use and the biota in agroecosysytem. However, the way forward is by utilizing biofertilizers for an effective and sustainable agriculture. That microorganisms like cyanobacteria, fungi, and bacteria have been known to promote plant growth as well as development of essential parts. In addition, they stated that studies have shown that biofertilizers have the ability of providing nutrients to plants in enormous amount that can elicit the yield of crops. The mechanisms of action using biofertilizers in eliciting plant development as well as growth were discussed. Highlights on the role of biofertilizers in various sectors like ecology and bioremediation were also discussed.

300  Biofertilizers Itelima et al. [8] in a review, looked at the major role of biofertilizers in terms of enhancement of crop productivity and soil fertility. They recounted that plant plays a major role in ecosystem restoration a part as been used as food. That nutrients derived from plants are very important for the sustenance of the world’s population. Hence, there is the need to harness several soil management methods in order to sustain plant continuous availability using biotechnology techniques. They reported that, of recent, the dire need to substitute the chemical fertilizer for biofertilizer is on the increase, because of the health and ecological concerns it portends. That biofertilizers are known for their notable function: enhancement of crop production and soil fruitfulness. The manipulation of microorganisms as biofertilizers in recent times has become very vital in the agrosector based on their possibility in sustainable farming and food safety, the authors opined. Microbes usually utilized for such purpose are cyanobacteria, fungi, ecto and endo mycorrhizal, rhizobacteria, and nitrogen fixing bacteria. In addition to their aforementioned functions, biofertilizers are also used to combat abiotic and biotic stressors acting as stress tolerance as well as enhance nutrient and water uptake in plants. In conclusion, they recommend biofertilizers as first class tiers in the management of soil functions and plant physiology. That they are cheap, eco-friendly, and non-toxic for organic farming activities. Kumar et al. [9] in a review, looked at the role and function of biofertilizers as sustainable tool in agriculture. The authors stated that population increase serve as a potential threat to food safety and security in terms of land scarcity and high demand for food. They pointed that the impacts of chemical fertilizers on the ecosystem and its over dependence of recent are alarming. Therefore, there is the need to improve the productivity and output of agricultural produce using effective biotechnology mechanism like biofertilizer to meet the demand of the evolving population in the nearest decades. They recounted that biofertilizers have several benefits over the chemical fertilizers which have make them good substitute over the latter. They stated that biofertilizers comprise of microbes which aid in the effective uptake of nutrients from the soil to the plants as well as the regulation and development of crop physiology. That fertilizers gotten from amalgam with microbes (biofertilizers) have come to stay as important sustainable tool for farming activities in the general sustainability and fertility of plant production. Mazid and Khan [15] in a review, looked at the prospects in the utilization of biofertilizers for farming in Indian. In the past times in India, chemical fertilizers use, affordability, and availability in farm activities have been warranted via subsidies and import. Of recent, biofertilizers

Biofertilizer Utilization in Agriculture  301 have materialized as a very effective substitute to conventional fertilizers because they are cheap, non-noxious, easy to be utilized, and eco-friendly. In addition, they act as additives to farm chemicals as well make nutrient available to plants in abundant ratio. They recounted that biofertilizers have come to stay as a promising commercial tool in the boosting of plants fruitfulness and development. The possible failures and successes in the utilization of biofertilizers were discussed. In conclusion, the authors recommended public enlightenment packages to improve the way farming activities are been done in the past in a sustainable pattern and also spur policy architects and private business to show more interest in this novel area of biotechnology. Fertilizers, herbicides, and pesticides have been known to cause deleterious influence when applied in the wrong proportion or over used persistently in the agroecosystem. Chemical constituents such as radicals from sulfuric and HCL acids sourced from artificial fertilizers increases negatively the level of acidity in the soil as well as impact on the physiology of the crops. Alori and Babalola [1] did a review of the use of inoculants made of microbes in the enhancement of crop worth and the health of humans. The authors stated that theses inoculants can act on biocontrol, biopesticides, bioherbercide, and biofertilizers as messengers which can be used to control weeds, disease, and pests, as well as improve the growth, health, and productivity of plant. They reported that the inoculants from microbes which include strain of algae, fungi and bacterial, are sustainable to use, cheap, and environmental friendly.

9.5 Management of Biotic and Abiotic Stress Bhardwaj et al. [4] did a review of the potential signature of biofertilizer as sustainable farming ad for the enhancement of crop fruitfulness, tolerance, and yield. They said that chemical fertilizers have resulted to serious problems to the health and ecology of soil, plants, and humans. These have warranted serious soil strategy management in the exploitation of novel, cheap, non-noxious, and environmentally friendly means such as biofertilizers of farming in order to provide sustainable production of crops and food security. The recounted some of the microorganisms use as biofertilizers such as cyanobacteria, ecto- and endomycorrhizal fungi, and PGRPs (plant growth-promoting rhizobacteria) and some other microbes that are beneficial which can enable plant withstand ecological stress, spur plant growth, and elicit nutrient uptake. The authors also highlighted the facilitated role of biofertilizers as functional personalities in the triggering of

302  Biofertilizers plant hormones, special defense against diseases, nutrient profiling, and so on. In conclusion, they recounted that knowledge achieved in the utilization of biofertilizers should be put into the reduction of issues provoked by the conventional fertilizers. Nowadays in the world, sustainable means of agriculture is trending. However, the growth and productivity of plant is been affected by certain ecological stressors. Selim and Zayed [17] in a review, looked at the sustainable function of biofertilizers in the management of ecological (biotic and abiotic) pressures in agriculture. They stated the living and nonliving (biotic and abiotic) stresses plants face in their thriving for survival. Examples of the living factors are humans, insects, plants, and pathogenic microbes, whereas the non-living are mineral nutrients, heavy metals, powerful light, wind, varied temperature, drought, waterlogging, and saline soil. They reported that most of these non-living factors are occasioned by climate change which can result to reversion of crop fruitfulness. That microorganisms that dominate the root of plants which are beneficial stimulate the plant to respond positively to ecological stress. However, different microbial strains have varied tolerance limits. Theses microbes are used as supplements in fertilizers called biofertilizers which can also fix and discharge siderophores for dinitrogen and Fe in plants, increase the nutrient contents via phosphate solubization, and enable polysaccharides and water uptake in the roots of plants and stimulation of phytohormones production and growth in plants. In conclusion, the authors stressed the importance of using microbial inoculants as a next generational biofertilizers in combating various ecological stresses. Chavoshi et al. [5] tested and evaluated the potential impacts of bioferilizers in remediating water stress of Phaseolus vulgaris in a field irrigation setting. Four subplots cases comprising of bacteria in soil, KSB (potassium solubilizing bacteria), PSB (phosphorus solubilizing bacteria), and control were used in the biological tested experiment. The outcome of the biological experiment showed that biofertilizers and the halt irrigation had significant collaboration on the biomass of Phaseolus vulgaris as well as high biomass (7,978 and 8,023 kg/ha) when PSB and KSB were applied, respectively. However, the utilization of biofertilizers as an alternative to the chemical fertilizers resulted to higher efficiency of water in reduced water settings. The final results of their experiment showed a high bio of 7,985 kg/ha noted in the biotic management of Phaseolus vulgaris under enough water. In conclusion, the authors reported that at the flowering stage, halt irrigation was boosted by the presence of Bio-K Bio-P fertilizers use which moderated and mitigated biomass reduction to almost 37%.

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9.6 Combinatory Effect of Biofertilizer With Other Substance and Their Effect on Crops Pereira [19] tested and evaluated the impacts of biofertilizers and biostimulants in the abatement of water stress and nutritional worth of Spinacia oleracea L. Four different types [TA (Twin Antistress), V (Veramin Ca), AM (Aminovert), and MEG (Megafol)] of biostimulants and two genotypes of spinach (Viroflay and Fuji F1), respectively, were employed in this study in two [W− (H2O-holding) and W+ (normal)] irrigation systems. The outcome from their study showed that the carbohydrate and fat contents were elevated under H2O stress when VW+ (Veramin) and MEGW+ (Megafol) were used on Fuji F1, whereas there was an increased in the calorific value when treated with MEGW+. In disparity, the ash and protein contents were elevated in the addition of TAW+ and AMW− on Viroflay. The richest sugar were observed to be glucose and Ra-nose; in addition, fructose and sucrose were observed to be the highest on Fuji F1 when Aminovert +, and TAW and CW were applied. Malic, oxalic, and organic acid contents were elevated in both examined plants correspondingly. VW− and MEGW− were observed to induced the level of α-tocopherol biosynthesis, whereas, the α–tocopherol level was elevated by AMW+ in Fuji plants. Linoleic and linolenic acids, the only fatty acids, were identified in an increasing level in TAW+, AMW+, and AMW− and in CW+, VW−, and AMW−, in the former and latter, respectively. 5,30,40-Trihydroxy-3methoxy-6:7-methylenedioxyflavone-40-Glucuronide, the phenolic compound at peak 12, was the most common compound in Viroflay in normal irrigation when CW− was added. The cytotoxic action and antioxidant of the samples tested did not reveal any capable results when liken to the +controls. In the other vein, the antibacterial action was noted based on the genotype, irrigation regime, and biostimulant tested. In conclusion, the authors stated that the differences of the impacts of the tested irrigation regimes and biostimulants were noted on chemical configuration and bioactive properties of both on the genotypes that highpoints the necessity for more research to be done in order to proffer possible abatement of water stress with biofertilizers and biostimulants. Mona et al. [20] tested and evaluated the potential impact of mineral N2 and bio fertilizers in eliciting various quality, yield, and growth in maize plants in a field setting. The biological controlled experiment consists of triplicates of factorial plan. Cultivar of Zea mays Giza10 were inseminated with NH4SO4 at different concentrations: 50, 100, and 125 kgfed−1 and treatments of strains of azotobacter at concentrations: 0, 5, and 10

304  Biofertilizers kgfed−1. The outcome from their study revealed that the biofertilizer and mineral N2 elicited the growth plant when compared with mineral N2. The maximum values of the morphometric and physiological parameters (dry weight, length, yield, height, area, and chemical level) of the plant’s grains and leaves were gotten by the utilization of 5 Kg/fed of azotobacter and 100 Kg/fed of NH4SO4 as when compared to the treatments. In conclusion, the authors stated that biofertilizer-azotobacter application at specific concentration aided in higher yield and quality of Zea mays. That biofertilizers can be used to lessen the cost of production of mineral fertilizer in in the next generation. Chemical fertilizers have been persistently in the increase of plant productivity in agriculture for many years. The incidence of ecological damage has been of great concern because of the potential health risk it portends. However, the utilization of biofertilization using specific microbes has been of recent in the limelight of abating chemicals in agroecosysytem. Sharma et al. [18] tested and evaluated the efficacy of varied N2 fixing bacteria with reference to the development and growth of leguminous plant by using Azotobacter and Rhizobium biofertilizers on Phaseolus vulgaris and Dolichos lablab in a controlled setting. Three treatments groups (Azotobacter (50%) + Rhizobium, Azotobacter only and Rhizobium only) including control were used in the biological controlled experiment. The outcome from their study after 1 month showed a 15% rise in the SFA (Saturated Fatty Acid) in Phaseolus vulgaris as well as a maximum constancy of secondary metabolites. They also reported that Azotobacter had a better impact on the plants when biofertilizers was applied. In conclusion, the authors recounted that Rhizobium and Azotobacter were in effect on the legumes toward the vigor and growth activities. That they also provided N2 fixing capability and protection against reactive oxygen species. Finally, they recommended both Rhizobium and Azotobacter biofertilizers for a large commercial production because of what they portend for agricultural purposes. Xu and Geelen [21] did a review of the utilizing of biostimulants from wastes and agricultural food as an eco-friendly approach in boosting crop fruitfulness and growth of plants (biofertilizers). The authors stated that modern farming seek to prom different ways of boosting crop production to meet the ever teeming populace. That the over reliance of chemical fertilizers which have done more harm compared to the advantages is alarming. However, a sustainable approach in the combing of biostimulant with biofertilizers in farming is the way forward in the quest for food security. They stated that biostimulants are natural substances that aid in the boosting of nutrient uptake and plant yields. They reported that wastes that are derived

Biofertilizer Utilization in Agriculture  305 from farm leftovers can serve as a media of biostimulating components. Apart from that, chistosan by-products, protein hydrolysate, derivatives of sludge municipal wastes, and vermin-compost. In conclusion, they stated that biostimulants are promising assets for future sustainable farming. The demand for food globally to meet the ever growing population as resulted to making alternatives in boosting the production farm produce. In line with this, Bargaz et al. [3] did a review of the integration of biostimulants (soil microbial resources) with biofertilizers in the improvement and management of farming efficiencies. The authors stated that modern farming has to be more eco-friendly, sustainable, and more fecund. That macronutrients like sulfur, potassium, phosphorus, and nitrogen and beneficial microbes have contributed immensely in the direct and indirect improvement of crops and the efficiency of fertilizers. They recounted that bioformulations from microbes can elicit the performance of plants. Bioformulations act as synergistic and synergistic influence with mineral fertilizers as co-amalgam in boosting fertility and yield of plants. The combine utilization microorganisms such as phosphate mobilizing bacteria, phosphate solubilizing bacteria, and N2-fixing bacteria with mineral properties are novel areas of biotechnology in farming. That microbial preparations are highly well-matched with mineral responses and have positive influence on the agroecosystem. In conclusion, they recommend continuous plant-soil management systems by testing, developing, and designing more novel ways in the biological enhancement of agriculture.

9.7 Conclusion and Recommendation to Knowledge This chapter has provided a detailed information on the practical utilization of biofertilizer as a sustainable biotechnological tool that could led to increase in agricultural production. Details information were provided on the modes of action of these biofertilizer while specific examples of cases where biofertilizer has been utilized for improving increase in agricultural production were also discussed. Recent advances in the application of microbial inoculum as a sustainable technology that could improve plant health were also provided. The application of genetic engineering, synthetic biology, nanotechnology, and bioinformatics could lead to increase in the production of biofertilizer. Moreover, the application of next generational sequencing and whole genome sequencing will play a crucial role toward the identification of significant gene of interest that could led produce high level of phosphorus and play a crucial role in the solubulization of complex phosphorus in the soil.

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References 1. Alori ET and Babalola OO. Microbial Inoculants for Improving Crop Quality and Human Health in Africa. Front. Microbiol. 9:2213, 2018. 2. Ajmal M, Hafiza Iqra Ali, Rashid Saeed, Asna Akhtar, Muniba Tahir, Muhammad Zain Mehboob and Aneesa Ayub 2018. Biofertilizer as an Alternative for Chemical Fertilizers. Research & Reviews: Journal of Agriculture and Allied Sciences. RRJAAS| Volume 7 | Issue 1 | January 2018. 3. Bargaz A, Lyamlouli K, Chtouki M, Zeroual Y and Dhiba D. Soil Microbial Resources for Improving Fertilizers Efficiency in an Integrated Plant Nutrient Management System. Front. Microbiol. 9:1606, 2018. 4. Bhardwaj D, Ansari MW, Sahoo RK, Tuteja N1. Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity. Microbial Cell Factories, 08 May 2014, 13:66, 2014. 5. Chavoshi, S., Gh. Nourmohamadi, H. Madani, H. Heidari Sharif Abad and M. Alavi Fazel. ‘The effect of biofertilizers on physiological traits and biomass accumulation of red beans (Phaseolus vulgaris cv.Goli) under water stress’. Iranian Journal of Plant Physiology, 8 (4), 2555-2562, 2018. 6. Carvajal-Muñoz J. and García CEC (201). Benefits and limitations of biofertilization in agricultural practices Livestock Research for Rural Development 24(3) 7. Girigiri, B., Ariole, C. N. and Stanley, H. O. ‘Bioremediation of Crude Oil Polluted Soil Using Biofertilizer from Nitrogen-fixing and Phosphatesolubilizing Bacteria’, 5(4), pp. 27–38, 2019. 8. Itelima JU, Bang WJ, Onyimba IA, et al. A review: biofertilizer; a key player in enhancing soil fertility and crop productivity. J. Microbiol. Biotechnol. Rep., 2(1):22-28, 2018. 9. Kumar MS, G Chandramohan Reddy, Mamta Phogat and Santosh Korav. Role of bio-fertilizers towards sustainable agricultural development: A review. Journal of Pharmacognosy and Phytochemistry; 7(6): 1915-1921, 2018. 10. Ma, H. et al. ‘Bioremediation of cadmium polluted soil using a novel cadmium immobilizing plant growth promotion strain Bacillus sp. TZ5 loaded on biochar’, Journal of Hazardous Materials. Elsevier B.V., (November 2019), p. 122065, 2020. 11. Mahanty, T., Bhattacharjee, S. and Goswami, M. ‘Biofertilizers: a potential approach for sustainable agriculture development’, Environmental Science and Pollution Research. 2016. 12. Mir, S., Sirousmehr, A. and Shirmohammadi, E. ‘Effect of nano and biological fertilizers on carbohydrate and chlorophyll content of forage sorghum (Speedfeed hybrid)’, 6655, pp. 157–164, 2015. 13. Mahdi S.S, G. I. Hassan2, S. A. Samoon3, H. A. Rather4, Showkat A. Dar5 and B. Zehra6. Bio-Fertilizers in Organic Agriculture, Journal of Phytology, 2(10): 42-54, 2010.

Biofertilizer Utilization in Agriculture  307 14. Mahanty T, & Surajit Bhattacharjee & Madhurankhi Goswami & Purnita Bhattacharyya & Bannhi Das & Abhrajyoti Ghosh & Prosun Tribedi. Environ Sci Pollut Res. Biofertilizers: a potential approach for sustainable agriculture development. Environ Sci Pollut Res Int., 24 (4), 3315-3335 Feb 2017. 15. Mazid M and Khan TA Future of Bio-fertilizers in Indian Agriculture: An Overview International Journal of Agricultural and Food Research, Vol. 3 No. 3, pp. 10-23, 2014. 16. Saeed KS, Sarkawt Abdulla Ahmed, Ismael Ahmaed Hassan and Pshtiwan Hamed Ahmed, Effect of Bio-fertilizer and Chemical Fertilizer on Growth and Yield in Cucumber (Cucumis sativus) in Green House Condition. Pakistan Journal of Biological Sciences, 18: 129-134, 2015. 17. Selim Sh M and Zayed Mona S. Role of Biofertilizers in Sustainable Agriculture Under Abiotic Stresses, 2017. 18. Sharma J, Tapasuita Gurung, Krittika Nandy and A.K, Mitra. Efficiency of different nitrogen fixing bacteria with respect to growth and development of legumes. Int.J. Curr. Microbiol. App. Sci. 3(10) 799-809, 2014. 19. Pereira C, Maria Inês Dias, Spyridon A. Petropoulos, Sofia Plexida, Antonios Chrysargyris, Nikos Tzortzakis, Ricardo C. Calhelha, Marija Ivanov, Dejan Stojkovi´c, Marina Sokovi´c, Lillian Barros and Isabel C.F.R. Ferreira, The Effects of Biostimulants, Biofertilizers and Water-Stress on Nutritional Value and Chemical Composition of Two Spinach Genotypes (Spinacia oleracea L.). Molecules 24, 4494, 2019. 20. Mona E. El- Azab* and Camilia Y. El-Dewiny. El-Azab and El-Dewiny, Effect of bio and mineral nitrogen fertilizer with different levels on growth, yield and quality of maize plants JIPBS, Vol 5 (2), 70-75, 2018. 21. Xu L and Geelen D. Developing Biostimulants From Agro-Food and Industrial By-Products Front. Plant Sci., 30 October 2018 | https://doi. org/10.3389/fpls.2018.01567, 2018. 22. Ye L, Xia Zhao, Encai Bao, Jianshe Li, Zhirong Zou & Kai Cao. Bio-organic fertilizer with reduced rates of chemical fertilization improves soil fertility and enhances tomato yield and quality. Scientific Reports volume 10, Article number: 177, 2020. 23. Hindersah, R., Nurhabibah, G., Asmiran, P., and Pratiwi, E. Antibiotic Resistance of Azotobacter Isolated from Mercury-Contaminated Area. Journal of Agricultural Studies. Vol 7, No 3, 2019. 24. Franchi, E., Rolli, E., Marasco, R., Agazzi, G., Borin, S., Cosmina, P., … Petruzzelli, G. Phytoremediation of a multi contaminated soil: mercury and arsenic phytoextraction assisted by mobilizing agent and plant growth promoting bacteria. Journal of Soils and Sediments, 17(5), 1224–1236, 2016. 25. Jiao, H., Luo, J., Zhang, Y., Xu, S., Bai, Z., and Huang, Z. Bioremediation of petroleum hydrocarbon contaminated soil by Rhodobacter sphaeroides biofertilizer and plants. Pak. J. Pharm. Sci., 28 (5), 1881–1886, 2015.

10 Azospirillum: A Salient Source for Sustainable Agriculture Rimjim Gogoi1, Sukanya Baruah2,3 and Jiban Saikia1* Department of Chemistry, Dibrugarh University, Dibrugarh, India 2 Vivekananda Kendra Vidyalaya, Laipuli, Tinsukia, India 3 Department of Chemistry, Silapathar College, Dhemaji, India

1

Abstract

Azospirillum is the most studied free-living nitrogen fixing bacterial genus among the plant-growth promoting rhizobacteria. It has various plant-growth stimulatory effects, governed by specific mechanistic routes. The association of this microorganism with the plant root system is highly interesting, and comprehensive studies are going on about it. Its survival in rhizosphere and bulk soil is a matter of concern to perform its anticipated effects in the first place. Along with agricultural applications for sustainable development, this organism shows usability in inhibiting accumulation of various hazardous substances in the plant roots. Thorough study on its genomic structure should be carried out to have a better understanding of its mechanistic pathways to expand its range of application. For enhanced large-scale applications of Azospirillum spp. as commercial inoculants, it is desirable to access improved methodologies, more funding, extended awareness and knowledge for production, and proper handling of bio inoculants with quality. Keywords:  Azospirillum, rhizobacteria, nitrogen fixing, plant-growth promotion, sustainable agriculture, bio inoculants

10.1 Introduction The global population is increasing in an exponential manner and estimated to reach 8 billion by 2025, according to the report of Food and *Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (309–334) © 2021 Scrivener Publishing LLC

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310  Biofertilizers Agricultural Organization [1]. This demands a comparable escalation in the global food production. As the cultivable land worldwide is declining with ever increasing population and rate of urbanization, the anticipated development in global agricultural yield has to be governed with an increase in food production in existing cultivable land, by employing advanced means [2]. This further exaggerates the dependence on chemical fertilizers, for increased food yield. By 2030, the extent of consumption of chemical fertilizers is estimated to rise by 54.6% [3]. However, this does not signify an increased uptake of nutrients by the plants, in fact, a major portion of the applied chemical fertilizers, especially Nitrogen fertilizers, is lost to the environment by “denitrification” and “leaching” [4], which is uneconomical, as well as hazardous to the environment. In order to minimize the precarious impact on the environment and for long-term food production, sustainable agricultural practices need to be employed [5]. In such a scenario, organic farming is gaining favorable attention in the last decades, in correspondence with growing health awareness and environmental safety precautions [6]. The application of microbial inoculants as bio-fertilizers is considered to hold crucial role, in this context, initiating a “microgreen revolution” [5]. The presence of an active community of soil microbes, such as bacteria, algae, fungi, and protozoa, residing in the rhizosphere, affects the growth and yield of crop plants [9]. The concept of rhizosphere was originally described by Hiltner, representing an “active layer of soil microbes in the area surrounding plant roots, which are activated by root products” [9]. Its composition is different from the rest of the soil, because of the root activity [8]. Among the different soil microbes present in the rhizosphere, the contribution and desired effect of plant growth-promoting rhizobacteria (PGPR) have been gaining enormous attention [6–9]. The PGPR are bacterial species, residing in the rhizosphere, which exhibit growth promoting properties of the plants and their roots by direct or combined mechanisms [5, 6, 9]. The most abundant microbial community present in the rhizosphere is bacteria, the genera Bacillus and Pseudomonas being the dominant ones [9]. Some PGBR contribute in plant growth directly by synthesizing and secreting growth stimulating substances, delivering readily available essential nutrient elements by the process of biological nitrogen fixation (BNF), phosphate solubilization, etc. Some PGPR promote plant growth by indirect ways, including prevention of hazardous effects, caused by plant pathogens [8], which causes leakage of nutrients [9]. Among the PGPR, the bacteria responsible for BNF are termed as “diazotrophs” [10], they convert elemental nitrogen to plant usable organic forms. Rhizobia are the most studied diazotrophic PGPR [11]. They fix atmospheric nitrogen by symbiosis with root legumes and actinomycetes [10, 12]. However,

Azospirillum for Sustainable Agriculture  311 in case of non-legumous plants, primarily forage and grain grasses are the main source of food in modern world [10]. Azospirillum serves as the principal alternative to the legume-Rhizobium symbiosis [11].

10.1.1 The Genus Azospirillum The genus Azospirillum is the second most studied and utilized diazotrophic PGPR after Rhizobium [5, 12, 14, 15]. The term “Azospirillum” refers to a small nitrogen spiral, with French-noun “Azote”, which means Nitrogen and Greek-noun “Spira”, indicating a spiral. The first Azospirillum species was reported by Beijerinck in 1925 from the Netherlands and was named Spirillum lipoferum, which, on revision of classification, was renamed by Tarrand et al. as Azospirillum lipoferum, indicating a fat bearing spiral [10, 14]. The genus was brought to attention when Dobereiner and Day isolated another species in 1974 from Brazil and named as Azospirillum brasilense [5, 10, 16]. The species name “brasilense” originated from “Brazil”, as it was isolated there [10]. The genus Azospirillum belongs to the Group 1, α-subclass of Proteobacteria, with order Rhodospirillales and family Rhodospirillaceae [16]. They are free-living, symbiotic, gram-negative, microaerophilic bacteria, having nitrogen-fixing ability without nodule formation. They are physically vibrio- or spirillum-shaped rods with diameter of 1 µm and length of 2–3 µm, which produces polar and peritrichous flagella [5, 8, 10, 13, 17]. Twenty species of Azospirillum genus are known till date (DSMZ 2018) [5]. Azospirillum brasilense and Azospirillum lipoferum are the two species of Azospirilla, on which most studies and researches have been conducted [10, 17, 18]. The third species of Azospirillum genus, Azospirillum amazonense, was reported in the year 1983 and was isolated from forage grass of Amazonian region [17, 19]. Another species of the genus was isolated from the paddy soil of a rice plant in China and later classified it as Azospirillum oryzae [17]. Then, Azospirillum halopraeferans was isolated from kellar grass in Pakistan. Azospirillum irakense was isolated from rice plants in Iraq [17]. In 1997, Ben Dekhil et al. suggested the conversion of the subspecies “Conglomeromonas largomobilis sp. Largomobilis” to the genus Azospirillum as Azospirillum largomobile, due to the pronounced similarity of the species to Azospirillum lipoferum and Azospirillum brasilense, which was then renamed as Azospirillum largimobile (Sly and Stackebrandt, 1999) [5, 10, 17]. In 2001, another Azospirillum species was described and classified as Azospirillum dobereinerae, in honor to Dr. Dobereiner, who familiarized the studies regarding the genus in Brazil [10, 17]. Strains of Azospirillum melinis and Azospirillum palatum were isolated from China. Strains of Azospirillum canadense and

312  Biofertilizers Azospirillum zeae were isolated from Ontario, Canada. Two species of this genus, Azospirillum rugosum and Azospirillum picis, were isolated from Taiwan. The other species of Azospirillum isolated are Azospirillum thiophilum, Azospirillum Agricola, Azospirillum fermentrium, Azospirillum formosense, Azospirillum humicireducens, Azospirillum ramasamyi, and Azospirillum soli [5, 20].

10.1.2 Properties of Azospirillum spp. Motility is an important taxonomic characteristic of the Azospirillum genus [21, 22]. This assists the bacterial cells to move toward abundant nutrient sources [7]. The Azospirillum cells are highly motile in liquid media, due to the presence of “single polar flagellum”, enabling the cells to swim [21–23]. The genus exhibits a versatile carbon and nitrogen metabolism [7]. The cells consume a variety of salts of organic acids, such as malate, oxalate, succinate, lactate, pyruvate, etc., and some sugars, such as fructose, glucose, and amino acids as the carbon sources, available in the rhizosphere and microbial sources [16, 21, 24]. Azospirillum irakense is the only species of Azospirillum genus, which has the ability to grow on pectin, and Azospirillum amazonense is the only species, able to survive on sucrose as the only carbon source [23, 24]. Ammonium ion, nitrate ion, nitrite ion, amino acids, and atmospheric nitrogen serves as the nitrogen source for the bacterial cells [7, 23]. The Azospirillum cells exhibit certain specific properties.

10.1.2.1 Chemotaxis Chemotaxis is the process of driving the bacteria to sense and move toward the most favorable niche, rich with the nutrient sources [7, 22]. This property helps the bacterial cells survive in the rhizosphere, amidst the plant-microbe interactions [7, 16, 22, 23]. Signal transduction system aids the cells to sense and adapt to these alterations by regulation of gene manifestation or modulation of the swimming pattern [8]. This chemotactic behavior is observed in Azospirillum spp. to several amino acids, monosaccharides, and disaccharides and various organic acids, exuded by plant roots [12, 23]. This property not only depends upon the attractant organic compounds, but also the motility of the bacterium [7]. In Azospirillum species, the chemotaxis was observed to be strain-specific. Plants such as wheat, rice, cotton, and sunflower use C3 cycle or the Calvin cycle for carrying out the dark reaction of photosynthesis, whereas plants such as kellar grass, sugarcane, and sorghum use C4 cycle or the Hatch-Slack pathway. The first category of plants is termed as the C3 plants, whereas the second

Azospirillum for Sustainable Agriculture  313 one as the C4 plants. While carrying out a study, it was observed that strains of Azospirillum isolated from C3 plants exhibited strong attraction toward malate, not to oxalate. In this case, malate is the major organic acid exuded by the C3 plant roots. These plant roots were observed to be preferentially colonized by Azospirillum brasilense. On the other hand, in case of the C4 plants, where oxalate is the major organic acid exuded, the strain isolated there performed intensive adherence to the oxalate. In this case, the plant roots were observed to be preferentially colonized by Azospirillum lipoferum [12, 16]. The principal chemotaxis pathway in Azospirillum is shown to be dependent on “proton-motive force-sensing”. This was established by the fact that changes in chemotactic behavior were directly related to changes in membrane potential, which is the prime constituent of “protonmotive-force” [21]. The chemotactic pattern in Azospirillum cells directs the host specificity in colonization by the Azospirillum species [12].

10.1.2.2 Aerotaxis Aerotaxis is the property specifically demonstrated in the genus Azospirillum, by virtue of which the Azospirillum bacterial cells move toward optimal oxygen concentration [7, 21]. This behavior aids the bacterial cells to be well adapted and versatile, prolonged-period survival, competing with other microorganisms, and thriving to carbon-rich and nitrogen-poor locations [12]. The rhizosphere pertaining low oxygen availability, creates a gradient, which acts as one of the principal stimuli in drawing the bacteria to the plant roots. This proves to be advantageous in guiding the bacteria to suitable destinations for fixation of atmospheric nitrogen [7, 21].

10.1.2.3 Formation of Cysts and Aggregates or Flocs In adverse conditions, such as withering and shortage of nutrients, Azospirillum cells, primarily vegetative Azospirillum cells, possess the ability to transform into inflated “cyst-like” forms. These forms are non-motile, extremely refractile, accompanied by the development of an outer-coating of polysaccharides and accretion of abundantly present poly-β-hydroxybutyrate (PHB) granules. The cells synthesize and store the polymer inside the cells. This formation is significant, because, in unfavorable starving conditions, the PHB granules act as carbon and energy sources for growth, proliferation, and nitrogen fixation [12, 16, 21, 22]. These are considered real cysts, as they highly resist withering, ultraviolet, and gamma radiation. Thus, the cyst formation helps the Azospirillum cells to survive under stressful conditions.

314  Biofertilizers Furthermore, Azospirillum cells possess the ability to aggregate, forming visible flocs, accompanied by loss of motility, under certain adverse physiological conditions, such as states with high pH, high carbon-tonitrogen ratio, which is unfavorable for growth of the bacteria. The flocs produced perform greater resistance to drought condition, compared to the free cells. These bacterial aggregations are formed by the congregation of the cells, forming stable, adjacent, and multicellular association. The natural polymers like polysaccharides and polyaminoacids, which are excreted at the bacterial cellular surfaces, are involved in cellular aggregation. These polymers have suitable length to form bridges between the microbial cells. The cellular aggregation taking place is highly specific and is reversible. These properties result from the high affinity existing between the complementary surfaces [10, 16, 21]. In general “microbial aggregation” system can be divided into three classes [21]: • A prelude to morphogenesis and differentiation. • A prelude to sexuality and parasexuality. • A mechanism for survival under diverse conditions [21]. Aggregates formed by Azospirillum, along with other free living nitrogenfixing bacteria such as Azotobacter and Klesbiella, are included in the third category. It has been established that the Azospirillum aggregates remain entrapped beneath the fibrillar material, associated with the plant roots [16, 21]. The principal existing interactions causing cell-to-cell adhesion can be Van der Waals interactions, hydrophobic interactions, ionic interactions, or hydrogen bonding, varying with the system. The process can be further facilitated by using culture media for growing the bacteria, rich in carbon sources, such as fructose, gluconate, malic acid, and PHB. Arabinose is the only sugar in existence, which has the ability to entirely inhibit the aggregation of Azospirillum brasilense cells [21].

10.1.2.4 Survivability in Rhizosphere and Bulk Soil In the rhizosphere, Azospirillum spp. encompass almost 10% of the total microbial population, whereas in bulk soil and tropical soil, the population declines to 0.2% of the total microbial population. A study revealed the numbers of Azospirillum brasilense to be 300 times higher in the rhizosphere [21]. In bulk soil, the soil water-holding capacity manifests the survival rate of the bacteria. The Azospirillum cells are generally irreversibly adsorbed by the upper surface of soil through a charge-charge interaction. The degree of adsorption is closely associated with the surface area

Azospirillum for Sustainable Agriculture  315 of the soil and the charge of surface particles [16, 22]. The important soil components are clay and organic matter. These strongly affect the extent of adsorption. In this context, it becomes imperative to understand how the charge-charge interaction takes place between the bacterial cell and the surface of clays and organic matter, as they all possess net negative surface charge, thus preventing any contact between the surfaces. This can be explained with the fact that there exist positively charged edges at the clay surface, to which the bacteria get adsorbed. The adsorption was found to be higher in microaerophilic conditions than in aerobic conditions. When a comparison was drawn between the ability of adsorption and motility of the Azospirillum cells, by introducing bacteria attractants, it was observed that the motility caused by the attractants to draw the bacteria was stronger than the bacterial adsorption to the soil. Adsorption of Azospirillum cells to sandy soils was found to be rather weak. A study revealed that Azospirillum brasilense Cd cells were strongly adsorbed to the surfaces of light and heavy-textured soils, but weakly to quartz sand [22]. In a study, it was concluded that the “partial failure” in root colonization after inoculation of Azospirillum spp. may arise when the bacterium is strongly attached to the soil surface, thus leading to poor availability of the cells prior colonization. The other two soil variables, upon which the extent of adsorption depends, are the cation exchange capacity (CEC) and redox potential of the soil. It was demonstrated that the overall percentage of adsorbed cells increases with increasing CEC, whereas the soil redox potential, which affects the extent of adsorption, directly influences the nitrogen fixation ability of the bacteria. The bacterial cells were not found to be homogeneously distributed throughout the soil surface. Their relative distribution is strongly influenced by their comparative movement through the capillaries or pore spaces. The exceptional case was observed for Azospirillum brasilense Cd, which was homogeneously distributed within the soil. Another important fact is that extent of adsorption was found to be higher in case of live bacteria, than dead bacteria. The factors exhibiting adverse effects on bacterial adsorption include agitation, application of ethylenediaminetetraacetic acid (EDTA), protease, bacterial inhibitors, and high temperature exposure. Too low temperature as well inhibits the growth of Azospirillum strains. The duration for survival in Azospirillum brasilense was diminished to a great extent, post removal of the plants, growing in the bacteria residing soil. This is attributed to the declining of nutrient sources provided by the plants for growth and survival of the bacteria. In India, introduction of rice straw to flooded soils was proven to extend the survival period and population of the bacterial cells of Azospirillum.

316  Biofertilizers

10.1.2.5 Competition With Other Soil Microorganisms The survival of the genus Azospirillum in rhizosphere is greatly influenced by another important factor, which is antagonism with other microorganisms, coexisting in the rhizosphere. In temperate and cold niches, the population of Azospirillum spp. was found to be much lower than other rhizosphere microorganisms. In case of nodule forming Rhizobia, the bacteria “infect” and “proliferate” inside the plant root tissue, thereby avoiding competition with other rhizosphere microbes for nutrients [10]. But, for freeliving Azospirilla, the bacteria experience the need to compete with other soil microbes in order to attain nutrient sources. In addition to the competition for nutrient sources, there exists another aspect, displaying competition for survival, as growth of some particular microbes vastly affects the growth of others. This can be understood with the fact that some strains of Azospirillum brasilense and Azospirillum lipoferum synthesize a particular class of compounds, termed as bacteriocins, which inhibit the growth of closely related bacteria in vitro [12]. In soil, the bacteria of the genus Azospirillum was shown to be parasitized by Bdellovibrio sp. and falls prey for the soil protozoa [16, 22]. Again, the strain of Azospirillum lipoferum stimulated the growth of B. japonicum, whereas growth of the B. japonicum strains did not affect the growth of Azospirillum lipoferum strains at all [22].

10.1.2.6 Association With Plant Roots The intimate association between Azospirillum spp. and plant roots is termed as “rhizocoenoses”. The organic compounds, exuded by the plant roots, create a gradient between the roots and their surroundings in soil. Chemotaxis and the motility of the bacteria make the bacteria to travel toward the plant roots, to get access to the root exudates, and to be used as carbon sources [7, 12, 15]. It has been demonstrated by the “in vitro binding assay” that the Azospirillum cells get attached to the plant roots following two phases: Phase I: Adhesion phase In the first phase, adhesion of the singular bacteria cells to the plant roots in a fast relatively weak and reversible manner occurs [7, 14, 16, 23]. It was believed that presence polar flagellum in the bacteria was required for such adhesive process. But, cases were encountered showing adhesion even without the presence of polar or lateral flagella in the bacteria. It was tested and established that exposure to heat or introducing acidic solutions caused disintegration of the bacterial flagella, thereby diminishing adhesion to wheat roots by Azospirillum brasilense [7, 16, 21].

Azospirillum for Sustainable Agriculture  317 Phase II: Anchoring phase In this phase, stable and irreversible anchoring takes place, accompanied by the formation of bacterial aggregates in the plant roots [7, 21], interceded by the formation of extracellular polysaccharides by the bacteria. This process coincides with the method of flocculation of the Azospirillum cells, which are analogous to the fibrillary substance, are formed during association of roots. Biphasic attachment of Azospirillum brasilense to plant root surface is shown in Figure 10.1. The association of the bacterial cells with the plant roots is followed by the colonization of the roots by the bacteria. Generally, bacterial colonization takes place in the root elongation zone [10, 18, 23]. Azospirillum cells possess the ability to colonize the plant roots, both externally and internally. The bacteria form small aggregates in the external colonization. Contrastingly, presence of single cells around the root surface was also observed. The bacteria of the genus Azospirillum, which colonize the live or dead plant roots externally, were found to be entrenched in the mucigel layers of the surface of the roots. The internal colonization has been demonstrated by only a few bacterial species; here, the bacterial cells penetrate into the intercellular spaces of the plant roots, but not through the root hairs. The cells also colonize internally the intercellular portion of the cortex. It has been established that a fibrillary network connects the cells of Azospirillum to each other and to the plant root surface. The specific mechanism of attachment is still not fully understood, but it is generally accepted that the attachment mechanism involves lectin binding, as well as possibility of involvement of agglutinins, found generally in the fibrillary

Phase II: Anchoring phase Slow process, strong, permanent, Fibrillar material

Phase I: Adhesion phase Fast process, weak reversible, Involvement of flagella

extracellular polysaccharides

Chemotaxis

Polar flagellum Lateral flagella Azospirillum towards root Plant cell wall

Figure 10.1  Biphasic attachment of Azospirillum brasilense to plant root surface [7].

318  Biofertilizers substance, assisting the cells to anchorage. The strain specificity also plays important role here in differing the various ways of root colonization. Along with changing bacterial strain, the variance in plant species and environmental conditions also effectively decide the mode of plant root colonization by the bacteria. However, there have been proposed different theories to comprehend the mode of penetration of the Azospirillum cells into the plant roots and subsequent colonization: i.

“Bacterial invasion via disrupted cortical tissues, from where, lateral roots branch out of the main roots.” ii. “Invasion through lysed root hairs and mechanical injuries occurring during plant growth.” iii. “Direct penetration through the middle lamella, following pectinolytic activity.”

10.2 Azospirillum and Induction of Stimulatory Effects for Promoting Plant Growth Inoculation with Azospirillum spp. induces various stimulatory effects in the inoculated plant. It includes healthy growth of leaves, increased dry matter, elongation in root, and improved yield. Increased mineral and water uptake was also observed in Azospirillum inoculated plants [16]. The beneficial effects exerted by the organism were initially believed to be induced by its ability of atmospheric nitrogen fixation [23]. But, it was later established that the contribution of the nitrogen content affixed by Azospirillum spp. is very less, as the plants do not take up major portion of the nitrogen converted by the organism. As a matter of fact, it was the production and secretion of some beneficial organic substances by Azospirillum spp. that actually uplift the overall plant growth [24, 25]. Various effects, combined with specific mechanisms, lead the route to growth promotion. The mechanisms used by Azospirillum spp. in promoting plant growth are discussed below. i.

Nitrogen fixation. It is the principal mechanism followed by Azospirillum, leading to growth induction. Though it has actually very less contribution in plant growth promotion, it still holds crucial importance in supplying the plants with essential nitrogen content. Acetylene reduction assay displayed the assimilation of atmospheric nitrogen into

Azospirillum for Sustainable Agriculture  319 host plants [7, 14, 16, 18, 23]. Azospirillum spp. converts atmospheric nitrogen, which cannot be used up by the plants directly into the plant usable ammonia form in micoaerobic conditions. The enzyme responsible for this action of Azospirillum spp. is nitrogenase. N2 + 8H + 8e– + 16 ATP

ii.

Nitrogenase

2NH3 + H2 + 16ADP + 16 Pi

Production of phytohormones. The plant hormones are termed as phytohormones. They are present in relatively small amounts, but are essential for the healthy growth and sustained metabolism of the plant. They are characterized in five categories: auxins, the most characterized one being indole-3-acetic acid (IAA), gibberellins, abscisic acid, cytokinins, and ethylene [7, 17]. All of these phytohormones contribute in plant regulatory system, enhancing the water and mineral uptake of plants, elongating the plant roots, introducing mechanisms, to defend against soil-borne pathogens. Azospirillum spp. has been demonstrated to produce all these hormones. However, the most studied and characterized one among them all is the auxin IAA. Azospirillum spp. produces IAA in three metabolic pathways; the first one comprises conversion of the precursor tryptophan into indole-3-pyruvic acid, followed by decarboxylation and then subsequent oxidation to finally obtain IAA [17]. The second mechanism involves conversion of tryptophan into indole-3acetamide, followed by hydrolysis to get IAA. The enzymes involved in these transformations are tryptophan-2monooxygenase and indole-3-acetamide hydrolase [17]. The third mechanism involves conversion of tryptophan into IAA, through the formation of tryptamine. IAA contributes in plant growth by regulating cell division and root elongation, tissue disintegration, and metabolism against plant pathogens and also by enhancing plant up take of essential minerals and water. Another important hormone produced by Azospirillum spp. which regulates plant growth is Gibberellins. They contribute in cell division and tissue elongation and stimulate the different stages of plant growth [7, 17, 21, 22].

320  Biofertilizers iii. Production of siderophore. The term “siderophore” is Greek noun, referring to iron carrier. These are low molecular weight compounds having high affinity for iron. Strains of Azospirillum brasilense isolated from strawberry were shown to have the ability to synthesize siderophore in cells. Another species Azospirillum lipoferum displays iron transport system, facilitated by siderophores. The siderophores possess the ability of solubilizing iron in stressed situations [17]. iv. Phosphate solubilization. Phosphate is solubilized in soil by microorganism through the release of phosphatases or formation of organic acids, which, in turn, releases inorganic phosphorus in soil. The species, Azospirillum halopraeferans, and two strains of Azospirillum brasilense, Cd and 8-I, have been reported to solubilize phosphate in soil [17]. v.

Bacterial nitrate reductase activity in roots. This property of Azospirillum spp. enhances the accumulation of nitrate in soil. This contributes to the total nitrogen uptake by the plants, in addition to nitrogen fixation. The reduction of nitrate in plant roots, carried out by the enzyme nitrate reductase (NR) improves nitrogen assimilation in shoots [16].

10.3 Applications in Various Fields Azospirillum spp. are the most studied free-living nitrogen fixing plantgrowth promoting rhizobacteria. The versatile beneficial effects exerted by the genus make it interesting and draws attention, in order to thrive deeper for better understanding of its properties and mechanisms of various actions, for enhanced future applications. The associative-symbiosis (in Azospirillum spp.) in the grass-bacteria systems can be distinguished from the legume symbiosis (in Rhizobia) by various techniques, including immunofluorescent technique, fluorescent antibody technique, and serological tests. It was possible to identify the individual cells by these techniques. Strains of Azospirillum brasilense were isolated from Ecuador, Florida, and Venezuela and established their nitrogen fixing ability. Then, by employing fluorescent antibody technique, the individual strains were

Azospirillum for Sustainable Agriculture  321 identified [25]. Bashan et al. carried out different investigations to comprehend poor survivability of Azospirillum spp. in 23 different types of soils using two strains of Azospirillum brasilense: Cd and Sp-245 [26]. In tropical seasons, the strains of Azospirillum inoculated were found to have not survived from one season to the next [27]. In Israeli soils, it was observed that the Azospirillum cells strongly adhered to the clay and organic matter in topsoil hardly eroded downward [28]. The formation of cysts [29], fibrillary materials [30], made the bacteria stand out among other rhizobacteria, which on experiment, were easily washed down when percolated [31]. Bashan et al. [26] found that enormous numbers of viable bacterial cells were observed in the vicinity of the root surface, regardless of the soil characteristics. But, as soon as the plants were drawn apart from the soil, the survival of the bacterial cells diminished. To use Azospirillum inoculants as commercial bio fertilizers in large-scale demands sufficient bacterial biomass. Fallik et al. [32] innovated technique for the production of Azospirillum biomass in a “fed-batch fermentor”. In their work, they used succinic acid as the source of carbon and liquid ammonia as the nitrogen source. It was observed that within 24 hours, the viable cell number became 1–3 × 1010 cfu/ml. As for the carrier of the Azospirillum species, ground or granular peat was recorded with highest number of viable cells, compared to talcum powder, bentonite, vermiculite, etc. Faure et al. [33] studied the activity of Azospirillum lipoferum laccase on phenolic derivatives. The laccases are the phenol oxidases, common in fungi and higher plants. This had been associated only with Azospirillum lipoferum, isolated from rice roots. The laccase components convert the ortho- and para-diphenols, to orthoand para-quinones, respectively. The result obtained displayed the conversion of phenolic compounds of aldehydic, acidic origin, or acetophenone type, were converted into 2,6-dimethyl-1,4-benzoquinone, by the activity of Azospirillum lipoferum laccase. Various works were carried out to demonstrate the capacity of Azospirillum spp. in increased growth of microalgae in freshwater surface. Gonzalez et al. [33] used strain of Azospirillum brasilense for enhanced growth of Chlorella vulgaris, a freshwater microalgae, essential in tertiary wastewater treatment. Their work reported inoculation of Chlorella sp. with a PGPR for the very first time. Alginate beads were used to immobilize the two co-inoculated species, ensuring close contiguity between them. The result obtained was quite satisfactory. The desired growth of the microalgae was observed, accompanied with significant increase in cell number, level of pigments, dry and fresh weight after inoculation with the Azospirillum species. The property of aggregation or flocculation of the genus Azospirillum had always attracted close attention of the researchers, comprising the facility to offer

322  Biofertilizers the cells better survival capacity. Burdman et al. [34] investigated the composition of exopolysaccharide (EPS) and capsular polysaccharide (CPS) of four Azospirillum brasilense strains having difference in aggregation ability. The cell aggregation property was analyzed by high performance anion exchange chromatography. They reported coinciding relationship between rate of aggregation and amount of arabinose present. Kefalogianni et al. [35] carried out a study to contextualize the biochemical activities of Azospirillum brasilense and Azospirillum lipoferum under controlled culture conditions. In both the species, production of EPS and CPS in large amounts was observed. After extinction of carbon sources from the culture, the polysaccharides were shown to be consumed. This implied that they were, in fact, used as carbon sources by the bacteria. The uptake of oxygen was found to be high during fructose assimilation and low during polysaccharide degradation. Herschkovitz et al. [36] utilized “molecular phylogenetic probes” and primers on 16S rRNA and rDNA of Azospirillum brasilense to investigate its impact on rhizosphere colonization and the composition of microbial community in two soil systems. It was observed that the species on inoculation did not affect the composition of the microbial community. The adhesion of the bacterial cells on solid substrates is related to the outer membrane of the microbial cells. The biofilm formation, resulting from bacterial colonization on solid surfaces in marine environments, leads to material degradation. To correlate these facts and to comprehend the control of this biofilm formation in sea water, Pradier et al. [37] carried out a study on the chemical composition of the bacterial surfaces in some marine bacterial strains. The D41 strain tested exhibited the presence of proteins. This strain gets attached to surfaces of stainless steel, glass, Teflon, etc. more in comparison to the strains DA and D01. The role of the protein present was demonstrated to have influence over the adhesion to a greater extent than the hydrophobicity. As the Azospirillumplant association leads to enhanced water uptake by the plant roots, inoculation with Azospirillum spp. is expected to reduce the water stress condition, thus promoting healthy plant growth. Pereyra et al. [38] performed an investigation, establishing the positive effects of Azospirillum spp. on plant water-stress condition. They inoculated Triticum aestivum seedlings with Azospirillum brasilense Sp-245, under supervised conditions of darkness and osmotic stress and tested for changes in composition of phospholipids and distribution of fatty acids. It was observed that inoculation with Azospirillum spp. prevented leakage of ions and enhanced 2,3,5-tripheniltetrazolium reducing capacity in the roots. The tolerance to water stress was found to be mediated by changes in the profile of fatty acid distribution of principal root phospholipids, phosphatidylcholine, and

Azospirillum for Sustainable Agriculture  323 phosphatidylethanolamine. Another important factor arising from the plant root association with Azospirillum is the production of phytohormone, which assist the growth promotion, alongside nitrogen fixation. Perrig et al. [39] carried out systematic investigation of phytohormone and polyamine production in the two Azospirillum strains: Azospirillum brasilense Az39 and Cd. The study also led to discovery of siderophore production and phosphate solubilization by these two strains. In controlled medium, the strains synthesized five major phytohormones, along with important growth regulator, cadaverine. The production of one of the most important growth regulator, IAA, was demonstrated by the work of Crozier and his co-workers [40]. They isolated some strains of Azospirillum brasilense and Azospirillum lipoferum from maize and teosinte roots proceeded for quantitative analysis of the synthesized IAA and similar indoles. It was reported that in the culture medium of Azospirillum brasilense 703Ebc strain, growth regulators such as IAA, indole-3-ethanol, and indole-3-methanol indole-3-lactic acid were present. However, the main source from where the IAA was originated, whether it was bacteria or the plant was not clarified. Due to the enormous plant growth promoting effect of Azospirillum spp., it has been marketed as commercial inoculants for agricultural applications. Most of the marketed inoculants are formulated as peat-based for seed coating or as pellets for sowing. However, in some cases, polymer coatings as carriers have been used in the formulations for better survival and activity of the inoculants in challenging environmental conditions. The polymer coatings help the inoculants in survival as well as optimize the application by controlled release from the coatings for a prolonged period. Bashan et al. [41] encapsulated two plant growth promoting rhizobacteria, Azospirillum brasilense Cd and Pseudomonas fluorescence 313 in two different alginate beads and stored for 14 years after drying. Though the initial population declined in both the beads, yet a significant number of cells survived, thereby demonstrating the ability of the polymer coating to protect the inoculants for a very prolonged period. Various applications of commercial Azospirillum inoculants were carried out in agricultural field, monitoring the growth of several crops. Piccinin and his collaborators [42] inoculated wheat seedlings with Azospirillum brasilense strain and investigated the overall growth and the agronomic yield. Positive results were obtained with enhanced growth and raised annual production. In place of conventional root application, Sudhakar et al. [43] carried out a study using Azospirillum, Azotobacter, and Beijerinckia bacterial strains on Morus alba, through foliar application. However, growth performance of Azotobacter was rather impressive compared to Azospirillum and Beijerinckia strains. The plant growth promoting effect of Azospirillum

324  Biofertilizers spp. can be used in solving environmental problems, other than in agricultural applications. For instance, mostly in arid or semi-arid environments, metalliferous mine tailings can cause long term threats, by running into the nearby water stream by erosion and distributing in the urban populations in vicinity as dust particles, produced from the tailings. Development of vegetative crops to stop the wind and water erosion and to precipitate the metals in the root zone is a probable way of preventing such hazardous state. The difficulty arising here is the poor chances of the tailings to act as growth substrates for vegetative plants. Inoculation with Azospirillum spp. plays a significant role in this context. When qualibush was inoculated with Azospirillum brasilense Sp6, significant growth was observed, accompanied by an increase in plant biomass, irrespective of the compost-amended, with high-metal content mine tailings surroundings [44]. Most of the studies on Azospirillum spp. were carried out on strains of Azospirillum brasilense and Azospirillum lipoferum. However, Sant’Anna et al. [45] in 2011 performed an investigative study on the physiology and biochemistry of the genus Azospirillum. Their work presented a genomic feature for the species. They observed that the species assist in phytohormone production, transport, “quorum sensing”, “antibiotic resistance”, and bacteriphytochrome biosynthesis. The nitrilase gene and the genes responsible for nitrogen fixation could be directly implemented in plant growth promotion.

10.4 Current Status Azospirillum genomes for different strains consist of multiple replicons, which may lead to “genome plasticity”. The rearrangements of the genomes take place spontaneously. However, on the formation of new megaplasmids, replicons might vanish. Genome sequencing of the Azospirillum spp. established that a major portion of the genome has been attained horizontally [46]. Several works have been performed recently to attain better comprehension about the genomic variance in the genus and its diverse applicability in the plant stimulatory effects. Kukolj et al. [46] inspected the proteomic and metabolistic benefits of the wild-type Azospirillum strain FP2 and its mutant ntrC, which was already demonstrated to play the central role in the nitrogen metabolism. The investigation led to the production of certain proteins, which are likely to be induced by ntrC. However, further experiments are needed to rationalize the mutual interactions of the protein candidates and the transcriptional activator ntrC mutant. Cecagno et al. [47] carried out a study on genetic variability on Azospirillum amazonenese by an “in silico comparative genomic analysis”, demonstrating

Azospirillum for Sustainable Agriculture  325 subtractive hybridization, utilizing “total coding sequence” (CDS). The comparative study was performed with certain closely related bacterial genomes. The ability of Azospirillum amazonenese to produce the plantgrowth promoting substances was successfully established, along with the fact that, similar to Azospirillum brasilense, the Azospirillum amazonenese species can alter genetic expression, stimulated by the presence of auxin. Works on investigating the metabolic routes and genomic studies on sulfur-oxidizing Azospirillum species, Azospirillum thiophilum, have been reported [20]. Orlova et al. [20] carried out a comprehensive study to have a better understanding on the detailed genomic structure and mechanisms followed by the metabolic pathways taken by the species. They reported the capacity of the species to metabolize C1 compounds, thiosulphates, and hydrogen gas and the mechanism involved genetic encoding of the enzymes of “carbon metabolism” through the processes of glycolysis and the cycles of tricarboxylic acid and glyxylate. Synergistic relationship between various rhizosphere bacteria and Leptospires, the organism possessing pathogenic and saprochytic properties, acts as environment protecting formations, through co-aggregation and biofilm production. Kumar and his coworkers [48] investigated the association of Azospirillum brasilense Sp245 with Leptospira and observed induced growth in static conditions. Another association benefiting the participating microorganisms is the “synthetic mutualism”. The synthetic mutualism of natural microalgae and various bacterial species has long been studied [50]. The resultant effect produced by such association is beneficial to the plants as well. Synthetic mutualism of Chlorella sorokiniana and Azospirillum brasilense was studied decades ago. It was understood that the Azospirillum species could use the exudates synthesized by the Chlorella species as nutrients. Azospirillum spp. then makes use of the exudates by producing IAA, which, in turn, is utilized by Chlorella spp. to induce growth. However, the mechanism followed and the compounds associated in the metabolism are not yet reported. Palacios et al. [49] investigated for the mechanism followed in such synthetic mutualism in Chlorella sorokiniana and Azospirillum brasilense. It was then satisfactorily established that the Chlorella spp. produces compounds such as tryptophan and thiamine, which are signal molecules for Azospirillum brasilense. These stimulate the synthesis of IAA, which counter stimulates the growth of the Chlorella spp. Counter productions of these signaling molecules support the synthetic mutualism between the two species and facilitates their combined growth. Co-inoculation of Azospirillum brasilense with another plant-growth promoting bacteria Pseudomonas fluorescence was expected to exhibit co-survival and mutual benefit in the mixed culture, along with better performance in growth

326  Biofertilizers promotion of plants. Various investigations were performed in this context. Maroniche et al. [50] co-inoculated different strains of Azospirillum brasilense and Pseudomonas fluorescence, through in vitro method. Their mutual interaction and the factors that affect their combined impact on rhizosphere colonization were studied. The Pseudomonas spp. was showed to induce “lethality” on the Azospirillum cells, when in direct contact by an “antibacterial type-6 secretion system”. They observed their growth in different cultures and detected that in King’s B medium, the Pseudomonas cells highly inhibited the growth of Azospirillum cells by a diffusible mechanism, stimulated by the production of siderophores. In Congo Red medium, mutual growth promoting effects were also observed along with inhibitory effects, the effect being different for different strains of the two bacteria. It was observed that Pseudomonas protegens CHA0 repressed the growth of Azospirillum brasilense Sp7 strain, but not Sp245 strain, by production of a Gac/Rsm-regulated antibiotic. Another work was carried out by Pagnussat et al. [51] to study the interaction of Pseudomonas protegens CHA0 and Azospirillum brasilense Sp245 under static conditions and in mixed biofilms. Under these controlled environments, the mutual growth of both the species was observed. The stimulating effect of Pseudomonas protegens CHA0 caused twofold increase in the biofilm that was formed by Azospirillum brasilense Sp245 in the plastic surfaces. The application of Azospirillum spp. as commercial inoculants has gained enormous attention due to its multiple stimulatory effects. Various experiments have been carried out recently to test the on-field applicability of the bacteria as commercial inoculants. Brazil being the largest producer of sugarcane [52], for micropropagation purpose, which refers to rapid growth of healthy crops on uniform plantation, inoculation with endophytic bacterial cultures has long been practiced. Barreto et al. [52] implemented Azospirillum inoculants to have witnessed the inoculants contributing almost 60% of the nitrogen uptake to the sugarcane plants, accompanied with elongated height and root length and increased biomass and dry matter production. Kundu et al. [53] performed a rather extensive experiment by taking the mixture of bio inoculants (Azospirillum spp., Azotobacter spp., Phosphate solubilizing bacteria, and Potash solubilizing bacteria) with inorganic fertilizer (NPK) and observed the growth of mango cv. Himsagar. By optimizing various field controls, it was concluded that the combined effect of the bio inoculants caused effective growth of the plant leaves and increase in quality and yield of fruits, dropping the NPK use up to 25%. Some recent studies exhibited certain significant environment beneficial activities, mediated by some regulatory

Azospirillum for Sustainable Agriculture  327 compounds secreted by Azospirillum spp. Xu et al. [54] studied the effect of Azospirillum brasilense and Bacillus subtilis, both of which produce exogenous abscisic acid (ABA) on plant accumulation of severe agricultural pollutant Cadmium (Cd). ABA was reported to have the ability of restricting Cd uptake by plants through IRT1 (Fe-regulated transporter 1) activity. The two bacterial species under observation produce ABA, thereby preventing Cd accumulation, by IRT1, mediated by ABA, on the wild-type plant species, Arabidopsis thaliana. The halogenated pesticides used by the farmers undoubtedly create harmful side effects for the environment. Bromoxynil is one of the highly utilized nitrile herbicide. Their soil contamination leads to various undesirable hazards. Knossow et al. [55] investigated the microbial degradation of this toxic compound by soil cultures under aerobic and anaerobic conditions. Chemical conversions, such as hydration, nitrilation, or hydroxylation of the nitrile group normally take place under aerobic conditions. On the other hand, reductive dehalogenation occurs under anaerobic conditions. By employing isotopic effects, Azospirillum, Pseudomonas, Stenotrophomonas, and Arcticibacter degradation products were found, indicating nitrile hydratase to be the principal route of degradation. It was demonstrated that nitrilase encoding sequence was present in Azospirillum genome. Thus, it was hypothesized that the reductive debromination of the nitrile group of Bromoxynil under aerobic condition was followed by nitrile hydratase, probably mixed with nitrilase [55].

10.5 Challenges in Large-Scale Commercial Applications of Azospirillum Inoculants The versatility of the Azospirillum genus in exhibiting critical plantgrowth promoting properties, either by nitrogen fixation or by secretion of essential compounds for growth stimulation is highly beneficial for plant growth. Certain substances produced and secreted by these organisms play important role in hazardous compound accumulation in the rhizosphere. Being the free-living non-legumous diazotroph, Azospirillum spp. turns out to be the satisfactory substitute for legumous Rhizobia, with the additional advantage of not having to be too precautious for the handling of the inoculant for field applications. For such numerous crucial advantages and usability of these organisms, they are being utilized in the agricultural field in large-scale commercially, enhancing the overall growth and yield

328  Biofertilizers of crop plants and environmental protection. But there exist certain constraints, which make the large-scale commercial applications of these inoculants troublesome. They are discussed below. i.

Resource constraints [56]: There exist problems including lack of suitable strain for inoculation, unavailability of quality supplies, and inaccessibility in proper infrastructure, added with the unawareness of the manufacturers for the value of products. ii. Production constrains [56]: The lack of knowledge, research on the quality of the inoculants as commercial products restraints the manufacturers to attain the products with superior quality. iii. Market constraints [56]: The ignorance of the manufacturers’ toward the marketing of the products, followed by the restraining the financial aspects. iv. Field constraints [56]: The practical implications of the inoculants on field are very different from the theoretical perspective in reality. This arises because of diverse environmental conditions, along with a huge population of indigenous rhizosphere microorganisms, which might adversely affect the growth and activity of the Azospirillum cells. v. Technical constraints [56]: Technical constraints can be crucial, when the source strain turns out to be unauthentic. Other technical problems may arise due to mishandling, inappropriate adoption of certain techniques, leading to contamination, destruction of the source strain and poor yield.

10.6 Programs Employed for Enhanced Applications of Azospirillum Inoculants Governments of many countries around the world have displayed close attention in practicing sustainable organic agricultural technologies, in correspondence to the global hiking of environmental pollutions, coming from utilization of conventional inorganic fertilizers. Many policies and funds have been introduced by the governments across countries in order to promote sustainable agricultural practices. “Common Agricultural

Azospirillum for Sustainable Agriculture  329 Policy” has been promoted by the European countries granting 30% of the funds to the farmers as a “green payment” [56]. The Taiwan government has introduced a policy to promote the use of bio inoculants for nitrogen fixation, phosphate solubilization, etc. They have engraved peer pressure in utilizing these bio products for soybean cultivation, which is traditionally consumed in the country as vegetable, peanut, and red bean, ensuring sustainability. The Indonesian Agricultural Sector also took up certain programs to improve the declined yield of crops by using bio products. The Indian Government also introduced certain policies to promote the bio product usage in agriculture throughout the country and also to enhance the market value for the bio products, to support the small-manufacturers. Policies such as “National Mission of Sustainable Development (NMSA)/Paramparagat Krishi Vikas Yojana”, “Rashtriya Krishi Vikas Yojana (RKVY)”, and “National Mission on Oilseeds and Oil Palm (NMOOP)” have been introduced for the sustainable agricultural purpose [56]. The Indian Council of Agricultural Sciences (ICAR) started the “National Organic Program (NOP)”, which was previously executed by the US Department of Agriculture (USDA) for improvement in the quality of the agricultural yield, obtained through organic manner [56].

10.7 Conclusion and Future Prospects In the context of increasing global awareness about environmental pollutions and worldwide “green” safety protocols, application of bio inoculants in agricultural field is accepted to be a much anticipated mode for sustainable agricultural development. The stimulatory effects of Azospirillum spp. as discussed are displaying its versatile applications ensuring environmental safety. However, for large-scale applications as commercial inoculants, there emerges certain complications regarding their production, long time survival, marketing, etc. Further studies on the genomic structure of Azospirillum spp. can lead to better understanding of the metabolic route taken by the organism and, hence, can create scope for innovated enhanced application in various fields. There is certain necessity in expanding the awareness about the bio inoculants worldwide, regarding their proper handling, proper technical application, and sophisticated storage methods. The agricultural societies and government across the countries should adopt further policies and awareness programs regarding the issue and grant more funds in this field to inspire further improvement and innovations of bio products.

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332  Biofertilizers 32. Fallik, E., Okon, Y., Inoculation of Azospirillum brasilense: Biomass Production, Survival and Growth Promotion of Sectaria italica and Zea Mays, Soil Biol. Biochem., 28, 123–126, 1996. 33. Gonzalez, L. E., Bashan, Y., Increased Growth of the Microalga Chlorella vulgaris when coimmobilized and coinoculated in Alginate beads with the plant-growth promoting bacterium Azospirillum brasilense, Appl. Environ. Microbiol., 66, 1527–1531, 2000. 34. Burdman, S., Jurkevitch, E., Soria-Diaz, M. E., Serrano, A. M. G., Okon, Y., Extracellular Polysaccharide Composition of Azospirillum brasilense and its Relation with Cell Aggregation, FEMS Microbiol. Lett., 189, 259–264, 2000. 35. Kefalogianni, I., Aggelis, G., Modeling growth and biochemical activities of Azospirillum spp., Appl. Microbiol. Biotechnol., 58, 352–357, 2002. 36. Herschkovitz, Y., Lerner, A., Davidov, Y., Rothballer, M., Hartmann, A., Okon, Y., Jurkevitch, E., Inoculation with the plant-growth promoting rhizobacteria Azospirillum brasilense causes little disturbance in the Rhizosphere and Rhizoplane of Maize (Zea mays), Microb. Ecol., 50, 277–288, 2005. 37. Pradier, C. M., Rubio, C., Poleunis, C., Bertrand, P., Marcus, P., Compere, C., Surface Characterization of three Marine Bacterial Strains by FTIR, XPES and TOF SIMS, Correlation with adhesion on stainless steel surfaces, J. Phys. Chem. B, 109, 9540–9549, 2005. 38. Pereyra, M. A., Zalazar, C. A., Barassi, C. A., Root Phospholipids in Azospirillum-inoculated wheat seedlings exposed to water stress, Plant Physiol Biochem., 44, 873–879, 2006. 39. Perrig, D., Boiero, M. L., Masciarelli, O. A., Penna, C., Ruiz, O. A., Cassan, F. D., Luna, M. V., Plant-growth promoting compounds produced by two agronomically important strains of Azospirillum brasilense and implications for inoculant formulation, Appl. Microbiol. Biotechnol., 75, 1143–1150, 2007. 40. Crozier, A., Arruda, P., Jasmim, J. M., Monteiro, A. M., Sandberg, G., Analysis of Indole-3-acetic acid and related indoles in culture medium from Azospirillum lipoferum and Azospirillum brasilense, Appl. Environ. Microbiol., 54, 2833–2837, 1988. 41. Bashan, Y., Gonzalez, L. E., Long-term survival of the plant-growth promoting bacteria Azospirillum brasilense Pseudomonas fluorescence in dry alginate inoculant, Appl. Microbiol. Biotechnol., 51, 262–266, 1999. 42. Piccinin, G. G., Braccini, A. L., Dan, L. G. M., Scapin, C. A., Ricci, T. T., Bazo, G. L., Efficiency of seed inoculation with Azospirillum brasilense on agronomic characteristics and yield of wheat, Ind. Crops Prod., 43, 393–397, 2013. 43. Sudhakar, P., Chattopadhayay, G. N., Gangwar, S. K., Ghosh, J. K., Effect of foliar application of Azotobacter, Azospirillum and Beijerinckia on leaf yield and quality of mulberry (Morus alba), J. Agric. Sci., 134, 227–234, 2000. 44. De-Bashan, L. E., Hernandez, J. P., Nelson, K. N., Bashan, Y., Maier, R. M., Growth of Qualibush in acidic, metalliferous Desert Mine Tailings: Effect

Azospirillum for Sustainable Agriculture  333 of Azospirillum brasilense Sp6 on Biomass Production and Rhizosphere Community Structure, Microb. Ecol., 60, 915–927, 2010. 45. Sant’Anna, F. M., Almeida, L. G. P., Cecagno, R., Reolon, L. A., Siqueira, F. M., Machado, M. R. S., Vasconcelos, A. T. R., Schrank, I. S., Genomic insights into the versatility of the plant-growth promoting bacterium Azospirillum, BMC Genom., 12, 1–14, 2011. 46. Kukolj, C. K., Pedrosa, F. O., de Souza, G. A., Sumner, L. W., Lei, Z., Sumner, B., do Amaral, F. P., Wang, J., Joshi, T., Huergo, L. F., Monteiro, R. A., Valdameri, G., Stacey, G., de Souza, E. M., Proteomic and metabolomic analysis of Azospirillum brasilense ntrC mutant under high and low nitrogen conditions, J. Proteome Res., 2019, https://pubs.acs.org/doi/ abs/10.1021/acs.jproteome.9b00397. 47. Cecagno, R., Fritsch, T. E., Schrank, I. S., The plant-growth promoting bacteria Azospirillum amazonenese: Genomic Versatility and Phytohormone Pathway, Bio. Med. Res. Int., 2015, 1–7, 2015. 48. Kumar, K. V., Lall, C., Raj, R. V., Vedhagiri, K., Vijayachari, P., Coexistence and survival of pathogenic Leptospires by formation of biofilm with Azospirillum, FEMS Microbiol. Ecol., 91, 1–11, 2015. 49. Palacios, O. A., Gomez-Anduro, G., Bashan, Y., de-Basha, L. E., Tryptophan, thiamine and indole-3-acetic acid exchange between Chlorella sorokiniana and the plant-growth promoting bacterium Azospirillum brasilense, FEMS Microbiol. Ecol., 92, 1–11, 2016. 50. Maroniche, G. A., Diaz, P. R., Borrajo, M. P., Claudio, F. V., Creus, C. M., Friends or foes in the rhizosphere: Traits of fluorescent Pseudomonas that hinder Azospirillum brasilense growth and root colonization, FEMS Microbiol. Ecol., 94, 1–10, 2018. 51. Pagnussat, L. A., Salcedo, F., Maroniche, G., Keel, C., Valverde, C., Creus, C. M., Interspecific Cooperation: Enhanced growth, attachment and strain-­ specific distribution in biofilms through Azospirillum brasilense-Pseudomonas protogens co-cultivation, FEMS MIcrobiol. Lett., 363, 1–9, 2016. 52. Barreto, M. C. S., Figueiredo, M. V. B., da Silva, M. V., de Andrade, A. G., de Oliveira, J. P., Almeida, C. M. A., de Araujo, L. C. A., Reis Junior, O. V., Ferreira Junior, M. U. F., da Costa, A. F., Lima, V. L. M., Biotechnological Potential of Endophytic Bacteria to improve the micropropagated seeding of variety RB92579 Sugarcane (Saccharum officinarum L.), Adv. Micobiol., 8, 859–873, 2018. 53. Kundu, S., Mishra, J., Effect of bio fertilizer and inorganic fertilizers on mango cv. Himsagar, J. Crop & Weed, 14, 100–105, 2018. 54. Xu, Q., Pan, W., Zhang, R., Lu, Q., Xue, W., Wu, C., Song, B., Du, S., Inoculation with Bacillus subtilis and Azospirillum brasilense produces abscisic acid that reduces IRT1-mediated Cadmium uptake of roots, J. Agric. Food Chem., 2018, https://pubs.acs.org/doi/10.1021/acs.jafc.8b00598.

334  Biofertilizers 55. Knossow, N., Siebner, H., Bernstein, A., Isotope Fractionation in the Microbial Degradation of Bromoxynil by Aerobic and Anaerobic Soil Enhancement Cultures, J. Agric. Food Chem., 2020, https://dx.doi.org/10.1021/acs. jafc.9b07653. 56. Saritha, M., Tollamadugu, N. V. K. V. P., The Status of Research and Application of Biofertilizers and Biopesticides: Global Scenario, Elsevier Inc., pp. 195–206, 2019.

11 Actinomycetes: Implications and Prospects in Sustainable Agriculture *

V. Shanthi

Department of Microbiology, St. Thomas College, Bhilai Nagar, Durg District Chhattisgarh, India

Abstract

Biofertilizers are being considered as a great gift of nature to mankind. The latent microorganisms being active ingredients of biofertilizers have served the purpose of solving environmental and health issues related to indiscriminate usage of harsh chemical fertilizers. Biofertlizers serve as important components of integrated nutrition management system for sustainable agriculture. Among the diverse types of microorganisms in nature, the role of actiomycetes cannot be underrated. Their large population in diverse soil types and their dynamic nature to maintain soil fertility and ecology is of prime importance in agriculture field. Biological management of soil ecosystem by actinomycetes through their diverse features like plant growth-promoting ability, managing plant health and vigor, and production of agro active compounds greatly contributes to the agricultural sector. The capacity of actinomycetes to mitigate unsafe and adverse effects of chemical fertilizers and also to promote positive effects in plants highlights their role in ecosystem resilience. These filamentous bacteria have immense beneficial effects in soil and hence open a great avenue for improved crop production in future. Keywords:  Actinomycetes, sustainable agriculture, agro active compounds, biofertilizer

*Email: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (335–370) © 2021 Scrivener Publishing LLC

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336  Biofertilizers

11.1 Introduction Nutrient management is a practice which deals with conservation of soil by managing essential soil nutrients at their best and optimum levels, and biofertilizers are considered as an important part of this management system. It is believed that biofertilizers have an edge over the chemical fertilizers. They are unique in the sense that they are safe, eco-friendly, and, at the same time, quite efficient [1]. In simple terms, biofertilizers can be described as substances containing living, efficient microorganisms that help upgrade and assist plant yield by enhanced extension of required nutrients to the host plant [2]. Their role in sustainable crop production and effective, ecofriendly nature is of paramount importance in agricultural field. These features of biofertilizers are inspired by the very nature of their constituent components—the microbes. The microbes tend to provide all the beneficial properties a biofertilizer should possess. Biofertilizers, as tools for food safety and sustainable agricultural practices, incorporate within them impressive features like nutrient mineralization and mobilization, serving as effective biocontrol agents against several plant diseases [3], sustaining plants under both biotic and abiotic stress and maintaining soil ecology and fertility by incorporating certain plant growth-promoting factors, etc. [4]. Microorganisms in biofertilizers are the ones which are creditable for all these features. Many different microorganisms are considered suitable for preparation of biofertilizers. But actinomycetes among them are unique. Characteristic distinctive features from bacteria and fungi: actinomycetes are unique by playing pivotal role in soil ecosystem, and their benefits being reaped not only in industries but in agriculture as well [5]. As prospective contenders for production of biofertilizers, actinomycetes possess the characteristic feature to secrete wide range of secondary metabolic derivatives and factors with antibacterial, antifungal, and antagonistic effects [6]. Actinomycetes rule the roost among the large diverse microbial population in terms of production of bioactive compounds. Reports confirm that of the 23,000 varieties of bioactive compounds produced by microbes; nearly 50% of them are produced by actinomycetes [7, 8]. Antibacterials, antivirals, and agrobiologicals like weedicides, insecticides, and growth regulatory compounds are some of them [9, 10]. Their ability to exist and survive in diverse soil conditions help themselves to indulge in various soil processes: nitrogen fixation, phosphate solubilization, and formation of

Actinomycetes and Sustainable Agriculture  337 humus to name a few [11]. Also, production of agro active substances and hence their role in sustainable agriculture arguably makes actinomycetes the most impressive and potent contenders for biofertilizer formulations. Among the vast assortment of microbes that exists on earth and in nature, actinomycetes are one of the most widely distributed organisms. Almost more than 100 genera of actinomycetes are natural inhabitants of soil [12]. Actinomycetes belong to the order Actinomycetetales and can be distinguished from other microbes with elevated levels of G+C content (74 mol %) [13, 14]. These gram-positive bacteria exist as saprophytes in the soil [15]. Being saprophytic soil inhabitants, they possess the ability to decompose organic matter like lignocelluloses and various types of complex carbohydrates like starch and chitin [16]. Morphologically, they show mycelia growth which culminates in sporulation. Actinomycetes usually exist in significantly high numbers in semi-arid soil environments although very little numbers of actinomycetes have also been known to exist in other climatic conditions too [17]. Actinomycetes usually prefer low level of moisture for growth and survival and are very well acclimatized to semi-dry conditions [18] probably due to their ability to sporulate even under dry conditions [19]. Actinomycetes are mesophilic in nature, though some species may be found in thermophilic habitats too [20]. They can also grow as epiphytes and have a wide host range [21, 22]. Recently, actinomycetes have garnered a lot of interest in microbiologists because of their diverse properties which makes them a good source for development of bioinoculants [23, 24]. Actinomycetes are very important microbes which can promote the overall vigor and yield of crop plants. They can be introduced directly into soil or can be applied to crop seeds [25, 26]. Actinomycetes are known to directly influence growth of plants by mechanisms like nitrogen fixation, phosphate solubilization, production of growth hormones, or indirectly by scavenging of iron by siderophore production and hence protection of plants from pathogens [27]. Also, they influence their growth by magnifying their sufferance to various stress conditions [28, 29]. A well-known genus of actinomycetes is Streptomyces [30]. Streptomyces and other actinomycetes genera contribute to over 75% of the many known active biological compounds [31]. Actinomycetes have long been inviting a lot of attention due to their capability and capacity to inhibit a broad range of fungal pathogens by producing antifungal agents, thereby protecting the plants from a variety of fungal diseases [32]. Actinomycetes are very good repositories of

338  Biofertilizers antibiotics and extracellular enzymes [33, 34]. Also, their prospective ability to serve as a source of biocontrol elements, biologically active agricultural agents and plant growth-enhancing ingredients cannot be ignored.

11.2 Role in Maintaining Soil Fertility 11.2.1 Nitrogen Fixation One of the critical element which limits plant growth is nitrogen. Availability and uptake of sufficient nitrogen by plants is crucial and indispensable for their growth and maturation [35]. The importance of nitrogen for plants lies in the fact that it is utilized for biosynthesis of important nitrogenous compounds like nucleic acids, amino acids, and proteins [36, 37]. It is a vital component of photosynthetic pigment; chlorophyll and also it is used for biosynthesis of ATP and nucleic acids [38]. However, the element nitrogen in spite of its abundance in earth’s atmosphere is not available to plants due to its inert nature unless it is converted to a more available form [39]. Elemental nitrogen is to be converted to either ammonium ion ( NH+4 ) or nitrate ( NO3− ) for it to be used conveniently by plants. There are a variety of microorganisms including actinomycetes with a potential to fix atmospheric nitrogen in both leguminous and non-leguminous plants, but when compared to other microbes, actinomycetes have a unique property of entering into a more collaborating relationship with a diverse group of plants [40] which other nitrogen fixing microbes lack. This feature of actinomycetes ensures its place as a top contender for production of biofertilizer in terms of nitrogen fixation ability. Actinomycetes can form nodule clusters and possess nitrogen fixing ability. Some actinomycetes enable their potential of nitrogen fixation by interacting with the plant while some help the plant by existing independently in the soil. In either case, the beneficial effect in plants is very much obvious. A considerable number of studies have evolved associated to the group of actinomycetes which interact with plants in some or the other way and increase soil fertility and hence affect the nature of plant growth. Some species of actinomycetes exhibit a very strong plantmicrobe interaction which is of great importance for the health of the plant. A unique and substantially strong interaction exists between Streptomyces lydicus WYEC108 which is a root-colonizing actinobacteria and Pisum sativum, a leguminous plant as reported by Tokala et al. [41]. This interaction between the pea and actinomycetes seems to be of utmost importance because the root-colonizing soil actinomycetes, Streptomyces

Actinomycetes and Sustainable Agriculture  339 lydicus WYEC108 not only promotes growth of healthy nodules in the legumes but also influences the pea root by greatly enhancing the frequency of root nodulation. Pea being a leguminous plant also is known to enter into symbiotic relation with bacteria (Rhizobium). Studies reveal that probably Streptomyces lydicus colonizes the pea root at a time when the common symbiotic nitrogen fixing Rhizobium species infects the pea plant to form root nodules. These active bacteria after colonization start to sporulate within the nodules and precisely within their cell layers [42]. So, such settlement by both Rhizobium and Streptomyces possibly causes an increase in nodule size which finally helps to improve the vigor of organisms inside the root nodules. This helps to enhance the assimilation of various essential soil nutrients by the nodules [43]. So, perhaps, it is quite clear that several important actinomycetes like Streoptomyces act as natural soil fertility enhancers for improved growth of not only pea but also for many other leguminous plants. Such study reports provide substantial evidence that many actinomycetes have nitrogen fixing ability and may work independently to promote plant growth and health but the fact that they are also quite compatible with other root nodulating organisms cannot be ignored. It is also believed that some actinomycetes have poor to average N2 fixing ability as independent organisms but, when combined with other nitrogen fixing microorganisms or bacteroides, show extraordinary improvement in nitrogen fixation. This has also been experimentally proved by Soe et al. [44]. The evaluation of the compatibility of Streptomyces griseoflavus P4 strain isolated from pea root nodules with Bradyrhizobium japonicum strain USDA 110 was successful. It was reported that the combination had noteworthy effects on the dry weight of nodules, shoot nitrogen accumulation, seed weight, etc. Individually, neither Bradyrhizobium japonicum nor Streptomyces griseoflavus P4 strains improved nodulation and seed weight but the two, when combined, had significant beneficial effects in soybean. Improved nodulation and hence enhanced nitrogen fixation were some of them. Actinomycetes not only interact symbiotically with leguminous plants but also fix atmospheric nitrogen by interacting with non-leguminous plants. In fact reports confirm that actinomycetes can fix comparably higher rates of atmospheric nitrogen with non-leguminous plants than leguminous plants [45]. Gautheir et al. [46] were the first to report the isolation of free-living actinobacteria from a nitrogen fixing non-leguminous tree (Casuarina equisetifolia) with the ability to form nodules. Two different actinomycetes were isolated from the nodules which were shown to possess nitrogenase activity based on acetylene reduction test [47]. The actinomycetes strains Streptomyces sp. D11 and Streptomyces sp. G2 although

340  Biofertilizers did not initiate nodulation in the host plant indicating the non-infective nature of these strains, these strains produced significant amount of nitrogenase which was found to be more sensitive to the presence of combined nitrogen. The influencing nature of Streptomyces sp. to nodulate pea root to a very high degree was reported by Tokala et al. [41]. Streptomyces sp. was also responsible for enhanced assimilation of iron by the root nodules and other soil nutrients. Non-legumes like Casuarina, Alnus, and Hippophae can symbiotically interact with the actinomycete called Frankia. These organisms have been reported to revive the soil fertility by fixing huge amounts of atmospheric nitrogen. Such interactions are believed to be very important for ecology also because such woody trees and shrubs are known to actively grow under stressful soil conditions [48, 49]. Frankia ceanothi has been reported to contain nitrogen fixing properties [50]. This actinomycete exists as an endophyte within the cortex of a shrub, Ceanothus greggii [51]. Nitrogen fixing ability was long assumed to be limited to the genus Frankia but there are non-Frankia actinomycetes which have shown to possess nif genes and catalyze atmospheric nitrogen to a more assimilable form. Gtari et al. [52] have reported presence of nif genes in non-Frankia actinomycetes like Slackia exigua, Rothia mucilaginosa, and Gordonobacter pamelaecae. It was verified by their capability to grow and multiply on a medium lacking nitrogen and also by their potential to reduce acetylene [53]. It has been reported that these nif gene sequences are transferred either vertically or horizontally [54, 55]. Several non-Frankia actinomycetes have been isolated till date with nitrogen fixing ability. All actinomycetes including Frankia show typical filamentous cellular morphology in their initial stage of their life cycle. Later on, these filaments show fragmentation. Apart from nitrogen fixation ability, actinomycetes species, viz., Streptomyces, Actinoplanes sp., and Micromonospora sp. reportedly have shown promotional effect on shoot growth and also enhanced actinorhizal symbiosis with Frankia. Also, these atinomycetes have been reported to produce bioactive metabolites [56].

11.2.2 Phosphate Solubilization Plant growth depends on availability of all essential nutrients of which phosphorus is one of them. Its essentiality to plants lies in the fact that it is basis for synthesis of several macromolecules, respiratory chain components, energy transduction processes, etc. [57]. Plants take up phosphorus only when available in soluble form, but unfortunately its insoluble nature in soil makes it unavailable to plants [58]. The conversion from soluble

Actinomycetes and Sustainable Agriculture  341 to insoluble form is aided by presence of large quantities of cations (Zn2+, Ca2+, etc.) [59, 60]. The problem of phosphorus precipitation has not only caused depletion in soil fertility but also great economic loss to farmers in terms of low crop productivity. These issues have instigated an urge in researchers to look out for probable solutions which culminated into the implementation of phosphate solubilizing microbes in crop farming. The category of phosphate solubilizing microbes includes a range of soil inhabiting microbes like bacteria, actinomycetes, fungi, etc. These organisms are believed to solubilize phosphorus by producing specific enzymes and making it available to plants [61, 62]. Among the different phosphorus solubilizing microbes, actinomycetes probably have an edge over other microbes firstly because of their large population numbers in rhizosphere soil than other microbes [63] and secondly because the slow, yet steady degradative activities of actinomycetes compared to bacteria or fungi [64] allow them to constantly release soluble phosphorus throughout the plant life. Actinomycetes are one such group of organisms which help improve plant development and hence crop productivity. The growth of plants and the agricultural yield from such plants finally depends on the soils’ nutritional quality and capacity of plants to uptake and assimilate those plant supplements. An important and commendable aspect of these organisms is that they have the ability to survive on the plant root exudates, which is their only source of nutrient supply [65]. In return, these actinomycetes help release soluble phosphate. The most important group of actinomycetes with phosphate solubilizing ability is Streptomyces but non-Streptomyces actinomycetes include Micromonospora endolithica [66], Gordonia, Rhodococcus, and Arthrobacter [65, 67, 68]. Actinomycetes have evolved to transform insoluble phosphates into soluble form by adapting different means. Some actinobacteria solubilize insoluble phosphates by secreting enzyme phytases. These extracellular enzymes enable degradation of phytate in a stepwise manner and belong to phosphomonoesterase group [69, 70]. Another method by which actinomycetes enable phosphate solubilization is by creating an acidic environment by producing a combination of acids like oxalic, malic, propionic acid, and gluconic acids. The acidic pH near the rhizosphere soil plays critical role in transformation of insoluble phosphates and making this essential nutrient available to plants [68, 71]. The phosphate solubilizing actinomycetes like Streptomyces griseus BH do not just act as soluble phosphate suppliers for the plants but also exhibit multitasking activities like inhibition of potential phyto pathogens including fungi, yeasts, and bacteria. Some strains of Streptomyces have shown to

342  Biofertilizers stimulate exorbitant increase in wheat aerial growth and biomass of nearly 70% in in vitro studies and to more than 30% in farm conditions when compared to controls. So, such reports are quite convincing for the development of biofertilizers and biocontrol agents from actinomycetes.

11.2.3 Potassium Solubilization Another essential element required for growth of plants is potassium. The ionic state of potassium usually absorbed by plants is required for opening and closing of stomata pores. Apart from decreasing stress effect in plants, potassium is also associated with plant detoxification process by disabling the toxic effect of reactive and unstable species of oxygen. Also, its role in various respiratory chain and metabolic processes is well known [72]. Deficiency of potassium is known to make the plant vulnerable to various plant pathogens including infestation by pests [73]. Depletion in potassium levels along with other essential plant macro nutrients is a common problem in the present situation citing reasons of intensive crop production system and use of high-yielding varieties [74]. An eco-friendly approach of integrated nutrient management system is to use potassium solubilizing microbes to alleviate the depleted potassium levels and revive the fertility of soil. These microbes enable this by using several mechanisms like exchange and complexation reaction, organic acid production followed by acidolysis, and chelation [75]. Various Streptomyces species have been reported to possess good potassium solubilizing ability, for example, Streptomyces sp. KNC-2 and Streptomyces sp. TNC-1 [76–78].

11.3 Role in Maintaining Soil Ecology Actinomycetes are known to be abundantly present in the root-soil interface of plants. These microorganisms are known to exercise favorable effects on plants. On the same note, the abundance can still be increased by application of biofertilizers prepared from actinomycetes. The approach of adding actinomycetes in form of biofertilizers would be beneficial to crop plants and enhance crop yields as demanded by the current status of agriculture which make indiscriminate application of chemicals as fertilizers leading to decline in fertility of soil and its nutritive value. Actinomycetes are known to possess certain compounds which upgrade plant growth ability and aid in maintaining ecology of the soil. These organisms have a unique potential of producing a variety of extracellular enzymes in soil like cellulase, amylase, protease, phytase, chitinase, and phosphatases [79].

Actinomycetes and Sustainable Agriculture  343 These exoenzymes help maintain the ecological health of soil by recycling the nutrients [80]. These enzymes help organisms to utilize the nutrients secreted by the plants in the plant rhizosphere soil. This activity helps to construct a nutritive pool of sugars, amino acids, peptides, organic phosphorus, and many other important supplements in the rhizosphere, hence enhancing both soil ecology and fertility. Such plant growth-promoting ability of actinomycetes can ensure extensive growth of plants and their development with respect to root length, plant height, vigor, and overall improved biomass [81]. Various species of actinomycetes are known to play critical role in rhizosphere by suppressing pathogenic species and, at the same time are also known to encourage both growth and multiplication of useful groups of soil microbes like nitrogen fixing microorganisms. For example, Streptomyces fumanus when applied or treated to wheat seeds in the form of biofertilizer, encouraged enhanced colonization of Azotobacter sp. contributing to additional inputs of nitrogen to the soil [82]. Also, actinomycetes have shown to create a balance between the rhizosphere inhabitants and stimulate secretion of growth stimulating compounds from these inhabitants which ultimately are reported to promote and improve soil ecology and hence improved plant growth. A good soil ecosystem ensures delivering all the essential services for overall plant growth and development with respect to enhanced division of root hair cells by producing growth stimulating compounds like auxins and cytokinins, thus leading to increment in numbers of lateral root hairs. This helps in developing a good and appropriate surface root system [83]. Actinomycetes are filamentous, thread-like organisms. Their intense hyphal networks allow them to penetrate through lignocellulosic organic materials like lignin and xylan and cause their degradation, thereby converting complex polymers to more simple assimilable form [84, 85]. Such degradative ability of actinomycetes plays crucial roles in production of compost from plant materials which are beneficial to plants [86]. Representatives of actinomycetes mainly Streptomyces like S. griseus, S. termoviolaceus, S. globisporus, S. albovinaceus, S. caviscabies S. setonni, S. virginiae, S. ruber, and S. viridosporus are known to secrete a combination of hydrolytic extracellular enzymes like chitinases [87], cellulases, glucanases [88], and peroxidases which aid in decomposition and degradation of polymers during composting [89, 90]. Such breakdown of polymers into small molecules helps turnover of essential nutrients like carbon and nitrogen [91]. Actinomycetes secreting extracellular enzymes like N-actyl glucosaminidase and urease help them to act on substrates like chitin and urea, enabling them to recycle soil nitrogen [92]. Their role in carbon

344  Biofertilizers cycling is with regard to their potential in solubilization of insect cuticles, exoskeleton of crustaceans, and cell walls of plants and fungi by secreting extracellular enzymes. Also, enzymes produced by these microbes cause breakdown of complex recalcitrant biomolecules like lignin and chitosan [93]. Actinomycetes are known to exert a positive effect on plants by intensifying their growth and overall performance. Biological nitrogen fixation, secretion of growth influencing elements, and production of micronutrients like vitamins are some mechanisms which actinomycetes are known to exert on plants as a part of their positive effect. Some variants of actinomycetes also appear to enhance both the quality and quantity of growthpromoting nutrients in the rhizosphere of plants which ultimately promotes growth and development [94]. There are reports which suggest that addition of actinomycetes into the plant root surroundings indeed enhances their growth and yield [95]. Inoculation of actinomycetes to plant rhizosphere is known to generate Indole Acetic Acid (IAA)—a phytohormone which acts on plant root system and stimulates nutrient consumption by the plants [31]. Also, Vurukonda et al. [96] have reported increased dry mass and higher protein content in peas when inoculated with Streptomyces. Berg et al. [97] reported that presence of actinomycetes especially certain species of Streptomyces in the strawberry plant rhizosphere reflected antagonistic effect in opposition to diverse number of fungal pathogens which usually infect and cause decline in strawberry yield. Several species of actinomycetes encourage plant growth promotion. They are believed to exhibit a positive and beneficial impact on the plant height and weight. Many species like Streptomyces sp 9K have shown to influence plant growth and dry mass of plants. Rhizosphere soil is rich in microflora. These microflora engage themselves in producing several plant growth influencing components resulting in enhanced productivity. The microbiological processes of these rhizosphere microorganisms can be additionally energized by applying biofertilizers. The application of biofertilizers stimulates microbial reproduction in soil and henceforth their magnified extracellular enzyme levels aids in maintaining influential levels of essential organic components in soil [98]. However, the impact of introducing bacterial inoculants and thereafter the consequential rise in microbiological activity in soil ultimately depends on several factors like condition and soil type, plant variety, adaptability, and survival rate of microorganisms introduced [99, 100]. There are different soil types where crops can be grown. Each soil type varies from other in its organic matter content. A good biofertilizer based on actinomycetes requires that it works under broad and wide varieties

Actinomycetes and Sustainable Agriculture  345 of soils and organic matter content. Several important representatives of actinomycetes have tremendous ability to promote growth of plants and are termed as plant growth-promoting rhizobacteria (PGPR). PGPR are believed to enhance growth of plants like sugar beet, both under green house conditions as well as under different soil types. These rhizobacteria are known to affect these promotional activities at early stages of plant growth where they have been proved to serve as suitable resources for organic and sustainable agriculture and farming [101]. Sugar beet seeds applied with nitrogen fixing and phosphate solubilizing rhizobacteria have shown to undergo drastic changes in their growth pattern and yield as compared to controls. Moreover, there was a tremendous increase in beet root weight to as high as 46.7% and also the beet sugar content by nearly 15% by such seed treatment.

11.4 Role as Biocontrol Agents The conventional pest management systems are presently facing a huge crisis [102]. The use of chemical insecticides and pesticides has not only caused serious environmental harm but also evolution of resistant pathovars and resurgence and ability to bioaccumulate are causing hazardous health issues. The recalcitrant property of these chemicals is also causing extensive damage to biodiversity. These and several other issues have prompted researchers to think of alternatives to pest control measures and thus the emergence of integrated pest management system. This system is conceptualized around biological control, which works on the principle of using living organisms to either completely inhibit or reduce the density of population of other living organisms [103]. Protection to crops throughout the crop period, environmentally safe, non-toxic to plants, and, above all, encouraging beneficial soil inhabitants are some of the commendable and impressive features of biocontrol agents [104]. Actinomycetes produce diverse chemical substances like β-lactums, peptides, and polyketides apart from a wide range of secondary metabolic compounds [24, 105]. Studies reveal that about 70% of biologically active agents are derived from actinomycetes [106, 107]. Diverse biological functions of actinomycetes to manage the plants in good health compel us to consider these organisms as good biological control agents [108, 109]. Another impressive feature of actinomycetes is that they exhibit their potential as biocontrol agent not only by existing independently in the plant rhizosphere but also as plant symbionts. So, obviously considering their bio controlling properties, various species of actinomycetes grow as

346  Biofertilizers symbionts in various plant organs as in case of rhododendron. This plant is commonly subjected to various fungal diseases just like any other plant, but experimental studies have proved rightly that rhododendron harbors various species of actinomycetes (Streptomyces sp.) as endosymbionts during its growth, helping the plant against major fungal pathogens especially Phytophthora cinnamomi and Pestalotiopsis syndowiana, suggesting the production of antifungal compounds by actinomycetes relieving the plant from attack by such fungal pathogens [110]. A simple yet impressive mechanism acquired by actinomycetes to enable their biocontrol potential for the plants is to colonize their internal structures. Most of the actinomycetes especially those belonging to Streptomyces genus enact their disease suppressing activity by occupying the intracellular plant structures as endophytes and prevent attack by phytopathogens [111].

11.4.1 Production of Antibiotics The secretion of variety of bioactive compounds by actinomycetes in rhizosphere soil of plants is believed to show pronounced positive outcome on productivity of crops. Enhanced defense mechanism and crop productivity by actinomycetes is made possible due to their mechanism of antibiosis which is enabled by production of several groups of antibiotics ranging from erythromycin, oleandromycin of macrolides group; streptomycin and kanamycin of aminoglycosides group; and nystatin and levorin belonging to polyene group [112, 113]. Some of these antibiotic producing actinomycetes genera include Streptomyces, Microtetraspora, and Actinoplanes [114, 115]. Biofertilizers based on certain actinomycetes like Streptomyces fumanus have divulged reports to successfully foster plant growth in extensive soil types and also in different host cultivars. Seeds pre-treated with actinomycetes have shown to stimulate growth and enhance yield even in low fertile soil and in low irrigated soil [116]. Many species of actinomycetes are very good antibiotic producers. The toxicological properties of these antibiotics against plant pathogens are abundant [117]. Such ability of these microorganisms probably can be exploited to inhibit specific plant diseases. The variants of actinomycetes used for such biological control depends on the plant disease. At this point, the ability of certain actinomycetes for use as both biocontrol agent and as a root-nodulating agent in leguminous plants should be well acknowledged. The actinomycetes have been known to exhibit these properties when the seeds are coated before planting or treating the seeds at the time of planting [118]. For example, antibiotic producing Streptomyces species when introduced around the alfalfa (Medicago

Actinomycetes and Sustainable Agriculture  347 sativa L.) seeds at the time of planting showed that those alfalfa seedlings which received Streptomyces amendments had produced hyphal filaments and spore chains as compared to control with no Streptomyces amendments. These strains of Streptomyces also enhanced root nodulation in the plant causing exemplary increase in nitrogen fixing ability. Moreover, the same Streptomyces species because of its antibiotic producing nature could inhibit the growth of Phoma medicaginis, causal organism of spring black stem and leaf spot disease in alfalfa. Many actinomycetes especially Streptomyces are known for their intimate relationship with plants by colonizing their internal tissues. For example, Streptomyces strains as endophytes in tomato roots have been acknowledged to produce antibacterial and antifungal elements and are responsible for impeding the growth of Rhizoctonia solani, a fungal pathogen of tomato affecting the crop and yield [50, 119]. Also, other actinobacterial strains like Microbispora sp. and Streptosporangium sp. are known to have strong relationship with maize roots and show antagonistic effect against many fungal and gram-positive bacterial pathogens [120, 121]. Alnus glutinosa is another plant which harbors Streptomyces sp. in its root nodules and this endophyte reportedly produces a novel antibiotic called naphthoquinone, which protects the host plant against attack from many bacterial and fungal pathogens suggesting the broad spectrum attribute of the antibiotic [122]. The intimate relationship of Streptomyces strains with plants like tomato and wheat suggest that harboring these organisms as endophytes within their tissues is only to derive some benefits of them, in form of secondary metabolites produced by endophytes. Also, these endophytes have an ecological edge over the competitive fungal pathogens [123]. These reports make it quite clear that many strains of actinomycetes play prominent role in plant development and also confer enhanced resistance to diseases hinting that these organisms indeed have an outstanding potential to serve as prspective biocontrol agents for many cash crops like wheat and maize [120, 124, 125]. As part of their ability to protect plants from various pathogens, reports of Streptoverticillium albireticuli possessing nematicidal activity are quite interesting. Innumerable Streptomyces representatives have been isolated and reported with ability to produce potent antihelminthic agents, thereby providing protection to plants from attack by various nematodes. For instance, Streptomyces avermitis has been disclosed to synthesize avermectins which showcase their potency against nematodic pathogens like Meloidogyne incognita [126] and Caenorhabditis elegans [127]. Avermectins have been classified as a new class of macrocyclic lactones.

348  Biofertilizers

11.4.2 Production of Siderophores Actinomycetes also possess another feature making it a unique contender as an active ingredient of biofertilizers. They synthesize small molecular weight and iron chelating molecules described as siderophores [128, 129]. Important representatives of actinomycetes producing siderophores belong to Streptomyces genus. For example, Steptomyces coelicolor is known to produce a peptide siderophore called coelichelin [130]. Another siderophore called enterobactin is reported to be produced by Streptomyces tendae [131]. There are many other representatives of actinomycetes which exhibit biocontrol activity through siderophore production. Some of which are Rhodococcus and Nocardia, which produce heterobactin type of siderophores [128]. These siderophores help scavenge ferric iron forming ferric-siderophore complexes [132]. The siderophores produced by actinomycetes are of hydroxymate type [133] possessing the capability to inhibit growth of a variety of phytopathogens. The pathogens succumb due to non-availability of iron in the rhizosphere soil environment [134] due to scavenging activity of actinomycetes like Streptomyces rochei IDWR19 and Pseudonocardia halophobica. Many different types of plants like Cicer arietinum L. (chick pea), Lens sativum (pea), and Vicia faba (faba bean) harbor different types of actinobacterial strains in their rhizoshere soil. Most of these actinomycetes are known to produce various kinds of antimicrobial components suggesting their role as biocontrol agents. Also, most of the actinomycetes obtained from these plants possess the ability to produce siderophores and phosphate solubilizing enzymes, thereby helping the concerned plants in improving their nutrient uptake from soil and thus enhanced yields. Many different pathogens like Phytophthora, Pythium irregulare, and Botrytis cinerea were suppressed when a combination of Streptomyces species strains was applied to seeds before sowing [135]. Such reports probably suggest that coinoculation of seeds with selected combination of actinomycetes possessing biocontrol activity can indeed prove to be beneficial to combat diverse plant diseases. The biocontrol action and other different attributes like growthpromoting activity and siderophore production by actinomycetes are not only restricted to cash crops like maize and wheat but also are very much prominent in fruit crops like guava [136]. Species of Streptomyces like S. canus, S. fradiae, S. avermitilis, S. Cinnamonensis, and Leifsonia poae are prominent in guava rhizoshere soil. Guava seedlings inoculated with a combination of these Streptomyces cultures reportedly caused considerable increment in both plant dry matter and height. Studies revealed that

Actinomycetes and Sustainable Agriculture  349 the culture combination of Streptomyces produced phosphate mobilizing enzymes, siderophores, and chitinases along with phytohormones like auxins and Gibberllic acid (GA3) [19, 22]. Crop plants are not only infected by primary pathogens like bacteria and fungi but are also prone to secondary infectious agents like nematodes which are commonly found in soil [137]. Several Streptomyces species are known to have brilliant antagonistic effect on nematodes as well. Streptomyces sp. CMU.MH021 is known to possess excellent potential to lessen the egg hatching rate of nematode pathogen like Meloidogyne incognita, and at the same, this nematophagous strain effectively promotes plant growth by producing IAA and hydroxamate siderophores.

11.4.3 Production of Hydrogen Cyanide Another mechanism of actinomycetes enabling it to act as a potential biocontrol agent is production of hydrogen cyanide (HCN). Several Streptomyces species have been recognized to produce HCN which inhibits the terminal electron acceptor in electron transport chain system of respiratory phyto pathogens and thereby inhibiting their growth and survival [138, 139]. Moreover, reports of actinomycetes producing HCN enhance phosphate and other mineral solubilization, thus enriching the fertility of soil is quite encouraging [140].

11.4.4 Production of Lytic Enzymes Actinomycetes have been acknowledged to secrete innumerable number of lytic enzymes [141] as one of their mechanisms to exhibit their roles as biocontrol agents. These lytic enzymes bring about the disintegration of cell wall material and hence the pathogens [142, 143]. For example, Pythium aphanidermatum, causal agent of damping-off disease in seedlings of cucumber (Cucumis sativus L.) can be inhibited by the exceptional and competent biocontrol activity of several strains of actinomycetes like Actinoplanes phillippinensis, Microbispora rosea, Micromonospora chalcea, and Streptomyces griseoloalbus. All these actinobacterial strains were shown to produce significant quantities of β-glucanases with the ability to lyse the fungal pathogen hyphae. Some strains produce diffusible inhibitory metabolites while a few have the ability to parasitize the oospores of pathogens [144]. Similarly, Streptomyces sp. 9P is another potent strain having broad spectrum antifungal properties. This strain secretes many important hydrolytic enzymes like chitinases, glucanases, and cellulases

350  Biofertilizers along with lipases and proteases, enabling it to inhibit a range of fungal phytopathogens like Alternaria brassiceae, which infects plant belonging to Brassica species; Collectotrichum gleosporioides, a common pathogen of perennial plants; Rhizoctonia solani, a pathogen with wide host range; and Phytophthora capsici, pathogen affecting peppers and other commercial crops [145]. There are many crop plants other than cash crops which add to the economy for the farmers and agriculturists. These include fruits and vegetables. Fruits and vegetables are probably more vulnerable to bacterial, fungal, and viral diseases, causing great economic loss probably due to their texture, nutritional content, etc. Considering the environmental issues and emergence of pathogen resistance to chemicals, natural and eco-friendly biocontrol agents are the need of the hour. In this very context, microbes like actinomycetes have garnered ample interest owing to their ability to produce volatile organic compounds. Although many soil inhabitants are known to produce these compounds which possess antifungal and antibacterial properties, but actinomycetes are considered good biocontrol agents with regard to this aspect as they show broad range antifungal activity and can control and sometimes prevent a variety of plant diseases [146]. The ability of actinomycetes originated volatile organic compounds to diffuse easily through the soil particles and cause severe morphological damage to the attacking pathogens [147] make these organisms to be used as a potent biocontrol agent over other conventional agents. There are several reports which corroborate with this remark. For example, volatile organic compounds produced by Streptomyces globisporus JK-1 have been reported to exert excellent antifungal activity against Botrytis cinerea, a pathogen attacking many plants like tobacco and tomato [148]. Similarly, Streptomyces coelicolor is known to inhibit spore germination of Penicillium chrysogenum and Botrytis cinerea [149]. The pathogenecity of Fusarium moniliformei can easily be damaged by volatile organic compounds from Streptomyces philanthi [73]. Another fungus Peronophythora litchii which causes litchi downy blight in litchi (Litchi chinensis Sonn) can be easily countered by volatile organic compounds produced by Streptomyces fimicarius BWL-H1 [150]. Studies reveal that Streptomyces fimicarius produces around 32 different volatile organic compounds of which important ones include phenyl ethylalcohol, caryophyllene, α-copaene, and methyl salicylate, each exerting its own systemic resistance to plants [151]. Studies confirm that volatile organic compounds like methyl 2,4,6-trichlorophenyl ether and methyl 2-methylpentanoate from Streptomyces have the ability to completely inhibit hyphal growth [152]. Also, the characteristic earthy smell of moist soil is due to volatile organic

Actinomycetes and Sustainable Agriculture  351 compounds-2-methylisoborneol and trans-1-10-dimethyl-trans-9-decalol produced by Streptomyces species [153].

11.5 Role as Plant Stress Busters Agricultural productivity depends on a number of factors including both biological and non-biological factors. The most important and significant factors which influence the crop productivity is the genetic makeup of the crop plant. The genetic makeup of a particular plant decides the productivity and hence yields of the plant, ability of the plant to resist attack of various pathogens and thus diseases, and also the genes enable the plants to resist various abiotic stresses like water scarcity, salinity, and drought conditions. Therefore, it is clear that genetic makeup of a plant is an asset as well as greatest limitation in the field of agriculture. But fortunately, the knowledge and techniques in genetic engineering have advanced leaps and bounds in containing these limitations. Development of new and better crop varieties has certainly solved these issues, of course, only when even the non-biological factors like average rainfall, humidity, and soil nutrition are present within the favorable limits considering the genetic makeup of the plant. The non-biological factors at times may limit the crop productivity and may prove to be stressful to the plants in spite of the intrinsic ability of plants to cope up with external environmental pressures. But not ignoring the power of nature, we have been provided with a diverse group of microorganisms with enormous ability. These natural inhabitants have evolved such that they can cope with a variety of adverse conditions and hence can be considered as natural mitigating agents for plants against abiotic stress. Animals including humans created a rather unique relationship with microorganisms. Plants also have shown great interaction with microbes. In fact, plant-microbe interactions are considered as important and integral in an ecosystem. Such integrated interaction between plants and microbes are believed to benefit both the partners. Plant rhizosphere creates a unique environment for microbial population while the microbes benefit the plant in various ways. They are also believed to offer defense against many abiotic stress conditions apart from overall development of [154, 155]. Plants are constantly confronted with innumerable stress factors including biotic and abiotic stress. Some of these stress elements include exposure of plants to high salinity conditions, infections from various pathogens including nematodes, attack from pests, water scarcity, and stress due to heavy metal contamination. Fortunately, plants have evolved such that

352  Biofertilizers they have an inbuilt mechanism to respond to all these stress conditions, the mechanism opted ultimately depends on type of stress. Plants produce an assortment of secondary metabolites [156] which help maintain their metabolism, and at the same time, their roots exude an important mixture of compounds into the root-soil interface which include amino acids, sugars, and organic acids. This is where the rhizosphere microbiome and plant interaction begins. The microbial population load in the plant rhizosphere interacts with roots and responds to stress by producing ethylene—a plant hormone for overall growth and development, in large quantities. Ethylene production in plants occurs at two successive phases [157, 158]. In the first phase, lower levels of ethylene production enable the plant to sense the stress and respond in a milder way. The second successive phase of ethylene peak helps the plant to overcome the stress, but such high levels also prove toxic to plants which can be seen in the form of low productivity, yellowing and falling of leaves, etc. [159]. It is at this point that the plant growth-promoting bacteria like actinomycetes help to bail out the plants from ethylene toxicity by producing 1-aminocyclopropane-1carboxylate deaminase (ACC deaminase) which acts by degradation of ethylene to non-toxic low molecular weight compound, leaving just enough ethylene to burst the stress and at the same time promote growth [160].

11.5.1 Resistance From Heavy Metal Toxicity Industrialization and persistent use of harmful chemicals have made man realize the seriousness of environment related issues. The indiscriminate use of chemicals containing heavy metals in industries and the release of hazardous industrial effluents without proper treatment into water bodies has caused serious implications on the ground water resources and to the environment as well. Heavy metal contaminated soil and water resources have taken a heavy toll on mankind. Of course, certain heavy metals like zinc, cobalt, iron, and chromium are considered as essential micronutrients necessary for growth of plants, microorganisms, and animals. But their presence exceeding even in millimolar concentrations than their required limits proves to be highly toxic to life forms. They cause various devastating effects on life forms like chromosomal aberrations and reduced growth rate. Plants usually are exposed to such heavy metal contamination stress in soils. Plants although have inbuilt mechanisms to wade off such stressful conditions, but only to a certain limit. There are some soil-borne microorganisms like actinomycetes which are naturally resistant to many heavy metals and have an excellent ability to either accumulate or remove such metals from soil, thereby serving as a

Actinomycetes and Sustainable Agriculture  353 unique heavy metal stress buster for plants [161]. The major and important representative genera of actinomycetes, i.e., Streptomyces have been reported to have an efficient bioaccumulation and biosorption mechanism for heavy metals, some of which are summarized in Table 11.1. This important characteristic feature of actinomycetes plays vital role in biological disposal of heavy metals from polluted or contaminated soils. Their ability to amass and remove particular heavy metals from soil is probably due to their Table 11.1  Actinomycetes with soil bioremediation properties. S. No.

Name

1.

Chromium

2.

3.

4.

References

Streptomyces sp. MC1

[163, 164]

Streptomyces rimosus

[165]

Amycolatopsis sp.

[166]

Zinc Streptomyces viridochromogenes

[167]

Streptomyces chromofuscus

[168]

Streptomyces ciscaucasicus

[169]

Streptomyces rimosus

[170]

Streptomyces zinciresistens

[171]

Streptomyces sp. K11

[172]

Copper Streptomyces coelicolor

[173]

Streptomyces fradiae

[174]

Streptomyces zinciresistens

[171]

Lead Streptomyces fradiae

[175]

Streptomyces rimosus

[176]

Streptomyces sp. VITSVK9

[177]

Streptomyces Sp. WW1

[178]

354  Biofertilizers extensive surface structures and enormous intracellular space [162] which other heavy metal resistant microbes lack. So, there is no reason to argue as to why actinomycetes cannot be exploited for agricultural purposes in the form of biofertilizer formulations owing to their soil bioremediation efficiency.

11.5.2 Resistance Against Drought/Water Deficit Lack of proper irrigation system and dependency on natural precipitation is a grave problem in many countries. Development of drought resistant crop varieties is of course a good solution to the problem. But developing new varieties do come with a cost. Sustainable agricultural practices using valuable microbes that may augment drought resistance of plants and enhancing crop yield even under acute water deficits may solve the issue [179]. Actinomycetes are known to grow and survive in dry and semi-dry conditions in contrast to other rhizosphere microorganisms. These organisms are adapted so well to the adverse climatic conditions that they may very well be used to compensate the crop loss due to such detrimental environmental conditions [180, 181]. Actinomycetes can colonize the roots or survive as endophytes and help the plants in increasing their survival rate and also enhance their resistance to various abiotic stresses simultaneously supporting the plants to acquire essential supplements efficiently from soil [182, 183]. Colonization of actinomycetes and other rhizosphere microbes in soil proves to be a cordial yet intense relationship which certainly enable the plants to establish properly even in water deficit soil conditions and improved fertility of soil help boost productivity [184]. So, role of such drought-tolerant actinomycetes, be it free-living, symbiotic, or endophytic [156], has caught the attention of agricultural research in recent years. Actinomycetes especially strains belonging to Streptomyces genus like S. coelicolor DE07, S. olivaceus DE10, and S. DE 27 obtained from rhizosphere soil are believed to be quite tolerant to drought and their plant growth-enhancing attributes and acute water stress tolerance attributes are quite commendable. Records of significant enhancement in wheat yields when seeds were grown in water stressed soil after treatment with these inoculants [185] provide enough evidence to prove the different attributes of actinomycetes. Many crop plants have to deal with erratic precipitation patterns. Global warming leading to lack of rains and drought-like conditions are common problems which many agriculture-based countries have to face. Lack of proper irrigation system further adds to the woes of farmers in most

Actinomycetes and Sustainable Agriculture  355 developing countries resulting in low productivity and yield. Many crop plants like onion (Allium sepa L.) have to deal with conditions like drought and osmotic stress. So, novel agricultural strategies need to be pulled up and the fact that microbes like osmotolerant actinomycetes can surely alleviate the crops from such problems [186] is quite encouraging. Osmotolerant actinobacterial strain, Citricoccus zhacaiensis B-4, has a plethora of traits ranging from plant growth promotion, secretion of zinc and phosphate solubilizing enzymes, and plant growth hormones like IAA, GA3, and ACC deaminase activity to tackle the osmotic stress conditions. The strain when applied to seeds caused a drastic increase in germination rate under stress conditions [187].

11.5.3 Resistance Toward Salinity Salinity, of the many environmental problems, is another stress factor which crop plants usually encounter. Loss in productivity due to reduced plant cell division is the common issue that plants face under salinity stress condition. Many plant crops like Oryza sativa L. have shown significant resistance by withstanding salinity stress conditions. Investigations have revealed that ability of these crop plants to encounter such stress was by the virtue of existence of endophytic actinomycetes with the characteristic feature to secrete several bioactive metabolites rendering the plants to sustain under such stressful conditions and at the same time enabling enhanced productivity. Studies reveal that actinomycetes like endophytic Streptomyces sp. GMKU 336 in plants with ability to produce ACC deaminase caused significant increase in potassium and calcium levels but decreased ethylene levels enabling Oryza sativa plants to encounter the salinity stress [188]. Additional traits like plant growth-promoting ability and siderophore production facilitated even more stability to the plants to survive under conditions like heavy metal toxicity.

11.6 Conclusion Actinomycetes by virtue of their capacity to influence plant growth and promote plant development have engrossed enormous consideration in recent years. Their interdependency with variety of plants and association with major soil inhabitants are the key factors which enables them to survive diverse and adverse environmental conditions. The remarkable characteristics of actinomycetes to create a healthy and conducive environment can be exploited and employed fruitfully for agricultural practices in

356  Biofertilizers future. Their role in nutrient recycling, providing defense to plants against pathogens and abiotic stress conditions, promoting plant growth is commendable, all of which ultimately targeted toward healthy crop plants and hence enhanced productivity. Furthermore, actinomycetes play significant role in maintaining soil equilibrium by possessing properties to remove harmful and destructive heavy metals in soil. Actinomycetes, with their competent caliber to produce several secondary metabolites like broad spectrum antibiotics, lytic enzymes, and volatile substances help in using them as potential biocontrol agents in agriculture. A dominant and principal feature of producing extracellular degradative enzymes and agroactive compounds by these organisms is effective in formation of humus and compost. The ability to confer innumerable advantages over plants and the multi-tasking actinomycetes can be efficiently used as a tool in alternate farming technique, thereby replacing harmful chemicals. It is obvious that all these qualities of actinomycetes are compelling enough to force our focus on these microbes and consider them as efficient and inevitable alternative for sustainable agricultural farming.

11.7 Future Perspectives Actinomycetes no doubt can be considered as rejuvenating agents for development of sustainable agriculture. Many important representatives of actinomycetes play critical role in healthy soil management process. The diverse characteristics of actinomycetes to benefit plant growth even under adverse environmental conditions are remarkable. Identification of effective and competitive strains of actinomycetes is the need of the hour. Also, multitasking actinobacterial strains need to be identified to cut down the economical burden in isolating and culturing single function varieties. Such actinobacterial consortium with most of the desirable characteristics will ensure improved assimilation of growth supporting nutrients by plants and hence productivity. Also, there is a need to evaluate the efficacy of these actinobacteria-based biofertilizers over a wide range of crop varieties. Needless to say, certain advanced methodological approaches are to be developed for further improving the dynamics of these organisms. Various advanced biotechnological tools can be employed to improve the standards of actinomycete-based biofertilizers. The shelf life, screening of potential carriers to support the credentials of actinomycetes, and hence the economic implications thereafter have to be evaluated and explore approaches for their mass production. The problem of creating awareness concerned to the concept of biofertilization and its beneficial effect over chemical

Actinomycetes and Sustainable Agriculture  357 fertilizers is to be properly addressed and above all taking the end-users into confidence and mobilizing them to switch over from using conventional chemical fertilizers to safe eco-friendly biofertilizers is an enormous task.

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12 Influence of Growth Pattern of Cyanobacterial Species on Biofertilizer Production Jasti Tejaswi, Kaligotla Venkata Subrahmanya Anirudh, Lalitha Rishika Majeti, Viswanatha Chaitanya Kolluru and Rajesh K. Srivastava* Department of Biotechnology, GITAM Institute of Technology, GITAM deemed to be University, Visakhapatnam, Andhra Pradesh, India

Abstract

Fertilizers play an important role in improving the yield of a crop plant. Fertilizers are needed for every type of soil as no soil will be embedded with all nutrients. Application of synthetic fertilizer will have immediate effect on plant growth and productivity along with many side effects. Application of biofertilizers will minimize the side effects and helps in increasing the fertility of the soil. Cyanobacteria are one of the most popular biofertilizers which are applied for improving the productivity of many crop plants. This chapter will focus on the morphology and growth conditions of cyanobacteria, their role as biofertilizers, and the advantages of applying the cyanobacteria and soil nitrogen fixing bacteria as fertilizers when compared with the synthetic fertilizers. Conditions for the in vitro culture of cyanobacteria and the mechanisms of gene transfer for their increased productivity were also discussed in detail. Keywords:  Biofertilizer, cyanobacteria, soil nutrition, in vitro growth conditions, gene transfer

12.1 Introduction Microalgae comprise a myriad of holophytic microorganisms with a polyphyletic and heterogeneous community of simple plants such as dinoflagellates *Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (371–392) © 2021 Scrivener Publishing LLC

371

372  Biofertilizers and blue-green algae. Excavation of the cyanobacterial fossils have shown their first occurrence on earth to be roughly around 3,500 million years ago [1]. With their global biomass constituting approximately 1,000 million tons, these photosynthetic gram-negative prokaryotes known as cyanobacteria are some of the most significant organisms inhabiting the face of the earth. The evolution of the present-day aerobically respiring living organisms was made possible gradually over a massive period of 2 billion years right from the time the first cyanobacterial species capable of performing oxygenic photosynthesis inhabited the face of the earth [2]. Cyanobacteria, also known as the blue-green algae, are some of the widely investigated organisms among the vast phytoplanktonic associations and are often found to exist in symbiotic relationships with other fungal species and plants [1]. The cyanobacteria were considered as a part of algae and were put under the division Cyanophyta since cyano in Greek meant blue-green. In concurrence with the endosymbiotic theory, the similarity between the rRNA of cyanobacteria and chloroplasts of the presentday algal and plant species proved the inheritance of chloroplasts from ancient cyanobacteria. These cyanobacteria contain cellular inclusions which perform functions similar to the plasmalemma, lamellae, and the nuclear region in the eukaryotes [3]. These organisms produce a watersoluble blue colored pigment named phycocyanins or phycobilins and also releases oxygen during the process of photosynthesis, which is comparatively more superior than bacterial photosynthesis by utilizing water as the electron donor and chlorophyll a as the primary pigment [4]. Owing to their ability of self-regulating their buoyancy and their extreme sensitivity to light, these cyanobacteria are often employed as natural indicators in water bodies. Cyanobacteria thrive optimally when the temperature lies in between 45°C and 70°C with the ideal pH range of 7.5 to 10 [5]. This trait can be fully exploited for augmenting the concentration of carbon, nitrogen, and phosphorous alongside mitigating the level of salinity in salt-affected soils. They also play a vital role in enhancing the quality of the inferior soil structure in many areas of arid and sub-arid regions [6]. These organisms are comparable to their bacterial counterparts in terms of their structure and internal physiology but their photosynthetic mechanism is analogous to aquatic plants due to the presence of photosynthetic pigments and enzymes [3]. Some of the distinguishing characteristics of cyanobacteria from other major algal groups such as diatoms, brown algae, and red algae are the absence of an outer chloroplast membrane and thylakoid associations along with the presence of cyanophycean starch and cell walls made up of a matrix of peptidoglycan [7]. Cyanobacteria largely hold two morphotypes: unicellular and filamentous forms. Both these forms give

Cyanobacteria on Biofertilizer Production  373 rise to structures that are either spherical in appearance or in the form of irregularly shaped colonies as found in Microcystis along with those that exist in bundles of filaments as observed in the genera Aphanizomenon. One defining feature of these filamentous forms is their ability to sustain hostile environmental extremities such as cold and drought through the formation of specialized spore staged differentiated cells named akinetes [1].

12.2 Habit and Habitat of Cyanobacteria Cyanobacterial species inhabit a diverse array of habitats including marine, brackish, or freshwater habitats and tend to thrive either in the form of mats, free-floating planktonic form, or also found attached to various surfaces as periphyton such as Lyngbya. Oscillatoria is one such cyanophyceae member, which flourishes near hot springs and survives up to a temperature of 62°C helping in nitrogen fixation and nutrient cycling processes. One unique property of these cyanobacteria is their ability to adjust and thrive in any soil type, thus making its distribution cosmopolitan. During the summer, spring, and early autumn, when the temperature of the water is high and there is a huge availability of nitrogen and phosphorous, these cyanobacteria spread extensively to form blooms that may either be floating (planktonic) like Pseudanabaena or found attached to the bottom of the water body (benthic) such as Phormidium. In instances where the cyanobacterial ability to remain at the high nutrient zones for its survival is threatened by high-velocity winds or water disturbances, they either float on the surface of water forming what is known as scums or may directly sink to the bottom of the water body due to the presence of air-filled vacuoles or vesicles in their cells thus adjusting their buoyancy [8]. During adverse conditions when sufficient nutrients, temperature and light intensity are lacking, the cells of cyanobacterial species such as Aphanizomenon, Microcystis, Nodularia, and Nostoc form blooms in nutrient-rich eutrophic water bodies, which tends to degenerate and die producing musty odor and clouding the surface of water bodies, resulting in the production of harmful cyanotoxins, that cause complications such as liver and neural damage, GI diseases, and a possibility of causing cancer when ingested [9].

12.3 Morphology and Mode of Reproduction Sheaths made of mucilage are primarily involved in enclosing the various forms cyanobacteria endure in such as unicellular, filamentous, or those

374  Biofertilizers that exist in colonies. Each cell is arranged in an end to end manner to form trichomes that are circumscribed by mucilaginous covering in filamentous bacteria like Tychonema. Unlike algae and other developed multicellular organisms, these cyanobacteria reproduce by simple binary fission mechanisms and not through the process of mitosis [10]. In many species of cyanobacteria, non-motile cells that are extremely resistant known as akinetes develop thick and augmented cell walls that tend to amass high quantities of protein-based reserves known as cyanophycean granules which germinate to form what is known as hormogonia or trichomes. Fragmentation of trichomes is the most common form of vegetative reproduction as seen in cyanobacteria and it also involves the differentiation of certain cells to perform functions for their propagation. When the cell division occurs perpendicular to the axial portion of the trichome, they are termed as uniseriate trichomes, whereas in those belonging to the family Stigonemataceae, the cell division occurs parallel to the longitudinal axis termed as multiseriate [11]. Hormogonia, the short and modified fragments of trichomes, may emerge inside the parental sheath and form multiple trichomes or may detach from the parental one and develops into a new filament exercising gliding movements. Essentially, two types of branching mechanisms occur based on the morphological characteristics of the external sheath and also the type of cellular division as mentioned above: true branching and false branching. True branching can be seen in Stigonema results in the formation of lateral branches, whereas, on the other hand, false branching results in diverging branches as observed in Tolypothrix. The nitrogen-fixing filamentous forms of cyanobacteria mainly consist of two distinct cell types: one which helps in the synthesis of ammonia called the heterocyst and the other which helps in reproduction and photosynthesis known as a vegetative cell. Except for Oscillatoria species, differentiated cells which act as the major sites for fixing nitrogen known as heterocysts occur in numerous filamentous species of cyanobacteria. These heterocysts are derived from vegetative cells and contain numerous minute pores called microplasmodesms that are devoid of cell envelope at both the ends of the cell helping in their connection to adjacent cells [12]. These minute pores serve as a conduit through which metabolic by-products pass through, whereas the thickened cell envelope works as a barrier for the diffusion of gases into and out of these cells. Antithetical to vegetative cells, these heterocysts lack the light-absorbing phycobilin pigment along with its photosynthetic mechanism not releasing oxygen resulting in an overall higher reducing nature of these cells. Alongside this, the presence of thick and waxy layers in the cell envelope hinders the diffusion of oxygen but allows the movement of nitrogen into the cell facilitating the process of nitrogen fixation. Heterocysts, by utilizing the organic carbon,

Cyanobacteria on Biofertilizer Production  375 transferred from adjacent cells as an energy source acts as an arena for nitrogen fixation as it provides a suitable internal environment for the action of the enzyme nitrogenase which gets hindered in the presence of oxygen [13].

12.4 Role of a Fertilizer in Plant Growth Plants obtain nutrients from the soil that are essential for the proper growth and physiological functioning of the cell. We hardly come across soils containing all the nutrients needed by the crop plants. It happens due to several reasons such as soil erosion, which results in the depletion of topsoil, therefore making the soil infertile. To overcome this problem, farmers across the globe began adding fertilizers to the soil to increase crop productivity [14]. A fertilizer is known to be a substance that comprises nitrogen, potassium, and phosphorous that are vital to improve the nutrient status and supplement the soil with all the nutrients it lacks earlier. Fertilizer, when added to the soil, nourishes it and supports the growth of a plant resulting in optimal crop yield. Besides, it helps in promoting the physical, biological, and chemical characteristics of the soil that are associated with porosity, microorganisms, and pH of the soil, respectively [15]. The addition of fertilizer in the required amount plays a crucial role in determining productivity. For instance, nitrogen, when added in low quantities, results in stunted growth whereas, on the other hand, it leads to poor leaf quality [16]. Fertilizers are broadly divided into three categories: 1. synthetic fertilizers, 2. organic fertilizers (Figure 12.1), and 3. biofertilizers. Fertilizers

Synthetic fertilizers

Organic fertilizers

Derivatives of manure Derivatives of compost

Synthetic nitrogen fertilizers

Synthetic potassium fertilizers

Derivatives of green manure

Figure 12.1  Types of fertilizers and their derivatives.

Synthetic phosphate fertilizers

NPK synthetic fertilizers

376  Biofertilizers

12.4.1 Synthetic Fertilizers These are also referred as man-made or chemical fertilizers, derived from non-living materials. They are being manufactured in factories by taking aid of the latest technology. It is non-biodegradable and can be absorbed by the plants immediately unlike those derived from natural sources [17]. It has both advantages and disadvantages. It is economical, readily available, and easily applicable. Moreover, they are prepared in different forms like pellets, granules, tablets, and spikes. Most importantly, minute quantities can be employed for obtaining the optimal levels of high yield [18]. Despite all these advantages, there might be a few limitations of artificial fertilizers that downgrade their market value. Some of the principal failures that may arise by employing specific fertilizers have been listed in Table 12.1. Since synthetic fertilizers are chemically a sort of salt, their over-application can lead to many detrimental consequences to the plants such as soil-water Table 12.1  Side effects of applying synthetic fertilizer. Type of fertilizer

Side effects

References

Synthetic nitrogen fertilizer

• Leaching of cations to lakes and rivers increases • Greenhouse gases are released • Results in natural transformation of nitrogen • Negative impact on nitrogen fixing bacteria • Causes nutrient disorders due to release of aluminium • Reduced quality of SOM • Leads to soil acidification

[16–18]

Synthetic phosphate fertilizer

• Saturation of sorption capacity of the soil • Leads to radioactivity • Has toxic elements like cadmium • A hazardous byproduct called phosphogypsum is released

[18]

Synthetic potassium fertilizer

• Releases chlorine gas into the soil that kills microbes • Causes calcium deficiency when given in excess • May lead to loss of plant structure

[17]

Cyanobacteria on Biofertilizer Production  377 pollution, susceptibility to infections along with a severe shift in its pH levels. All these negative effects can be cumulatively known as salt burn risk [19]. Leaching of the soil acts as a harmful factor as it results in the loss of more than 30% of the nutrients, which are soluble in water thus not accessible to the plants for their nutrition. Moreover, an overgrowth of vegetation in the water bodies resulting in increased levels of nitrogen leads to a phenomenon known as Eutrophication. Apart from this, several chemical fertilizers being constituted by acids such as sulfuric acid and hydrochloric acid tend to decrease the quality of the soil and cause a surge in its acidic content further causing stunted plant growth [20]. Iron being water soluble and a constituent of the fertilizer has the potential to cause rust stains on concrete if mishandled. Another major disadvantage is the unnecessary hardening and caking of the fertilizer due to its nature of absorbing moisture content in the atmosphere, therefore, emphasizing its need to be stored in dry conditions [21].

12.4.2 Organic Fertilizers To overcome these difficulties, farmers started using organic fertilizers derived from living materials. They are available in different forms such as compost, manure, and green manure. Compost is obtained from waste organic material like decayed waste, whereas manure is derived from cows, chickens, goats, and many others. Green manure is taken from growing plants, primarily diverse types of legumes. Even though they act in similar ways to synthetic fertilizer in developing the quality of the soil and yield in maximum crop productivity, they do not cause pollution in the surrounding environment. Additionally, they assist in augmenting soil composition, water retention capabilities, and resistance to soil erosion. Moreover, it helps in preventing diseases by reaching the nutritional requirements of the plant and enhancing tolerance [22]. But the requirement of it in large quantities and its slow release into the soil is the only disadvantage of organic fertilizer. Therefore, it became difficult for the farmers as they had to purchase the fertilizer in large quantities.

12.4.3 Biofertilizer The conventional farming methods included the usage of compost and manure to provide nutrients to the plant. However, they are not as powerful as synthetic or chemical fertilizers that immediately act in the soil. But the high usage of synthetic fertilizers caused environmental pollution and ended in the discharge of cancer-causing agents into the atmosphere in addition to contaminating the water bodies, which is detrimental to human beings.

378  Biofertilizers So,  later on, biofertilizers have been developed to cut down the levels of pollution and are used as a great alternative to traditional fertilizers. Biofertilizers contain living microorganisms that serve in boosting the supply or availability of principal nutrients to attain maximum productivity [23]. They are cheaper, eco-friendly, effective, and easily available to farmers. The microorganism used in biofertilizers makes the soil fertile by solubilizing the insoluble phosphates and also by fixing the nitrogen present in the atmosphere. They produce a lot of substances required for the growth of plants into the soil [24]. Firstly, nitrogen fixation is the process that involves the conversion of nitrogen present in the air into ammonia biologically, which is then converted into other compounds like nitrates and nitrites by various microorganisms present in the soil. Some of the major nitrogenfixing microorganisms available in the soil include Azotobacter, Azospirillum, and Rhizobium species [25]. Rhizobium, a symbiotic bacteria belonging to the family Rhizobiaceae, involves fixing nitrogen for leguminous plants like chickpea, and soybean. The production of ammonia occurs due to the formation of outgrowths called root nodules when these bacteria invade the roots. Also, the amount of bacteria existing in the soil is proportionate to the availability of legumes in that particular area [26]. Unlike Rhizobium, Azospirillum species do not produce any root nodules but are involved in fixing nitrogen on salts of organic acids. Moreover, they enter into the roots of plants like maize and sorghum to produce certain plant growth substances, Table 12.2  List of bacteria capable of nitrogen fixation. Funciton

Family

Species

Nitrogen Fixation

Rhizobiaceae

Rhizobium spp. Azorhizobium spp. Bradyrhizobium spp. Mesorhizobium spp. Sinorhizobium spp.

Azotobacteraceae

A. salinestri A. chroococcum A. vinelandii A. armeniacus A. beijerinckii A. paspali A. nigricans

Spirilaceae

A. amazonense A. halopraeferens A. brasilense

Cyanobacteria on Biofertilizer Production  379 whereas Azotobacter species produces antibiotics against fungal growth inside the roots that prevent various infections. The list diverse microbial species involved in the nitrogen fixation process other than cyanobacteria is provided in Table 12.2. Microorganisms that have the capacity to solubilize inorganic compounds of phosphate like hydroxyapatite, dicalcium, and tricalcium phosphate are referred to as phosphate solubilizers. Species of pseudomonas, Bacillus, Rhizobium, and Agrobacterium are the potential phosphate solubilizers. All of these microbes that come under nitrogen fixers and phosphate solubilizers can undoubtedly supply nutrients such as nitrogen, phosphorus, and potassium. But there has always been a huge requirement for micronutrients like zinc. It has been found that Bacillus species help in solubilizing insoluble compounds of zinc such as zinc oxide and zinc carbonate [22]. These are the various microbes used as biofertilizers for their respective advantages.

12.5 Cyanobacteria as Biofertilizer Cyanobacteria, also known as blue-green algae, are a collection of microorganisms having all the three fundamental characteristics of a biofertilizer. They take part in fixing nitrogen to ammonia, solubilizing phosphate compounds, and producing plant growth substances into the soil. Production of high ammonia quantities can be achieved by using cyanobacteria as biofertilizer when compared with other natural sources. So, cyanobacteria were claimed to be an effective fertilizer when compared to the ones that use other microbes [27]. Some examples of blue-green algae are Anabaena, Nostoc, Plectonema, and Oscillatoria. The activity of nitrogen fixation by cyanobacteria was witnessed only during nitrogen-deficient conditions in the soil. Under nitrogen-rich conditions, the nitrogenase enzyme required for the nitrogen fixation process remains inactive. The activity of the nitrogenase enzyme remains active up to the availability of 40-ppm nitrogen in the form of ammonia. Due to the toxicity of nitrogen as ammonia at 75 ppm, there has been repression in the activity of nitrogenase enzyme to fix nitrogen as well as to the growth of cyanobacteria. Nitrogen in the form of nitrate was not harmful and also acceptable by blue-green algae even at a concentration of 100 ppm. Organic carbon, the most significant component, is a determining factor to check the fertility or richness of the soil. In states like Punjab and Haryana, the constant usage of inorganic fertilizers has led to the exhaustion of carbon resources that resulted in leaving the soil inappropriate for cultivating crops. Later on, the researcher’s De and Sulaiman reported that microorganisms such as algae particularly

380  Biofertilizers cyanobacteria play a crucial role in building up soil organic matter [24]. Moreover, they aid in increasing the porosity of the soil due to the presence of a filamentous structure and also produce hormones such as auxins, gibberellins, amino acids, abscisic acids, and cytokinins. They aid in improving the growth of root and shoot, germination of the seed, and increasing the weight of the fruit and grain. They also help in maintaining and advancing the fertility of the soil augmenting the growth of the rice crops [28]. There are several benefits of cyanobacteria not only on rice crops but also on barley, tomato, etc., in reducing the overall salinity and increasing the quantity of phosphorous nature of the soil apart from countering the growth of weeds [29]. After nitrogen, phosphorus is considered to be the vital nutrient required by the plant in large quantities. With the use of cyanobacteria as a fertilizer, the availability of phosphorus to the plant has been more, due to the production of acidic metabolites and enzymes by cyanobacteria, which help in enhancing phosphorus content. Additionally, cyanobacteria deliver various macromolecules such as carbohydrates, proteins, and lipids into the soil during their different stages of growth. These molecules involve interacting with the particles of soil and help in tying them together. This property results in the formation of small micro aggregates, which later on form clusters. These compounds gradually develop macroaggregates that determine the stabilization of the soil [30]. In addition to this, cyanobacteria have the ability to be unaffected by any environmental pollutants and also acts as a biodegrading enzyme. Some examples of nitrogen-fixing blue-green algae that were isolated from diverse agricultural and ecological zones owning to its ability to augment rice production are Nostoc linkia, Calothrix, and Anabaena. Another plant species that exist in a symbiotic relationship with the blue-green algae is Azolla, a fern which is found in rice fields and other shallow aquatic zones, which helps in dual cropping. These ferns live independently and manufacture their food due to the presence of chloroplasts [28]. These blue-green algae also produce important organic compounds apart from their role as a biofertilizer. Anabaena and Azolla aids in spore production through the process of lignolysis, releasing phenolic compounds. Some organisms such as Nostoc and Aulosira that have ability to fix approximately 25 kg/ha of nitrogen are often utilized in paddy fields as inoculants. On the other hand, Anabaena in symbiosis with the aquatic fern Azolla has the ability to fix roughly 60 kg/ha in particular seasons [29]. Not only do cyanobacteria form symbiotic associations with ferns but they also associate with bacteria, diatoms, mosses, and animals like marine sponges. The dry green algae with their high content of macro, micronutrients, and other essential amino acids can be used in place of chemical fertilizers

Cyanobacteria on Biofertilizer Production  381 and be produced economically using sewage and brackish water. A part from acting as a nitrogen source, cyanobacteria also provide resistance to plants from harmful fungicides and pesticides and also help in restoring the quality of the soil. Based on the study conducted on soils of Ethiopia, the cyanobacterial strain inoculated in the lettuce plant could improve the growth performance and overall productivity of the crop [31]. Upon getting a collective idea on the functions of cyanobacteria, the best utilization of cyanobacteria as biofertilizers will decrease the heavy dependence on fertilizer employment and could further encourage efforts for a sustainable environment.

12.6 Production of Cyanobacteria Initially, the culturing of cyanobacteria involved its growth in association with the soil that acts as a carrier. It requires the addition of the soil-based inoculums to the rice fields. Due to its commercial benefits and simplicity, the majority of the farmers in the country utilized this method often referred to as algalization. But, this method resulted in high contamination levels and poor quality of the inoculums. To overcome the problem, glasshouse and lighter carriers like straw instead of soil have been employed. Different technologies to attain production of inoculum in large quantities have been made available. The present day technologies are soil based and straw based methods in which the growth of algae is medium specific. They include usage of trough or tank method, pit method, and field method, nursery cum algal production method [28]. The process involved in trough or tank method is explained through a flowchart (Figure 12.2). In the pit method, pits are dug instead of troughs or tanks in the ground and layered with dense polythene sheet to retain the water. Other than this, the remaining procedure is the same as in the trough method. This method is accessible and cheaper to run by small producers. When it comes to the application of the produced BGA biofertilizer, it is recommended to use half a KG of the fertilizer to an acre of rice or any other growing field. It is suggested to be used only after mixing with sufficient amount of soil. Re-application of biofertilizer should be done only after three to four hours of time. To attain maximum results half a portion of any synthetic fertilizer can be added to the rice crop. Many other advances are being made in the production technology and more reliable establishment of an inoculated organism in different soils and in proving their profitable impact on the different crop yields.

382  Biofertilizers Prepare a permanent tank

Spread soil and add water in required quantities

Add mother culture of blue green algae after 8 hours

Maintain pH depending on the nature of the soil

Allow the bacteria to grow for 10-15 days

Due to evaporation hard flakes of algae are seen

Algal flakes are collected and stored in plastic bag

They are ready to be used in the agricultural fields

Figure 12.2  Culturing of cyanobacteria through tank method.

12.7 Methods for In Vitro Culture of Cyanobacteria Cyanobacteria usually grow at a very slow rate and it is a tedious task to maintain their culture. So, in order to meet the required biomass productivity of cyanobacteria under in vitro conditions, a set of suitable and appropriate conditions need to be fulfilled. These conditions can affect the growth and composition. Some of the factors are need to be considered while culturing the cyanobacteria are provided below.

12.7.1 Macro- and Microelements Carbon is the most required nutrient, which can be taken as both inorganic and organic forms. Formation of inorganic carbon species is considered to

Cyanobacteria on Biofertilizer Production  383 be a function of both temperature and pH. Some of the sources are sugars, fatty acids, and amino acids. Cyanobacteria can be grown in a culture media containing 18% dissolved CO2. Nitrogen form in which it should be present is guided by pH and temperature, if the pH level is high, ammonia is dominant. This nutrient can have impact on composition of biomass [32]. Phosphorous is a necessary macro-nutrient for the growth, which act as growth limiting factor. Cyanobacteria can store excess amount of phosphorous in the form of polyphosphate reserves [33]. Cyanobacteria also require other macro-nutrients in significant amounts, such as sulfur (S), calcium (Ca), magnesium (Mg), and potassium (K). Micronutrients including molybdenum (Mo), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), cobalt (Co), boron (B), manganese (Mn), and chloride (Cl) are needed for their in vitro growth.

12.7.2 Temperature Temperature is an important physical factor, which have a huge impact on the oxygen evolving activity of the photosystem II (PSII), and has effects on the cyanobacterial membranes and guide nutrient availability along its uptake. Optimum temperature for biomass production varies among different genera and strain [32].

12.7.3 Light and Cell Density Light is the main source of energy for growth of cyanobacteria and helps to carry out required metabolic processes. Cyanobacterial growth rates are increased with increased light intensity but up to the point of light saturation [34]. High light intensities lead to the phenomena called photoinhibition. Mixotrophic cultures are more advantageous due to their less sensitive to light oversaturation, require less light for growth, and have higher metabolic activity.

12.7.4 Media There are different types of media used for culturing of cyanobacteria such as Hughes medium comprising of NaNO3 (1.5 g/L), K2HPO4 (0.039), MgSO4.7H2O (0.075), Na2CO3 (0.02), CaCl2 (0.027), Aedta (0.001), Citric acid (0.006), Fe citrarte (0.006), and microelements (1 ml) [35]; Chu10 medium [37] [Ca (N) 2 (0.04), K2HPO4 (0.01), MgSO4 7H20 (0.025), Na2CO3 (0.002), Na2SiO3 (0.025), Ferric citrate (0.003), and Citric acid (0.003)]; and Erdschreiber medium [NaN03 (0.1 g), Na2HPO4 (0.02 g), soil

384  Biofertilizers Table 12.3  Specific conditions for the culture of cyanobacteria. Organism

Specific culture conditions

A. variabilis

Optimum temperature is 35°C

Nannochloropsis species

Higher biomass production is observed under monochromatic LED source of blue light than red and green

Microcystis aeruginosa (UV-006, UV-010)

Showed highest growth rate at pH 9

Spirulina strains

Optimum temperatures 24–28°C and 40–42°C

Spirulina platensis

Growth at pH between 9.0 and 10.0, but grows well at 11.5

extract (50 ml) (good estuarine soft), sea water 1000 c3 (33% 0 S) (unpolluted water), vitamin B12 (0.01 to 0.05 ~g/100 ml), gibberellic acid (2.0 mg/L), streptomycin (6 mg/100 ml), tetracycline (5 mg/100 ml), sulfadiazine (4 rag/100 ml)] [36]. BG-11 medium, C-N mediums [37], and Zarouk medium are most commonly used for the in vitro growth of cyanobacteria. List of specific conditions for the growth of cyanobacteria has been listed in the Table 12.3.

12.8 Methods for Gene Transfer into Cyanobacteria Cyanobacteria are the phylum which can perform oxygenic photosynthesis falls under prokaryotes. These are visible to naked eye and contain pigments for absorption of light and present everywhere [38]. For the diversification of these kind of group inheritance of genes naturally or genes acquired by both vertical and lateral transfer methods are necessary [39]. To increase their productivity, we need to construct or develop new methods for culture and genetic manipulation are mandatory for certain strains [40]. Transformation is of two types one is Chromosomal transformation and plasmid transformation (involves introduction of foreign gene followed by cloning) [41, 42]. Cyanobacteria have also been used as biofertilizers due to their unique nature of naturally fixing N2 to the habitat [43]. We can also add other resistances to these strains using the genetic recombination [44]. Different gene transfer methods applied for gene transfer into cyanobacteria are as discussed.

Cyanobacteria on Biofertilizer Production  385

12.8.1 DNA-Mediated Transformation DNA-mediated transformation of auxotrophic valine requires (val) marker with resistance to p-fluorophenylalanine (fpar) in N. muscorum [45]. N. muscorum was grown in BG-11 medium and were tested repeatedly for purity. Followed by cloning where either spores or single cells are used, DNA was extracted from suitable donor strain. The growth of val mutant was observed along with wild type. Harvesting the recipient cells by centrifugation succeeded by washing and suspension. DNA was added and incubated under light. DNA uptake is ended by chilling the test tubes, the cells were pelleted and finally incubated. As the results indicate, maximum number of transformants was observed during the exponential period of 8–28 h. Method of transferring the herbicidal resistant gens Machete resistance (Matr) and Basalin resistance (Basr) from Gloeocapsa to N. muscorum was done by Singh et al. [51]. Here, the transformation technique of Daniell and McFadden [55] was followed along with genetic cross. Crossing experiment include Matr, Basr, Strs and Mats, Bass, Strr of both strains are taken in equal proportion. The results showed that DNAase activity had some effect on this transformation experiment compared with cross and transformation obtained by unicellular cyanobacteria [46–48]. It was proved that DNAase treatment reduces the formation of recombinants by 97%. Naturally occurring transformation was observed in Thermosynechococcus elongates, which is a thermophilic cyanobacteria. Transformation of exogenous DNA into the cyanobacterial genome by constructing suitable vectors without any damage to the host genome and also advanced new selectable marker for T. elongatus by optimizing the codons, which code for the gene kanamycin nucleotidyltransferase [49].

12.8.2 Electroporation This involves the usage of high-intensity electric field, which creates pores in cell membrane of the recipient organism, so that it can make a way for the entry of the macromolecule such as DNA [50, 51]. Electroporation has many advantages like used for large amount of cells in short period, highly efficient, less amount DNA is required, can used for non-competent cells, andis also simple [52]. By using electroporation method, shuttle vector pRL6 is introduced into Anabaena sp. Strain M131 by using plasmid RP4 for a period of 2.5-ms time constant, 8 kV cm−1 field strength maximum transformants are obtained [53]. To transform the Oscillatoria MKU 277 by introducing shuttle plasmid PRL489, 250, 500,750, and 1,000 V cm−1 electric pulses were given for 2 or 4 ms [54].

386  Biofertilizers

12.8.3 Conjugation For filamentous, N2-fixing cyanobacteria, which show heterocyst development, the method for transformation is through conjugation by using a plasmid from E. coli to the different cyanobacterial strains [32]. Conjugation technique involves three necessary plasmids independent of host and recipient, they are: 1. Conjugal Plasmids: These contains genes required for forming contact between cells and can control the moment of other two plasmids, and these can be of two types both large and small plasmids, e.g., RP4 and pRL433. 2. Helper Plasmids: To overcome the restriction endonucleases released by cyanobacteria, helper plasmids such as pRL518 and pRL623 code for methylase and mob genes (cut at bom site), which are prerequisites for conjugational transfer. This plasmid initiates the transfer. Cargo Plasmids: These are the ones that are used to transport the suitable DNA to the recipient. The main features of this plasmid are presence of bom site. There are two kinds of cargo plasmids (i) Shuttle vectors such as pRL6 has replicon from cyanobacteria and E. coli and can replicate independently and used for new gene expression [43]. (ii) Suicide vectors are used for developing knockout cyanobacteria by transposon mutagenesis or gene replacement mutagenesis by homologous recombination [54]. Anabaena PCC7120 strain is conjugated by using RP4 (conjugal plasmid) along with other two types of plasmids. This type of method is also seen in Plectonema boryanum [55] and Fremyella diplosiphon [32].

12.8.4 Biolistic Method This method is used primarily in eukaryotes cells and tissues. In this technique, DNA of interest is coated on particles of gold and tungsten of micrometer size, and these particles are targeted to the cells with high pressure caused by a gun or pressure gas. Bacterial magnetic particles (BMPs) of size 0.1- to 0.5-µm diameter are used instead of the gold or tungsten particles because BMPs can bind large quantities of DNA than the later one. BMPs coated with pSUP1021 (kmr) were bombarded at Synechococcus sp. NKBG 15041 at certain velocities by particle gun. It was noted that dry BMPs can spread well than the wet ones [56, 57].

12.9 Conclusion and Future Prospects Exhaustion of soil productivity and rising environmental pollution is a primary concern to cultivation in terms of crop yield. Cyanobacterial

Cyanobacteria on Biofertilizer Production  387 biofertilizers have the potential to act as a supplement to chemical fertilizers, thus providing an economic benefit. The use of cyanobacterial biofertilizers for rice crops would make an overall improvement in both the physicochemical properties of the soil and also increased availability of biologically fixed phosphorus and nitrogen. Besides, some of these biofertilizers also produce growth-promoting substances that help to increase the quality and grain yield. Various gene transfer methods have been described, out of which the most widely employed techniques are DNA mediated gene transfer and conjugation. Here, three types of plasmids work together for transforming the recipient organism into a transformant. By utilizing other procedures such as electroporation and biolistic (gene gun method), the insertion of a foreign gene into that organism can be accomplished, thus overcoming its natural incompetence to transform. Currently, several experimental investigations are being carried out on biological phenomena like transduction for the sole purpose of uncovering the most advanced and efficient transfer methods. Apart from this, optimal and standardized culture conditions such as pH and temperature for growing cyanobacteria in vitro needs to be understood for obtaining desirable outcomes. Many biologically active compounds such as various pigments, steroids, vitamins, amino acids, and carbohydrates have been isolated from blue-green algae since the origin of the human civilization and are now being extensively accepted as a part of pharmaceutical and nutraceutical formulations. Many of these secondary metabolites produced by cyanobacteria have medical importance due to their bioremediating potential, anti-microbial, anti-cancerous, and anti-viral drug formulating capability. Metagenomics also presents itself as a viable strategy for evading the dilemma of axenically cultivating cyanobacteria for isolating metabolites by the creation of metagenomic clone libraries of cyanobacterial nucleic acids screened for the presence of desirable genes. Even though there has been a phenomenal advancement in the way these cyanobacterial secondary metabolites are being utilized, the very thought of creating previously uncultivable strains of transgenic cyanobacteria with abilities to produce novel metabolites that may open new gates for the progression of humanity as a whole enticed several researchers toward this field.

12.10 Abbreviations B, Boron; Bas, Basalin sensitive; Basr, Basalin resistance; BGA, Blue green alga; BMPs, Bacterial magnetic particles; Ca, Calcium; CaCl2, Calcium chloride; Cl, Chloride; Co, Cobalt; Cu, Copper; DNA, Deoxynucleic acid;

388  Biofertilizers Fe, Iron; fpar, Fluorophenylalanine; K, Potassium; K2HPO4, Dipotassium hydrogen phosphate; Matr, Machete resistance; Mats, Machete sensitive; Mg, Magnesium; MgSO4.7H2O, Aqueous magnesium sulfate; Mn, Manganese; Mo, Molybdenum; Na2CO3-Sodium carbonate; Na2HPO4, Disodium hydrogen phosphate; Na2SiO3, Sodium silicate; NaN03, Sodium Nitrate; NaNO3, Sodium nitrate; Ni, Nickel; ppm, Particle per million; PSII, Photosystem II; RP4: Plasmid type; rRNA, ribosomal ribonucleic acid; S, Sulfur; Strr: Streptomycin resistance; Strs, Streptomycin sensitive; val, Valine; Zn, Zinc.

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Cyanobacteria on Biofertilizer Production  389 14. Sharma, A., Chetani, R.; A review on the effect of organic and chemical fertilizers on plants. Internat. J. Res. Appl. Sci.Eng. Technol.(IJRASET), 2017, 5, 677–680. 15. Chukwu, L. I., Ano, A. O. and Asawalam, D. O.; Effects of poultry manure and NPK fertilizer on soil properties and nutrient uptake of maize (Zea mays L.) plants growth in an Ultisol. Proceedings of the 36th Annual Conference of the Soil Science Society of Nigeria (SSSN) on 7th – 11th March. University of Nigeria Nsukka, 2012 16. Ewulo, B.S., Ojeniyi, S.O., Akanni, D.A.; Effect of poultry manure on selected soil physical and chemical properties, growth, yield and nutrient status of tomato. African Journal of Agricultural Research, 2008, 3, 612–616. 17. Liu, Y.L., Zhang, B., Cheng-liang, L., Feng, H., Velde, B.; Long term fertilization influences on clay mineral composition and ammonium adsorption in rice paddy soil. Soil Sci. Soc. Am. J., 2007. 72, 1580–1590, 18. Madani, M.J.H., Mobasser, H.R.; Effects of nitrogen fertilization and rice harvest height on agronomic yield indices of ratoon rice-berseem clover intercropping system. Aust. J. Crop Sci., 2011, 5, 566–574. 19. Guo, J.H., Liu, X.J., Zhang, Y., Shen, J.L., Han, W.X., Zhang, W.F. ; Significant acidification in major Chinese croplands. Sci., 2010, 327, 1008–1010. 20. Venterea, R.T., Groffman, P.M., Verchot, L.V., Magill, A.H., Aber, J.D., Gross nitrogen process rates in temperate forest soils exhibiting symptoms of nitrogen saturation. Forest Ecol. Manag., 2004, 196: 129–142. 21. Tripathi, S., Srivastava, P., Devi, R. S., Bhadouria, R.; Influence of synthetic fertilizers and pesticides on soil health and soil microbiology. In: Prasad, M.N.V. (eds) Agrochemicals Detection, Treatment and Remediation, Butterworth-Heinemann, 2020, pp. 25–54 22. Tirado, R., Allsopp, M.; Phosphorus in agriculture: problems and solutions. Greenp. Res. Lab. Techn. Rep., 2012, 2, 1–4. 23. Hazra, G.; Slow or controlled release fertilizers for the holistic approach to economical and environmental issues: A review. IJMER., 2014, 3, 190–208. 24. Choi, W.J., Kwak, J.H., Lim, S.S., Park, H.J., Chang, S.X., Lee, S.M., Kim, H.Y.; Synthetic fertilizer and livestock manure differently affect δ15N in the agricultural landscape: a review. Agricult. Ecosy. Environ., 2017, 237, 1–15. 25. Hazra, G.; Different types of eco-friendly fertilizers: An overview. Sustain. Environ., 2016, 1, 54. 26. Berova, M., Karanatsidis, G., Sapundzhieva, K., Nikolova, V.; Effect of organic fertilization on growth and yield of pepper plants (Capsicum annuum L.). Fol. Horticult. Ann., 2017, 22, 3–7. 27. Mishra, D., Rajvir, S., Mishra, U., Kumar, S.S.; Role of bio-fertilizer in organic agriculture: a review. Res. J. Recent Sci., 2013, 2, 39–41. 28. Venkatashwarlu, B.; Role of bio-fertilizers in organic farming: Organic farming in rain fed agriculture: Centr. instit. for dry land agricul. Hyderabad, 2008, pp.85–95.

390  Biofertilizers 29. Al, A., Ghany, M.A., Alawlaqi, M.M.; Role of biofertilizers in agriculture: a brief review. Mycopath, 2014, 11(2), 2–5. 30. Farrand, S.K., Van Berkum, P.B., Oger, P.; Agrobacterium is a definable genus of the family Rhizobiaceae. Int. J. Syst. Evol. Microbiol., 2003, 53,1681–1687. 31. Doroshenko, E.V., Boulygina, E.S., Spiridonova, E.M., Tourova, T.P., Kravchenko, I.K., Isolation and characterization of nitrogen-fixing bacteria of the genus Azospirillum from the soil of a Sphagnum peat bog. Microbiol., 2007, 76, 93–101. 32. Majeed, A., Muhammad, Z., Islam, S., Ullah, Z., Ullah, R.; Cyanobacterial application as bio-fertilizers in rice fields: role in growth promotion and crop productivity. PSM Microbiology, 2017, 2, 47–50. 33. Song, T., Martensson, L., Eriksson, T., Zheng, W., Rasmussen, U.; Biodiversity and seasonal variation of the cyanobacterial assemblage in a rice paddy field in Fujian, China. The Federat. Europ. Mater. Societ. Microbiol. Ecol. 2005, 54, 131–140. 34. Thajuddin, N., Subramanian, G.; Cyanobacterial biodiversity and potential applications in biotechnology. Curr.Sci., 2005, 89, 47–57. 35. Prasad, R.C., Prasad, B.N.; Cyanobacteria as a source Biofertilizer for sustainable agriculture in Nepal. J. Plant Sci. Botanica Orient., 2001, 127–133. 36. Sahu, D., Priyadarshani, I., Rath, I.; Cyanobacteria as potential biofertilizer. CIBTech. J. Microbiol., 2012, 1, 20–26. 37. Kaushik, B.D.; Developments in cyanobacterial biofertilizer. In Proc. Indian. Nat. Sci. Acad., 2014, 80,379–388. 38. Osman, M.E.H., El-Sheekh, M.M., El-Naggar, A.H., Gheda, S.F.; Effect of two species of cyanobacteria as biofertilizers on some metabolic activities, growth, and yield of pea plant. Biology and fertility of soils, 2010, 46, 861–875. 39. Menamo, M., Wolde, Z.; Effect of cyanobacteria application as biofertilizer on growth, yield and yield components of romaine lettuce (Lactuca sativa L.) on soils of Ethiopia. American Sci. Res. J. Eng. Technol. Sci., 2013, 4, 50–58. 40. Giorgos, M., Dimitris, G.; Cultivation of filamentous cyanobacteria (bluegreen algae) in agro-industrial wastes and wastewater. Appl. Energ., 2011, 88, 3389–3401. 41. Grobbelaar, J.U., Algal nutrition: mineral nutrition. In: Richmond, A., (eds) Handbook of micro algal culture: biotechnology and applied phycology. Oxford: Blackwell Publishing Ltd., 2004, pp.97–115. 42. Abu, G.O., Ogbonda, K.H., Aminigo, E., Optimization studies of biomass production and protein biosynthesis in a Spirulina sp. isolated from an oil-polluted flame pit in the Niger Delta. Afr. J. Biotech., 2007, 6, 2550–2554. 43. Hughese, D., Gorhamp, R., Zehnder, A.; Toxicity of a unialgal culture of Microcystis nerupinosu. Can. J. Microbiol., 1958, 4, 225–36. 44. 4mamijrthy, V.D.; Procedures adopted for the laboratory cultivation of Trichodesmium erythraeum. Marine Biology, 1974, 14, 232–234.

Cyanobacteria on Biofertilizer Production  391 45. Das., B, Singh, P.K.; The effect of 2, 4-dichlorophenoxyaceticacid on growth and nitrogen-fixation of blue-green alga Anabaenopsis raciborskii 1977, 5, 437–445. 46. Enrique, F., Alicia, M., Muro, p., John, C.M.; The Cyanobacteria: Molecular Biology, Genomics and Evolution. Caister Academic Press, 2008, pp.1–200. 47. Novichkov, P.S., Omelchenko, M.V., Gelfand, M.S., Mironov, A.A., Wolf, Y.I., Koonin, E.V.; Genome-wide molecular clock and horizontal gene transfer in bacterial evolution. J. Bacteriol., 2004, 186, 6575–6585. 48. Takano. H, Takeyama, H., Nakamura, N., Sode, K., Burgess, J.G., Manabe, E., Hirano, M., Matsunaga, T.; CO2 removal by high density culture of a marine cyanobacterium Synechococcus sp. using an improved photo bioreactor employing light-diffusing optical fibres. Applic. Biochemis. Biotechnol., 1992,34/35, 449–458. 49. Golden, J.W., Wiest, D.R.; Genome rearrangement and nitrogen fixation in Anabaena blocked by inactivation of xisA gene. Sci. 1988, 242, 1421–1423. 50. Elhai, J., Wolk, C.P.; A versatile class of positive-selection vectors based on the non-viability of palindrome-containing plasmids that allow cloning into long polylinkers. Gene, 1988, 68, 119–138. 51. Singh, R.N.; In: The role of blue green algae in nitrogen economy of Indian agriculture. Ind. Counc.Agricul.l Res., New Delhi, India, 1961, pp. 17–19. 52. Singh, P.K.; Algicidal effect of 2, 4-dichlorophenoxy acetic acid on blue green alga Cylindrospermum sp. Arch. Microbiology 1974, 97, 69–82. 53. Keshav, T., Umakant, S.; DNA-mediated transformation in Nostoc muscorum, a nitrogen-fixing Cyanobacterium. Aust. J. Biol. Sci., 1982, 35, 573–577. 54. Singh, D.T., Nirmala, K., Modi, D.R., Katiyar, S., Singh, H.N.; Genetic transfer of herbicide resistance gene(s) from Gloeocapsa sp. to Nostoc muscorum. Molec. Gen. Genet., 1987, 208, 436–438. 55. Daniell, H., Mc Fadden, B.A.; Characterization of DNA uptake by the cyanobacterium Anacystis nidulans. Molec. Gen. Genet., 1986, 204, 243–248. 56. Keshav, T., Umakant, S.; Genetic transfer in a nitrogen-fixing filamentous cyanobacterium. J. Gen. Microbiol., 1981.124,349–352. 57. George, J., Sreekala, M.S., Thomas, S.A.; A review on interface modification and characterization of natural fiber reinforced plastic composites. Polym. Eng. Sci., 2001, 41, 1471.

13 Biofertilizers Application in Agriculture: A Viable Option to Chemical Fertilizers Rajesh K. Srivastava

*

Department of Biotechnology, GIT, Gandhi Institute of Technology and Management (GITAM) Deemed to be University, Rushikonda, Visakhapatnam, India

Abstract

In the current period, the use of beneficial microbial strains (such as cyanobacteria) has been exploited as a biofertilizer for maintenance of crop growth and production with good soil structure and compositions. Biofertilizer application in our agricultural sectors has now gained more attention of many farmers to minimize or to stop the adverse effects of chemical fertilizers. In modern agriculture practices, lot of chemical fertilizers and other soil nutrients are added for better crops productivity that has caused the gradual loss of soil health. People have developed the practices for soil management for maintaining the healthy soil conditions for crop growth, and in this regard, cyanobacterial strain as biofertilizer has been added for more food safety and sustainable crops production. As biofertilizers, plant growth-­inducing or promotion capability rhizobacteria, ecto as well as endo mycorrhizal fungal species, and cyanobacterial strain and other useful microorganisms are applied in soil fertility maintenance that can help in improving in nutrient uptake capability, induced plant growth, and plant tolerance capacity to biotic or abiotic nature of stresses in soil. Further, biofertilizer can enhance the plant defense system with its protection. Further biofertilizer functions are reported to gene regulation signaling network for cellular pathways or cellular responses that help in crop productivity improvement. The author will emphasize the different factors that can develop the various types of biofertilizers for crop improvement. Keywords:  Biofertilizer, cyanobacteria, factors, soil, fertility, adverse effect, chemical fertilizer Email: [email protected]

*

Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (393–412) © 2021 Scrivener Publishing LLC

393

394  Biofertilizers

13.1 Introduction Chemical fertilizer is made from inorganic compound and petroleum products, rocks, other organic sources, and it can help in improvement in soil fertility in fast ways and it can monitor of significant advantage of chemical fertilizer that produced desired nutrient in low cost. Chemical fertilizer can be manipulated at industrial level and is basically contained of known amount or weight of nitrogen, potassium, and phosphorus elements, and their exploitation is reported to cause air or ground water pollution with promotion of eutrophication processes in water bodies [1]. Recent efforts have done to channelize the production of nutrient rich and high quality of food in sustainable mode the production of nutrient-­ rich high quality food in sustainable manner to confirm or ensure more biosafety. Various types of innovative farm production strategies has been found to more demands of organic and biological-based fertilizers alternative to chemical or agro-fertilizers. It has reported that soil fertility can rely on organic matters input for improving of nutrient supply with conserving of soil or field management. In this regard, one innovative approach (organic farming) is applied for ensuring food safety and enhanced soil biodiversity that can achieve additional advantage (shelf life without any adverse effects to ecosystem) from application of biofertilizers [2]. This organic farming can dependent on presence of natural microflora of soil [beneficial bacterial species or fungal species with include of arbuscular mycorrhizal fungi species (AMF)]. This special type of fungi is called as plant growth promoting capability rhizobacteria (PGGR). The biofertilizer can maintain the good health condition and also maintained the soil environment more nutritive (means of richness in all the types of micro- or macroelements as nutritive sources) via helping in N2-fixation process as well as solubilization or mineralization processes of P or K elements in soils. These processes can induce or promote the release of growth regulating substance as well as antibiotics production. Further, biodegradation process of organic matters or compounds is reported in biofertilizer added in the soils. Biofertilizers can be added as inoculums in soils and they can go for multiplication processes via participation in nutrient cycling processes with benefits of enhanced crop productivity. In case of added chemical fertilizer in soil, it has reported that 60% to 90% of total applied  chemical fertilizers is found to loss outside the soil surfaces and residual chemical fertilizer is reported to use by plants for their growth [3]. There are many microbial strains such as Azotobacter, Azosprillum, Rhizobium, and Cyanobacteria that are used as biofertilizer microbial

Biofertilizers Application in Agriculture  395 inoculums with potassium or potassium solubilizing microbial strain and also mycorrhizae. These strains are known as PGPR group and is found to enhance their number in soil under or below without any tillage material or least tillage treatment. Effective microbial strains of Azotobacter, Azosprillum, Phosphobacter, or strains of Rhizobacter can help for providing the N2-element to some plants such as Helianthus annus that helped to enhance the height of plant, with its leave numbers and stem diameter. Further, % of seed filling capacity and more seed dry weight is also reported from these strains application. These biofertilizers influences have seen in rice crop with promotion of physiology and improvement in root morphology from Azotobacter, Azosprillum or Rhizobacter application in soil [4]. Biofertilizers are organic substrates, containing beneficial microbes, and these have helped in promoting the plants or tree growth via increased supply of nutrients to plants. Microbial strains in biofertilizer mycorrhizal fungi, blue-green algae, microalgae, and bacteria in lived condition. It has found that mycorrhizal groups fungi has capability to preferential withdraw the mineral from organic matter for plant growth and cyanobacteria is exhibited by property of nitrogen fixation. In nitrogen fixation, dinitrogen molecules are converted into nitrogen compounds and some bacteria has capability to convert insoluble phosphate of soil into soluble forms [5, 6]. Rhizobium is one good example of vital symbiotic nitrogen fixing bacteria and this bacterium can get shelter with food from plants, and then, it returns the fixation of nitrogen to plants. Another nitrogen fixing bacteria is Azospirillum that lived in roots of higher plants without developing any relation to plants and it is called as rhizosphere association with collection of plant extrude that is used as food by them [7]. Rhizosphere association processes is known as associative mutualism. It has seen that cyanobacteria (i.e., blue-green bacteria) are found from symbiotic association with several plants (such as Liversworts, cycad root, fern, or lichen). Nitrogen fixing cyanobacteria is reported as Anabaena species that is found at leaf cavities of fern with responsible for nitrogen fixation. It is found that decay of fern plants (Azolla pinnate) can release it and utilized its residue by rice plants without any regulation of plant growth [7]. It has found as free-living soil bacteria with performing of nitrogen fixation. There are saprotrophic anaerobes bacteria such as Clostridium, Beijerinckii, Azobacteria, or Bacillus polymyxin that are used as inoculums of biofertilizer and Azobacteria and Azospirillum are widely used microbial in biofertilizer preparation or production [7, 8]. There are four species or strain Trichoderma that was isolated from natural sources (like wood) and is applied to make the biofertilizer. It has

396  Biofertilizers shown some good results after 30 days treatment in plant (flowering Cheese cabbage) growth and development such as enhanced rate of seed germination (22.5%), more plant height (24.4%), fresh plant weight ( 42%), as well as crop yields (37.4%) after comparing to control plant growth profiles [9]. Further improvement in soluble sugar contents (2.04%), soluble protein (6%), and chlorophyll contents (2.8 mg/g) in same plants is reported with this biofertilizer for 30 days compared to control (1.74%, 5.61%, and 2.64 mg/g). Content of nitric has showed in small size in blue area than control plant surfaces study and is concluded that biofertilizer can enhance the tolerance capacity of flowering of Chinese cabbage against to environmental stresses [9, 10]. Increased enzymes activity in soil at 30th days of treatment is reported that enhanced activity of urease enzyme (25%), phosphatase (13%), and also catalyze enzymes (14%) in soil environment with providing of more inorganic N and P element with reduction of harm effects to flowering Chinese cabbage. Addition of Trichoderma as biofertlizers agent has enhanced nutrient uptake and tolerance capacity against stresses with improved quality, antioxidant activity as well as production in flowering Chinese cabbage. Further, this biofertilizer improved or promoted the plant growth or crops growth and its yield of this cabbage plants with maintaining of soil quality [10, 11]. Excessive application of biofertilizer in soil can reduce the use of chemical fertilizer (after excessive amount in soil) with reduction of pollution in environment component (such as water resources). There are various impacts of Trichoderma-enrich biofertilizer (BioF) reported. For example, BioF/­compost that contained household or kitchen wastes with Trichoderma harzaianum T22 and BioF/liquid form that contained T. harzaianum T22 strain culture in liquid nature media solution (broth) have been checked their impacts on growth profile, yield capacity, or nutrition rich with quality of Lycopersicon esculentum growth, yield, or nutritional quality of Lycopersicon esculentum (tomato plant) in field trial studies [12, 13]. It has shown the encouraged responses with enhanced 200% to 336% of yield by use of BioF/compost in alone form (T3) or combination with some elements N, P, K applications (T4), respectively [13]. Further, application of 50% BioF/compost as well as 50% BioF/liquid form with 50% combination of some elements such N, P, or K has reported has shown significant (P < 0.05) performance over control. Higher content of total organic or inorganic soluble solid matter including sugar, ascorbic acids, β-carotene, lycopene, or Mn in tomato with improved tomato productivity is reported with application of BioF/compost. Further, protein content and some essential mineral sources were also increased by 50% BioF/compost

Biofertilizers Application in Agriculture  397 with 50% combination of some useful elements like N, P, or K [14]. Author will discuss the different factors of biofertilizer productions and its beneficial impacts on soil fertility and food safety for world.

13.2 Chemical Fertilizer Chemical fertilizers are sources of some essential elements (N, P, K, or others) in high concentration, reported as compounds form, and these compounds are needed for promotion of plant growth. This fertilizer contained the three important elements C, H, or O in their structure or compositions and is utilized in more quantity as nutrients for plant growth. Plant nutrients are reported as primary or secondary nutrient and micronutrients. These fertilizers are involved in production of primary nutrient suitable for healthy soil conditions and are shown in Table 13.1. In category of nitrogen fertilizer, nitrogen or N-element is found as ammoniacal nitrogen (i.e., ammonium sulfate, or ammonium chloride), nitrate nitrogen (calcium ammonium nitrates), or amide nitrogen (urea) and is shown in Table 13.1 [15]. Another category of chemical fertilizer is phosphatic fertilizer and it contained available phosphate forms [i.e., single super phosphates and triple super phosphate (TSP)]. Potassium containing fertilizer is muriate (potassium chloride) as well as potash sulfate and these helped to provide the K-element to soil. Fertilizers are grouped. Sometimes, farmer used the chemical fertilizers in complex or mixed form that can help in providing more than one nutrients to soil [16]. Application of chemical fertilizers is found for food production, and these fertilizer industries growths are dependent on effective forms of Table 13.1  Various forms of chemical fertilizers used in crops production [15]. S. no.

Types of chemical fertilizer

Examples

1.

Nitrogen straight fertilizer

Urea, Ammonium sulfate, Ammonium chloride, Calcium ammonium nitrate (CNS)

2.

Phosphatic straight fertilizer

Single super phosphate (SSP), Triple super phosphate (TSP)

3.

Complex and mixed fertilizers

Di-ammonium phosphate, Nitrophosphate (NP), different blends of NPK fertilizers

398  Biofertilizers respective available nutrient to the crops growth. The manufacture and usage of chemical fertilizers have shown more impacts on polluting our environment and also its low efficiency operation. Enhanced rate of chemical fertilizer production and its usage in crop lands is responsible for releasing its residual parts to the environment including water sources. So, effective control in the fertilizer industry can be found the proper measures or approaches for healthy environment. It is needed for some effective recommendation for implementation of control measures as well as waste reduction potentials and is shown in Table 13.2 [17]. In India, its government agency has taken the many procedures for providing the financial benefits or aids to farmer people for this country development. These steps are given below. Promotion for biofertilizers production is done with financial supports for establishing the biofertilizers synthesis plants or unit, and subsidy was given at the rate of 25% of the total financial benefits or supports up to the value of maximum Rs. 40 lakhs via government agency such as NABARD. They have also supported the financial assistance or benefits up to 50% of total cost or Rs. 100/hectare rate for farmers that promoted the biofertilizers through scheme of integrated scheme for supports to crops of oilseeds, oil palm and maize crops (ISOPOM) [18]. It is also reported that they provided the financial assistant of Rs. 150/ hectare under the scheme of accelerated pulses crops productions via program through National Food Security Mission (NFSM). Government has also encouraged the producer of organic biofertilizers with support of scheme for financial benefits or assistance to set up the production plants or unit with following guidelines: Table 13.2  The details of sales of chemical fertilizers during two years 2012–2013 and 2013–2014 (values in lakh Metric tons) [17]. S. no

Products

Years

Sales

1.

Urea

2012–2013 2013–2014

301.6 173.9

2.

DAP

2012–2013 2013–2014

92.3 40

3.

MOP

2012–2013 2013–2014

21.3 12.4

4.

NPK

2012–2013 2013–2014

77.3 39.3

Biofertilizers Application in Agriculture  399 a) Government has provided the financial assistance to farmers under the National Project on Organic Farming (NPOF) scheme as credit link back subsidy via NABARD. Farmer can set up the fruit or vegetable processed wastes or agrowastes components via compost unit with providing of subsidy benefits up to 33% of total cost with maximum limits supports to Rs. 60 lakhs per unit. b) Government has provided the financial assistance with 50% subsidy to farmer under National Horticulture and they can set up the vermin-compost units up to maximum support of Rs. 30,000 per beneficiary in India. c) National project scheme of Indian government for management of soil health and its fertility has supported with financial aids or assistance to most the farmers for promotion of biofertilizers via integrated nutrient management (INM) scheme [19]. Government of India has also promoted the balance use of chemical fertilizers with above-mentioned subsidy to farmers for development of biofertilizers. In year of 2008–2009, promotion of balanced or controlled with judicious guideline for application of chemical fertilizers in combination with organic nutrient matter or manures is provided on the basis of test reports of different soil profiles under the scheme of national project on management of soil health and their fertility. For this task, soil tests are done or set up under new static soil testing laboratory and also new mobile testing laboratory facility that can provide the strength to existing soil testing program under existing soil test laboratory facilities. These have some major components under this scheme [18, 19]. ◦◦ It can promote the strengthening of soil testing service. ◦◦ It has induced for setup or strengthens the facility of static/ mobile soil testing laboratory (STLs). ◦◦ It promoted the training or field demonstration on balance use of fertilizers. ◦◦ Industrial government has promoted the INM for improvement and maintenance of soil healthy and crop or productivity. ◦◦ Indian Institute of Soil Science (IISS) has promoted the scheme or approaches for preparation of geo-referenced maps facility for interlinking approaches for testing the soil

400  Biofertilizers Table 13.3  Sales of urea and N or K fertilizers shown for year of 2010–2012–2013 (in Rs. Crores) in India [21]. S. no.

Products

Years (2010–2011)

Years (2011–2012)

Years (2012–2013)

1.

Urea

24,336

37,760

40,016

2.

P and K

41,500

36,809

30,576

3.

Total

65,836

74,569

70,592

fertility status through the scheme of soil test crop residues data (STCR) basis with implementation of rules or recommendation for site specific field locations or major cities in India [20]. Author will discuss some important information regarding the fertilizer nature.

13.2.1 Customized Fertilizers It has shown some aims for promotion of site specific with nutrient management procedure to achieve the maximum quantity of uses of chemical fertilizer efficiency. These chemical fertilizers are reported for carrier of multi-nutrient sources with designed to consist of micro- or macronutrient elements sources for application in soil to promote the crops growth and this is found to basis of formulation of nutrients combinations with gained of results via soil test. In this promotion of customized fertilizer, the Department of Agriculture and Cooperation (DAC) has been surveyed for the 25 fertilizers’ impacts via soil testing [21].

13.2.2 Fortified Fertilizer It can encourage the micronutrient use in fertilizer, and DAC has promoted the fortified fertilizers with micronutrients such as Zn, B, along with NPK, and is shown in Table 13.3 [20].

13.3 Biofertilizers Biofertilizer can be any substance that contained microbial cells with capacity to promote plant or tree growths via helping the increased supply

Biofertilizers Application in Agriculture  401 of nutrients to plants. Lots of living organisms are reported that mycorrhizal fungi, blue-green alga, and bacteria. Mycorrhizal fungi exhibited their capability to withdraw the minerals components from organic matters for plant growth [12]. Efficient and suitable microbial strains can be exploited for biofertilizer production tasks via providing the alternative option to chemical fertilizers in modern agricultural sector with highly potential agents and maintaining of soil health. Currently, procedure or methods for biofertilizer development is costly or expensive. But, biofertilizers application is crop production can enhance crop productivity with ensuring of food safety. For production of biofertilizers, selection of some microbial strains that have developed plant growth promoting capacity and bacteria, fungi, or cyanobacteria is utilized for efficient biofertilizers development in farming fields. In this regard, lots of extensive works on biofertilizers production are done that have supported the information for good crops yield and can be seen in Figure 13.1 [22]. There are various mechanisms involved in effectiveness of biofertilizers application in crop cultivation can enhance the crop yield and food safety with promotion of plant growth. It has also provided the protections

Isolation microoganism (Rhizobium, Azotobacter, Azosprillum or Cyanobacteria) from soils In-vitro screening of microbial isolates for plant growth potentials or capability Screening for microbial isolates of plant growth in plotted soils under green house conditions Field trails of highly potential or efficient microbial isolates in farming or cropping soils Refinement of highly potential or efficient microbial isolates Biofertilizers production for crops

Figure 13.1  Schematic diagram of biofertilizer manufactures.

402  Biofertilizers against different plants protection. Biofertilizers have exhibited critical roles or impacts and its application in broad sectors (includes bioremediations, ecology, or agriculture). Control of chemical fertilizer can be done by using microbial bioagents and is found to eco-friendly components under crop management programs [23]. It has shown the major problems of inconsistence nature during its application in field due to their effect in microbial growth pattern and it is dependent on multiples biotic or abiotic parameters or factors (i.e., low concentration of organic matter, soil moisture percentages, salinity level, pH effect, or temperature). These factors in soil structure or composition are utilized by several individual microbial strains that can apply as biocontrol agent against plant-pathogens [24]. Several individual microbial strains are reported to apply as biocontrol agents that can be controlled against plant pathogens. In cropping field, it has shown the limited application due their results as inconsistence effective as biocontrol agent, and sometimes, it has reported unpredictable outcome or results due to different soil conditions. It has found that application of two or more types of microbial strains (that show the capability of fungal and rhizobacterial strains that can promote the plant growth) against to plant pathogens. These effective microbial strains can provide the better options for improving the performance of biocontrol agents against the pathogens in natural soil habitat (i.e., R. solani) and be seen in Figure 13.2 [25]. The Rhizosphere relation in soils and roots can develop the interface to promote the microbial activity as hub centre. It can provide the better mechanisms that can help for understanding the effects of inoculums of microbial strains in host rhizospheres on pathogen and its effects as biocontrol agents. The rhizosphere can exhibit the direct antagonism via enhancing the growth of plants and levels of induced or enhanced resistances properties that can be found by using of microbial strains

Protect the environment from pollutants as natural fertilizers. Eco-friendly and cost-effective Destroy many harmful substances present in the soil, causing plant diseases

Biofertilizers properties

Improve soil texture and yield of plants

Proved to be effective even under semi-arid conditions

Not allow pathogens to flourish

Figure 13.2  Properties of biofertilizer exhibited for plant growth.

Biofertilizers Application in Agriculture  403 (i.e., species of Trichoderma, Hypocrea, or Gliocladium) against several crops pathogenic agents [26]. In modern agriculture practices, various types of inorganic or organic fertilizers are used with pesticides or insecticides. It required effective microbial inoculants and good impacts of microbial structures such as Trichoderma species that has shown capability of higher reproduction rate or growth stability in extreme conditions [27]. It has reported that this microbial strain exhibited broad range of substrate utilization efficiency and capability to transform the rhizosphere and other existing banks and control the pathogen by antagonism. Endophytic bacterial species with plant association are reported to fixation of atmospheric nitrogen with help in enhanced growth of plants. Inoculations with diazotrophic bacteria can also provide the alternative options with dropping of N-fertilizer needs [28]. It is also needed to understand the plant-­microbial interaction for development of bacteria based fertilizers. Advanced research works are focusing on morphological characters of microbial MYSP113 with evaluation of its beneficial interactions with sugarcane plants. It needed to study the mechanism of plant growth promotion (PGP) processes with their interaction. In this regard, two big groups of antagonistic nature bacterial strain (i.e., Bacillus and Pseudomonas species) are generally applied microbial system against plant pathogens system [29]. These strains of bacteria is reported to inhibit the growth of pathogen agents in crops via various ways of antimicrobial nature exopolysaccharides (ExoPS) or antibiotics compounds secretions. The Bacillus species or strains can produce with secretion of an antibiotics compounds (e.g., bacillomycin, surfactin, fengycin, or iturin types of antimicrobial agents) that can apply against growth of pathogenic agents. From last few decades in agriculture history, usage of chemical fertilizers and pesticides were found in boosting of agro-production [30]. Their special attributes or properties are quick, non-specifics actions, and less expensive or low cost production and storage that made the convenience to farmer. Resistance cultivars can be found most economic manner and long-term approaches to control the diseases of plant during its growth, and it can be found to boost or promote the process with application of inoculums of microbial strains [30, 31]. Biofertilizer is made up of the following types.

13.3.1 Biocompost It is eco-friendly biofertilizers and is composed of waste matters from sugar industries and allowed for decomposition. It is enriched by human friendly bacteria, fungi, and various plants. Application of biocompost in soil can help full or complete complement of nutritive nourishment with

404  Biofertilizers improvement in oil plant health in sustainable mode growth promotion of cropped plants. It has helped in providing of micro- and macronutrients with high organic matter contents and higher microbial activity [32, 33]. This biocompost has enhanced the enzymes activities of the plants growth via increasing rate of chlorophyll biosynthesis that can be resultant in high productivity and quality of fruits. It has increased the uptake rate of micronutrient with increased levels of NPK in biocompost treated field [34]. This biocompost is prepared from distillery effluents in agriculture via lowering its cost of production. It has facilitated in reduction of pollution loads on aquatic ecosystem. Effect of biocompost application is reported on potato plant growth. It has shown the slight changes over chemical fertilizers (100% NPK) affect quantity of copper, iron, manganese, and zinc due to application of biocompost in field [35].

13.3.2 Trichocard This biofertlizers agent is reported to eco-friendly and nonpathogenic matters or substances, used for various types of plants, horticulture, or ornamental plant growth. Paddy, apple, sugarcane, brinjal, corn, cottons, and vegetables are main plants that used Trichocard type of fertilizer for insect control and growth promotion of plants. This biofertilizer is functioned as destroyer or antagonistic hyper parasitic against eggs of several bores, fruit, leaves, fruits eaters, or others pathogens in the cropping field [36]. In Trichocard types of biofertilizer, Trichogramma species are used, and it is small-group wasps that are endoparasites of lipidopteran eggs. This species has shown majors advantages to exhibit the biological controls agents and is shown their very small size of adult eggs (their parasitoids size measures ~ 0.3 mm in length) that made it invisible to the casual observer [37]. This species has shown the best agents for prevention mode treatment on reinfested packaged products materials. It has capability to lay of their eggs on lepidopteran insect that killed the developing moth embryo before it batched and it has helped in prevention with damaging of larval stages. It has found that parasitoid larva is reported to consume the contents of the eggs or pupates that emerged as a wasps in 7 to 14 days [38]. It has reported that adult parasitoids are mated in very short periods after emergence and a single female wasp is shown the capability of parasiting up to 50 or more eggs in her adults for life span of 3 to 14 days. Trichogramma species is reported to forage while walking on a substrates and wasps are reported to release their parasitoids eggs that affixed to a card via storing at temperature of 8°C to 12°C for periods of 7 days [39].

Biofertilizers Application in Agriculture  405

13.3.3 Trichocard Production • In Trichocards formation, female Trichogramma is laid her eggs. These eggs can be grown there and completed their life cycles. Then insect is reported to hatch the eggs and came out [38]. • Later, Trichogramma is supplemented in the form of cards and one card can contain around 20,000 eggs and is used to destroy shoot borer, fruit borer, or leaf borer insects of paddy, maze, sugarcane sunflower, cotton, pulses fruits or vegetables that can prevent nearly 80% to 90% losses [39]. • It is necessary to keep the Trichogramma species at 5 to 10 °C in ice box or refrigerator before applying to fields. Later is applied for different type of crops 3 to 4 times at the interval of 10-15 days. It can work as soon as possible once eggs of harmful insects appears in fields that tied in small pieces of cards immediately in different parts of the fields on the lower surfaces of leaves or at joints of leaf or stems. • It can use the five cards per hectare in normal crops and then 10 cards per hectare in bigs (i.e., in sugarcane) and it is applied in evening in the fields but do not spray any chemical pesticides before during or after its use [37].

13.3.4 Azotobacter This fertilizer protects the roots from pathogens from soil environment and play important roles in N2− fixation. N2 (nitrogen) is reported a important element or nutrient components for plant and is reported the 75% nitrogen is comprised of total atmosphere. Eco-friendly nature of biofertilizer is found to use as alternative to chemical fertilizers in fish culture or aquaculture, and it has represented to an important aim or target to minimize the water pollution and also improved the water quality via enhancing the growth rate of fishes. Azotobacter species is free living microbial cells that exhibited the N2− fixation ability of diazotroph. It has shown the several beneficial or positive effects on the crop growth or its yield in agriculture sector. This can be helped in biosynthesis of growth regulatory substances such as auxin, cyokinin, and Giberellic acid (GA). It can stimulate the rhizospheric microbes growth via protecting the plant from phytopathogen via improving the nutrient uptakes and boosting of nitrogen fixation processes. It can promote the quantity or number of these bacterial species with their abundance in soil with many factors such as soil pH or soil fertility [40]. In Azobacter species, there is type of biofertilizer

406  Biofertilizers that has shown the impact of inoculation mode or age and it is two types of strains that are Azospirillum brasilense and Azotobacterium chroocococcum; and it is reported on bacterial counts, chemical characteristics of water, specific growth rate (SGR), Aspartate aminotransferase activity (AST), Alaninine aminotransferase profile (ALT), and mode of histopathological changes [41]. It has reported that profile of water activity analysis results such as consumption of dissolved oxygen (DO) profile in cultivation media, biochemical oxygen demand (BOD) profile, or chemical oxygen demand (COS) profile with nitrogen phosphorus profile, NO3-N or O-phosphate groups levels are found to increase by treatment of Azotobacter microbial strain. Azosprillum treatment has given lower levels compared to Azotobacter. It was also reported that mixed or single bacterial treatment can increase the fish SGR with mixed or single bacterial treatments increased (35% more). Mixed bacterial treatment can improve water parameters and ALT and AST level with some histopathological lesions in fishes than single treatment. It can conclude that single inoculation of Azotobacter as biofertilizers can be suitable probiotics and reported in use in Orechromis niloticus aquacultures [40, 41].

13.3.5 Phosphorus Phosphorus-based fertilizers is used for determination of total nitrogen levels in the soil, and, sometimes, soil needs more quantity of nitrogen and less quantity of nitrogen. It released the phosphorus into soil the settle in clay minerals. Phosphorus can be found to play contribution or role in plant growth and its development with reduction of deficiency in plant growth. Soil is reported to posses the total quantity of phosphorus element in form of inorganic or organic matters that were present in inactive form and it not available to plant [42]. It has found that many farmers did not afford the use of P-fertilizers in case of deficiency. So, it can provide the alternative technique to provide the phosphorus. For this phosphorus availability, phosphate solubilization microbes (PSMs) are used that can help in hydrolyzing the organic or inorganic insoluble phosphorus to soluble P form and can easily assimilated by plants. These PSM group microbes are beneficial groups in eco-friendly and economically sound approaches for overcoming the P-scarcity and its uptake by plants [42, 43].

13.3.6 Vermicompost This fertilizer is reported to eco-friendly organic matters with comprises of vitamins, hormones, organic carbons, sulfur, or antibiotics that helped

Biofertilizers Application in Agriculture  407 in increasing in quantity and quality of yield. It has shown the capacity of quick fixation to improve the fertility of soil. Vermicomposts are reported as sources of necessary organic matters that are plant and or animal origin residual parts and also reported to consist of fine divided earthworm coating and produced nonthermophically processes with biooxidation as well as stabilization of the organic material. This biofertilizer is produced due to interactions between aerobic nature of microbial strains and earthworms, as this material passed through the earthgut [44] and is shown in Figure 13.3. Vermicompost can provide or supply the nutrients and growth-­ inducing hormones to plants with improvement in the soil structure, leading to increase supply of nutrient and water holding capacity to soil. Fruit, flower, vegetables, and other plant products are reported to found in growing or production vermicompost. It can help in improving better quality of soil structures [45].

Selection of suitable earthworm (Red worms (Eisenia foetida) and African earthworm (Eudrillus engenae)

Selection of site (with place with shade, high humidity and cool)

Different structure of vermicompost production (height of 2.0-2.5 feet and a breadth of 3 feet)

Waste selection for vermicompost production (Cattle dung farm wastes, crop residues, vegetable waste, agro industrial waste, fruit market waste and all other bio degradable waste)

Figure 13.3  Steps for vermicompost formation that used as biofertilizers.

408  Biofertilizers

13.4 Conclusion This chapter has discussed the biofertilizer application for crop growth and their productivity. This biofertilizer is found the eco-friendly nature and also easily helped in providing necessary nutrients to plants. It has provided the soil fertility with bioagents for plant pathogen for many crops such as cotton plant and sugarcane. This fertilizer has helped to provide the alternative option to chemical fertilizers that enhanced the problem in water bodies such weed growth. It helped in reduction of pollution in water sources because 80% to 90% chemical are not utilized by plant and come to water bodies and created the problems to aquatics. A lot of microbial strains has been discussed in this chapter that has formed interaction with plant and helped in nitrogen fixation processes. This chapter also emphasized the merit and demerits of biofertilizers, and it is one best option for more safe foods for nation.

13.5 Abbreviations ALT, Alaninine aminotransferase; AMF, Arbuscular miycorrhiza fungi; AST, Aspartate aminotransferase; BioF, Trichoderma-enrich biofertilizer; DAC, Agriculture and Cooperation; ExoPS, Exopolysaccharides; GA, Giberellic acid; IISS, Indian Institute of Soil Science; INM, Integrated Nutrient Management; ISOPOM, Integrated scheme for crops of oilseeds plants, oil palm plant and maize crops plant; NABARD; NPK; Nitrogen, Phosphorus and Potassium; NPOF, National Project on Organic Farming; PGP, Scheme of plant growth promotion activity; PGPR, Plant growth promoting capacity of rhizobacteria stains; PSM, Phosphate solubilization microbes; SGR, Specific growth rate; STCR, Soil Test Crop Residues Data; TSP, triple super phosphate.

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Biofertilizers Application in Agriculture  409 4. Choudhury, M.A., Kennedy, I.R.; Prospects and potentials for system of biological nitrogen fixation in sustainable rice production. Biol. Fertil. Soils, 2004, 39, 219–227. 5. Berg, G., Zachow, C., Müller, H., Phillips, J., Tilcher, R.; Next-generation bio-products sowing the seeds of success for sustainable agriculture. Agrono., 2013, 3, 648–656. 6. Nina. K., Thomas, W.K., Prem, S.B.; Beneficial organisms for nutrient uptake. VFRC report 2014/1, virtual fertilizer research center. Washington, DC: Wageningen Academic Publishers; 2014, pp.63–69. 7. Bardi, L,, Malusà, E.; Drought and nutritional stresses in plant: alleviating role of rhizospheric microorganisms. abiotic stress: new research. Nova Science Publishers Inc., Hauppauge, 2012, pp. 1–57. 8. Dogan, K., Kamail, Celik, I., Gok, M.M, Ali, C.; Effect of different soil tillage methods on rhizobial nodulation, biyomas and nitrogen content of second crop soybean. Afr. J. Microbiol. Res., 2011, 5, 3186–3194. 9. Hermosa, R., Viterbo, A., Chet, I., Monte, E.; Plant-beneficial effects of Trichoderma and of its genes. Microbiol., 2012, 158(1), 17–25. 10. Molla, A.H., Haque, M.M., Haque M.A., Ilias, G.N.M., TrichodermaEnriched Biofertilizer Enhances Production and Nutritional Quality of Tomato (Lycopersicon esculentum Mill.) and Minimizes NPK Fertilizer Use. Agri. Res., 2012, 1, 265–272. 11. Ji, S., Liu, Z., Liu, B., Wang, Y., Wang, J.; The effect of Trichoderma biofertilizer on the quality of flowering Chinese cabbage and the soil environment. Scientia Horticul., 2020, 262, 109067. 12. Khan, Z., Tiyagi, S.A., Mahmood, I., Rizvi, R.; EffectsofNfertilization, organic matter, andbiofertilizers onthe growth and yield of chilli in relation to management of plant-parasitic nematodes. Turk J. Bot., 2012, 36(1), 73–78. 13. Shoresh, M., Harman, G.E.; The molecular basis of shoot responses of maize seedlings to Trichoderma harzianum T22 inoculation of the root: a proteomic approach. Plant Physiol., 2008, 147: 2147–2163. 14. Vinale, F., Ghisalberti, E.L., Sivasithamparam, K., Marra, R., Ritieni, A., Ferracane, R., Woo, S, L, Lorito, M.; Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with plant pathogens. Lett. Appl. Microbiol., 2009, 48, 705–711. 15. Zhang, Q.C., Shamsi, I.H., Xu, D.T., Wang, G.H., Lin, X.Y., Jilani, G.; Chemical fertilizer and organic manure inputs in soil exhibit a vice versa pattern of microbial community structure. Appl. Soil Ecol., 2012, 57, 1–8. 16. Chang, K.H., Wu, R.Y., Chuang, K.C., Hsieh, T.F., Chung, R.S.; Effects of chemical and organic fertilizers on the growth, flower quality and nutrient uptake of Anthurium andreanum, cultivated for cut flower production. Sci. Hortic-Amsterdam, 2010, 125, 434–441. 17. Yan, X., Gong, W.; The role of chemical and organic fertilizers on yield, yield variability and carbon sequestration results of a 19-year experiment. Plant Soil, 2010, 331, 471–480.

410  Biofertilizers 18. Sun. R., Zhang, X.X., Guo, X., Wang, D., Chu, H.; Bacterial diversity in soils subjected to long-term chemical fertilization can be more stably maintained with the addition of livestock manure than wheat straw. Soil Biol. Biochem., 2015, 88, 9–18. 19. Savci S.; An agricultural pollutant: chemical fertilizer. Int. J. Environ. Sci. Te., 2012, 3, 77–80. 20. Yang, S.X., Liao, B., Li, J.T., Guo, T., Shu, W.S.; Acidification, heavy metal mobility and nutrient accumulation in the soil–plant system of a revegetated acid mine wasteland. Chemosphere, 2010, 80, 85. 21. Hossain, M.A., Piyatida, P., da Silva, J.A., Fujita, M.; Molecular mechanism of heavy metal toxicityandtolerancein plants: central role of glutathione in detoxification of reactive oxygen species and methylglyoxal and in heavy metal chelation. J. Botany, 2012, 2012, 37. 22. Mahanty, T., Bhattacharjee, S., Goswami, M., Bhattacharyya, P., Das, B., Ghosh, A., Tribedi, P.; Biofertilizers: A potential approach for sustainable agriculture development. Environ. Sci. Pollut. Res., 2017, 24, 3315–3335. 23. Gupta, G., Parihar, S.S., Ahirwar, N.K., Snehi, S.K., Singh, V.; Plant growth promoting rhizobacteria (PGPR): current and future prospects for development of sustainable agriculture. J. Microb. Biochem. Technol., 2015, 7, 2. 24. Glick, B.R.; Plant growth promoting bacteria: Mechanisms and applications. Scientifica 2012, 2012, 15. 25. Kang. B.G., Kim, W.T., Yun, H.S., Chang, S.C.; Use of plant growth promoting rhizobacteria to control stress responses of plant roots. Plant Biotechnology Report 2010, 4(3), 179–183. 26. Khan, M.Z, Zaidi, A., Wani, P.A., Oves, M.; Role of plant growth promoting rhizobacteria in the remediation of meta contaminated soils. Environ. Chem. Lett., 2009, 7(1), 1–19. 27. Khan, A.L., Waqas, M., Kang, S.M.; Bacterial endophytes Sphingomonas sp LK11 produces gibberellins and IAA and promotes tomato plant growth. J. Microbiol., 2014, 52, 689–695. 28. Dormaar, J.F., Lindwall, C.W., Kozub, G.C.; Effectiveness of manure and commercial fertilizer in restoring productivity of an artificially eroded dark brown chernozemic soil under dryland conditions. Can. J. Soil Sci., 1988, 68, 669–679. 29. Cheng, Y., Wang, J., Zhang, J.B., Muller, C., Wang, S.Q.; Mechanistic insights into the effects of N fertilizer application on N2O-emission pathways in acidic soil of a tea plantation. Plant Soil, 2015, 389, 45–57. 30. Babalola, OO.; Beneficial bacteria of agricultural importance. Biotechnol. Lett., 2010, 32(11), 1559–1570. 31. Afzal, A., Bano, A.; Rhizobium and phosphate solubilising bacteria improve the yield and phosphorus uptake in wheat (Triticum aestivum L.). Internat J. Agricul. Biol., 2008, 10, 85– 88. 32. Shahi, U.P., Mamta, Dwivedi, A., Dhyani, B.P., Kumar, A., Kumar,  Y.; Assessment of biocompost for chemical fertilizers substitution by

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14 Quality Control of Biofertilizers Swati Agarwal, Sonu Kumari and Suphiya Khan* Department of Bioscience and Biotechnology, Banasthali Vidyapith Rajasthan, India

Abstract

Biofertilizers are one of the essential components of the organic farming and sustainable agriculture. These are low cost, environmental friendly, and improve soil health. Their effect on plant growth may not be spectacular as chemical fertilizers, being biological material. Their use is of great significance in developing countries because even the marginal farmers can afford to buy and use them. Awareness among farmers is increasing on the biological fertilizers. But the accomplishment of the biofertilizer inoculation is based on the nature of inoculants used. Hence, it is essential the manufacture of biofertilizers adhere to the quality control protocols in the production units ensuring a quality product. Care should also be taken to overcome other constraints coming in the way of biofertilizer technology. We should recognize that in adopting a rational approach to the use of biofertilizers in sustainable agriculture, the microbial labor force holds a vast potential for the future. Keywords:  Agriculture, biofertilizer, microbes, fertilizers

14.1 Introduction Biofertilizers are the products developed through biotechnology utilizing the active organisms from plant’s ecosystem [1]. These microbes present in soil and root zones of the flora have the capacity to mobilize nutrients from non-usable to usable nature for enhancing the productivity of soil and crops. Pathway utilized by these microbes includes nitrogen fixation, phosphorus solubilization, and nutrient mobilization [2]. These microbes *Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (413–428) © 2021 Scrivener Publishing LLC

413

414  Biofertilizers are considered as microbial fertilizers which have a property to survive in synthetic media having stable genetics. These microbes survive upto their shelf life time as these are carrier dependent that can be solid or liquid. These microbes utilized as biofertilizer are resistant to different ecosystem and soil stresses [3]. Initially, these biofertilizers showed low efficiency as quality checks were absent and diverse efficiency products were flooded in market. In 1964, Nitragin first synthesized the biofertilizers using Rhizobium and transported to India [4]. In 1990, widespread project of biofertilizers was started and further exhibited at large scale [5]. After this, government showed attention in the synthesis of biofertilizers and initiated different production units [6]. The functioning of the biofertilizers had also faced many limitations [7]. Lignite was utilized as the carrier for the biofertilizers, and constant sterility was upheld. However, ambient temperatures are sky-­ scraping, and low temperature storage systems are required until they are delivered to the fields [8]. Other than this, time consuming nature as compared to chemical fertilizers is the major limitation that makes it restricted to reach to customers [9]. The professed amount and practicality was never a fact in the biofertilizer and it was polluted with other microbes. Amendments were done in biofertilizers as incorporation of disinfected aqueous carriers, temperature-resistant verities, consortia of inoculants, and improved solutions results in elevated shelf life [10]. Other than this, government enhanced grants to the manufacturing units developing sterilized aqueous inoculants and offer a subsidy if the established unit synthesized biopesticides efficiently [11]. Thus, these actions facilitated the development and ensured the utilization of biofertilizers to 20,000 MT; still, quality problem is always present as major problem [12].

14.2 Biofertilizer Requirement and Supply The increase in requirement for natural goods is flourishing, not only because of the  elevation in meticulous clients but also due to growing earnings, alongside the enhanced agricultural application that creates organic products more strong [13]. Thus, the enhanced requirement of natural goods is reported globally and resulted in the elevated organic agricultural practices (Figure 14.1 and Table 14.1). The agricultural field area of global organic farming is elevated from 37.5 to 57.8 million hectares [14]. The above-mentioned data signify that the enhanced natural

Quality Control of Biofertilizers  415

Regional Growth Rates High Mid Low

Figure 14.1  Global presentation of biofertilizer market.

Table 14.1  Year-wise demand and supply chart for biofertilizers. Year

1990

2000

2011

2031

2041

Demand

16.0

19.0

20.2

27.3

31.1

Supply

12.8

14.9

15.8

20.9

23.9

Gap

3.2

4.1

4.4

6.4

7.2

farming application  enhanced the soil fertility due to the utilization of biofertilizers. This has promoted the requirement for biofertilizer food stuffs, as these are natural and eco-friendly [15]. Almost 28% of biofertilizers world market share belongs to North America [16]. Among this, the United States covers the highest shares that are more than half of shares of North America [17]. The market for biofertilizers has also increased in both United States and Canada because of its natural and eco-friendly property. Still, Mexico is a budding market in North American biofertilizer taxonomy [16]. As a result, the diverse benefits of biofertilizers result to their extensive utilization and enhanced acceptance and utilization in sustainable farming [18].  The affirmative agricultural attitude in the country has increased the market of bio-based foodstuffs in the area. In the future, North American area is estimated to maintain its market, because of enhanced utilization of organic crop due to nutrition and demand of high quality food values [19].

416  Biofertilizers The approximate existing requirement for biofertilizers is 18,500 tonnes per year in India, while predictable manufacturing is approximately 10,000 tonnes per year [20]. Furthermore, Government of India is focusing on creating extra requirement by appropriate expansion and encouragement by frequently organizing seminars on biofertilizers [21].

14.3 Process of Biofertilizer Quality Control Quality supervision is especially crucial and should be achieved constantly to manage the microbial products in support of the clients [22]. The rules applied for calculating the quality are restricted to the concentration of the accessible microbes and viability and conservation of the definite microbes [23]. It is vital to locate control plots that do not include existing microbes, however, whose other composition is similar as the ultimate microbial products. In addition, it is extremely advantageous that the biofertilizer manifests the key effects for quality organization of the ultimate biofertilizer products. The main effects are utilized as a sign for the biofertilizer. Moreover, the effects are incorporated as assured actions of the biofertilizer. It is a necessary condition to differentiate among the existing microbes and the additional compositions on the effects of the biofertilizer assured by the manufacturer [24]. If the concluding outcomes of the two experimental plots are similar or cannot be confirmed statistically, then the product is simply an organic material. This shows that the effects of microbial products have to be initiated from the certain microbes, and the goal of the matters should be accessible in details as an instruction [25]. Biofertilizers, recognized as microbial products, function as nutrient provider and soil conditioners that reduced the agricultural load and preserve the eco-system [26]. High-quality soil state is essential to improved crop production, as well as human or animal wellbeing interests. Therefore, the resources utilized to maintain high-quality soil state are treated as ecological materials. On the other hand, as stated previously, there are still a number of problems to be met on the application of microbial products. More accurate quality control must be made in support of the clients. While the effects of biofertilizers are diverse among countries because of the variances in weather and soil environment, the significance of biofertilizer on ecological preservation in the 21st century must not be overlooked [27]. In the similar way, a variety of biotechnologies should be accepted for enhancing the biofertilizer effects with concern

Quality Control of Biofertilizers  417 Identification of strains guaranteed (genus, species) Regular inspection for quality control by authority under acts

Density of strains guaranteed (colony forming unit)

Assessment of main activity as effect indicators of biofertilizers

Evaluation of effect for target crops (growth rate, nutrient adsorption etc)

Registration under the regulation

Figure 14.2  Process for quality control of biofertilizers.

for the environment. Biofertilizers minimize the ecological load emanating from the chemical compounds [28]. Our viewpoints on biofertilizers are similar for biocontrol and bioremediation, since we are members of an environment connected to the world wide web of foods. The following chart showed the process layout for quality control of biofertilizers (Figure 14.2).

14.4 Requirement of Quality Control Identifying the imperative requirement for not only elevating the manufacturing but of producing better quality biofertilizers, the Government of India included biofertilizers under statutory control system by modifying the Fertilizer Control Order (FCO) in 2005 [29]. This confirmed the vital rules that the biofertilizer product and the procedure of manufacturing would have to follow [30]. Terms that required to be adhered to by diverse biofertilizers, the least shelf life period, and tolerance border are mentioned [31]. The relevant state directors of agriculture are regulator of the Act in that exact state [32]. Ground officers are selected, who can assemble the sample

418  Biofertilizers from production location, seller, or farmers and post the samples to laboratories for examination. Seven laboratories under the central government and eight laboratories under the state governments have been recognized to perform the examination [33]. Action in opposition to defaulter has been given as caution with instructions to extract that batch, suspension of license, termination of license, prosecution, and fine or detention or both as penalty. Harsh observance to the rules by manufacturing units, monitoring of product and procedure by the examiner and the consequent penalties meted out to debtor required to be followed. The marketplace is flooded with biofertilizer goods, which, at times, live up to the declaration of the manufacturer, but by and large these claims are not substantiated by the performance of the product, building the requirement for rules and quality management even extra crucial [34]. The constant belief of farmers in the profit of biofertilizers requires to be continued and the responsibility lies with the manufacturer to guarantee the product getting in the marketplace is that of the confirmed quality. While the government has now placed out rules and terms, confirming to their obedience is the matter that has

Media composition

Large Scale Production

Growth condition Purity Cost

Packaging

Carrier Type Carrier sterilization Peat: Water content Granules: Drying speed Other additives Level of contamination Drying

Storage and Transport

Sterilized: If contaminated Temperature: 4°C Time 109 cells/g carrier or /ml liquid inoculants. Azotobacter when applies to soil at 10 kg/ha (carrier-based inoculant) and 625 and 1,250 ml/ha (liquid inoculant) along with 75% nitrogen phosphorus increase plant height by 7.4%, 6.6%, and

440  Biofertilizers 7.6%, respectively, of wheat plant. It can fix nitrogen upto 35.08 mg/gram of carbon [124]. Azotobacter along with Azospirillum helps in root development in eucalyptus [125]. Azotobacter along with phosphate solubilizing bacteria and 75% nitrogen + phosphorus increase the plant dry matter, grain, and straw yield. This combination of Azotobacter and phosphate solubilizing bacteria is also effective for yields of maize and strawberry. Azotobacter enhances the nutrient absorption from the soil also because it releases growth regulators like auxins and gibberellins [126, 127]. Liquid inoculants increase microbial population in the soil compared to carrier inoculants. Azotobacter and phosphate solubilizing bacteria when added to the soil show dehydrogenase activity. Dehydrogenase enzymes help in oxidation of organic matter of the soil. This activity is more with liquid inoculants than carrier inoculants [115]. Azotobacter and phosphate solubilizing bacteria (12.5 kg/ha each) when added along with farmyard manure (10 t/ha) and soil tested based recommended dose of fertilizer to sugarcane give better growth response [128, 129]. It can also help cane to grow in low phosphate soil [130]. These treatments not only provide useful minerals for growth of plant but also add nutrient to the soil and make it fertile after crop harvest [131].

15.4.2 Azospirillum It is free living and ubiquitous in nature. It colonizes plant and helps them to grow [132]. It can fix nitrogen [133] and phosphorus [134], produce phytohormones [135] and siderophores [132], and help tolerate stress situation [136, 137]. It can be used as a biofertilizer for wide range of crop plant. A. brasilense Ab-V5 and Ab-V6 strains are used for maize and wheat biofertilizer by increasing its productivity [138]. A. brasilense HM053 is a mutant that has the capacity to excrete ammonium and fix nitrogen in high NH +4 concentration [139]. A. brasilense HM053 when applied to the soil along with fertilizer N (30kg) produces larger maize spikes, larger spikes diameter, and more number of grains per row. It also increases yield of maize (10.3%) and nutritional status and content of the leaves [140]. A. brasilence along with A. lipoferum when inoculated in maize more amount of nutrients found in leaves and grains [138].

15.4.3 Paenibacillus It is a group of Gram-positive, aerobic, or facultative anaerobic, rod-shaped, endospore-forming bacteria. These bacteria can grow and tolerate extreme environmental conditions [141]. These are potent biofertilizer because

Biofertilizers: Features and Applications  441 they can fix nitrogen and phosphorus and can secrete various antimicrobial compounds and phytohormone [142–144]. Paenibacilli species having nitrogen-fixing capabilities are Paenibacillus polymyxa, Paenibacillus macerans, Paenibacillus azotofixans, Paenibacillus sabinae, Paenibacillus sonchi, Paenibacillus forsythia, Paenibacillus sophorae, Paenibacillus taohuashanense, and Paenibacillus beijingensis [145–153]. Paenibacillus can produce antibacterial and antifungal substances [154–157]. They can also produce IAA to a maximum of 7.19 mg/L. It can work as a biofertilizer for wheat, cucumber, and tomato. It has the potential to increase the height to the maximum of wheat seedlings by 30.9%, cucumber seedling by 50.0%, and tomato seedling by 64.6%, and root of wheat by 54.2%, cucumber by 94.4%, and tomato by 55.2%. These bacteria also show positive result toward dry weight of root and shoot. The results showed that Paenibacillus can promote plant growth directly by producing hormones and solubilizing nutrient as well as indirectly by killing plant pathogen by antifungal compounds [158]. P. brasilensis PB172 (NR025106) can protect seeds and roots of wheat from pathogenic fungi like Fusarium moniliforme and Diplodia macrospora [159, 160].

15.4.4 Phyllosphere Associated Methylobacterium Not only rhizobacteria but also phyllobacteria can be a potent candidate for biofertilizer. Methylobacterium isolated from phyllosphere of ginger has the capabilities like mineral solubility, production of IAA, siderophores and hydrolytic enzymes like chitinase. It increases photosynthetic activity of crops [161]. Plant phyllosphere releases methanol because of degradation of pectin during its growth [162]. Methylobacterium grows on phyllopshere by utilizing methanol as a carbon source [163]. It has antifungal property due to which it can control fungal infection in ginger plant. It helps plant to grow under stress situation [164]. Methylobacterium showed positive response for phosphate, potassium and zinc solubilization [165]. It can produce IAA upto 7.5µg/ml. Hence it can be a potential biofertilizer for crop plants [166].

15.4.5 MO Plus Biofertilizer MO plus biofertilizer is an alliance of microorganism that helps to enhance crop production. It consists of Bradyrhizobium japonicum, which is found in root nodule of soybean and helps to fix nitrogen directly from atmosphere, and Streptomycetes, which helps to control plant diseases [167]. When this group mixed with bacterium Paenibacillus polymyxa, it gives

442  Biofertilizers better results in terms of rice germination and control of diseases. MO plus biofertilizer secrets IAA and gibberellic acid that helps in growth of plants. MO plus biofertilizer can secret 0.095 mg/L of IAA and 2,225 mg/L of gibberellic acid [168].

15.5 Effect of Biofertilizer on Various Plants (Experimental Design) 15.5.1 Azotobacter spp. (AZT) and Azospirillum spp. (AZP) on Eucalyptus grandis Characteristics: The colony morphology of AZT is cream colored, irregular, and bright and of AZP is flat, bright, large morphology, scarlet red color, with circular and regular edges [169]. AZT and AZP strains are gram negative bacillus. Mechanism: AZT enhances plant growth by producing growth hormones, fungicide, siderophore, and by fixing N and P [120, 121]. AZP enhances plant growth by producing growth hormones and nitric oxide [170]. Production of biofertilizer: For preparation of biofertilizer, nitrogen source used is chicken manure in low dose of 100 g and high dose of 200g; carbon source used is eucalyptus leaf litter in low dose of 300 g and high dose of 500 g; and micronutrient source used is rhizospheric soil in low dose of 50 g and high dose of 100 g. These organic substrates are dried, crushed, and sieved, and then, it is autoclaved. Azotobacter and Azospirillum consortium were added keeping the moisture content upto 32%. After mixing of organic substrate and microbe, the resultant product was allowed to stand for 60 days for the production of biofertilizer. When the pH, carbon content, and nitrogen content were measured, it was found that pH decreases with time interval 0, 30, and 60 days. pH stabilizes at 6.4–6.2 for AZP and 6.4–6.1 for AZT; for carbon content, it was found that it decreases with time by 11.0% for AZP and 10.7% for AZT; and nitrogen content first increases then decrease to some level and become constant [122, 171]. At the start of the process, C/N ratio was 17.2, and at the end of 30 and 60 days, it was 12.3 and 12.9, respectively. Number of inoculants also increases in first 30 days and then decreases. Therefore, it was observed that biofertilizer stabilized after 30 days of incubation. Outcome: Azotobacter spp. AZT+ and Azospirillum spp. AZP (1:1) were mixed and used for Eucalyptus grandis in doses of 200 g (T1), 300 g (T2), 400 g (T3), and 500 g (T4) and urea (T5) and control (T6) [172]. It was

Biofertilizers: Features and Applications  443 observed that T4 gives better results in all the growth parameters compared to rest including urea and control [125, 173–175]. It was found that AZP is dominant in the rhizosphere of E. grandis soil [176–178]. IAA, which helps in growth of the plant, is produced by these microbes; for AZT, it is 10.8 to 40.2 µg/ml; and for AZP, it is 7.9 to 42.8 µg/ml [122]. Biofertilizer has positive response in case of various growth parameters. For seedling height: T4 increases it by 0.7 cm/unit time, whereas control increases 0.4 cm per unit time. For stem diameter: T4 increases by 0.08 mm per unit time, whereas control increases by 0.03mm; in case of mean leaf number: T4 is 288.0% of control; fresh aerial biomass: T4 92.0 g/plant, whereas control 19.5 g/plant; Aerial dry biomass: T4 51.9 g/plant, whereas control 19.0 g/plant; root dry biomass: T4 9.2 g/plant, whereas control 2.4 g/plant; lateral root: more in T4 than control; total nitrogen: T4 26.7%; and total phosphorus: T4 57.2% [122].

15.5.2 Bradyrhizobium Strains and Streptomyces griseoflavus on Some Leguminous, Cereal, and Vegetable Crops Characteristics: The Rhizobium, Bradyrhizobium, Streptomyces, Bacillus, and Azotobacter are the PGPR [179]. These are those bacteria that are present in the rhizosphere of the plant and help in enhancement of growth. The symbiotic association of these bacteria can enhance the development better for leguminous and some cereals. Bradyrhizobium are present in root nodule of soybean and help in fixing nitrogen to the plant. Streptomyces are important bioprotectant. They help to survive plant by protecting them from diseases. It can produce important compounds like vitamins, alkaloids, plant growth factors, enzymes, and enzyme inhibitors [180, 181]. Production of biofertilizer: Biofertilizer is produced by using B. japonicum SAY3-7 plus B. elkanii BLY3-8 and S.griseoflavus P4 with peat soil. To produce biofertilizer, 100 g of sterilized peat is first kept in a polythene bag. To it, 20 ml each of Bradyrhizobium strains (B. japonicum SAY3-7 and B. elkanii BLY3-8) were added. Then, over it, spores of S. griseoflavus P4 were added. At last, this bag is kept in a black polythene bag; this will prevent the bacteria from direct light [19]. The population density of final fertilizer was maintained to a level of 108 cells/g soil. Outcome: Biofertilizer was found to be effective in increasing shoot and root growth of mung bean, cowpea, and soybean compared to control under N deficient condition. With addition of nitrogen, biofertilizer increases the shoot and root growth of mung bean, cowpea, and soybean compared with the control [19, 182]. This biofertilizer was also found efficient in forming nodule and nitrogen fixing [183]. S.griseoflavus P4 with

444  Biofertilizers B.yuanmingense increase nodule formation for soybean [184]. It also effective in increasing shoots biomass of mung beans and soybeans as compared to control. In case of cowpea, it increases shoot biomass only during early growth stages. Nitrogen uptake and phosphorus uptake also increase by addition of biofertilizer [185, 186]. Nodule formation is more in mung bean and soybean during full bloom stage and early-pod fill stage. In case of nitrogen uptake, it is more during three unfolded trifoliate leaf stage, full bloom stage, and early-pod fill stage of mung bean and soybean. But in case of cowpea, N uptake is more during unfolded three trifoliate leaf stage. Phosphorus uptake in case of soybean, it is in all stages. For cowpea, phosphorus uptake is more in three unfolded trifoliate leaves stage. Biofertilizer can also increase the number of pods per pot in mung bean, cowpea, and soybean. Therefore, the use of combination of microbes as a biofertilizer is very much effective in plant growth and development [19]. Streptomyces spp. MBR-52 help increase the adventitious root of plant [187].

15.5.3 Rhizobium and Rhizobacteria on Trifolium repens Characteristics: In leguminous plants, low soil phosphorus is a major limitation. So, it is very important to improve its growth in phosphorus deficient conditions. Preparation of biofertilizer: This consists of two Rhizobium spp. strains (CHB1120 and CHB1121) and their combination with two PGPR strains (Bacillus arybhattai strain Sb and Azotobacter vinelandii strain G31). These strains are grown in different growth medium, and cell density was adjusted to 109 cells/ml. For coinoculation, these suspensions were mixed in 1:1 ratio, and 1 ml of mixed culture was added to the sterilized clover seeds [188]. Outcome: Rhizobium and PGPR strains can solubilize calcium phosphate and produce siderophores. But it cannot solubilize iron phosphate. IAA production is more in case of rhizobial strains [189]. The coinoculation of CHB1121 and G31 significantly increased shoot fresh weight by 59.3% and root fresh weight by 27.0% compared to the single inoculation of CHB1121 alone. Rhizobium legumonosarum when coinoculated with Bacillus subtilis or B. megaterium increase chlorophyll content, shoot, and nodule dry weight [190]. Inoculation of CHB1121 in combination with G31 proved to be the best treatment because it helps to increase shoot and root fresh as well as dry weight of clove [191]. Rhizobium strain CHB1121 significantly increases the number of nodules per plant by 42.9% in comparison with strain CHB1120. When compared with control (noninoculated), both the individual inoculation of the two Rhizobium strains

Biofertilizers: Features and Applications  445 and their coinoculation with PGPR significantly increased the nodule numbers [192]. Coinoculation gives better response in terms of number of nodule when compared with individual inoculation. For nitrogenase activity, it was found that when Rhizobium was coinoculated with Sb gives higher nitrogenase activity when compared to Rhizobium and G31 inoculation [188, 193]. Rhizobium and PGPR promote plant growth through the process of nitrogenase [194–196]. This coinoculation is also important in case of nutrient uptake [197–199]. N uptake is more when Rhizobium is coinoculated with PGPR. Coinoculation of CHB1120 and Sb resulted in higher nitrogen uptake compared to control and CHB1120 alone. The single inoculation of CHB1121 resulted in a significantly higher P content in shoots of white clover by 341% in comparison with the uninoculated control. CHB1120 and G31 coinoculation also useful in Ca absorption and helpful in increasing Mg content by 44.0%–85.8% [188]. This suggests that compared to single inoculation coinoculation gives better results related to growth parameters and nutrient uptake in low phosphorus soil.

15.5.4 Arbuscular Mycorrhizal and Phosphate Solubilizing Fungi on Coffee Plants Characteristics: For the evaluation of growth parameters and nutrient solubilization and uptake for the coffee, interaction of arbuscular mycorrhizal and phosphate-solubilizing (P-solubilizing) fungi was tested. AMF and phosphate solubilizing bacteria solubilize phosphate more effectively and hence help plants to grow better [200, 201]. For this, consortium of arbuscular mycorrhizal fungi (CAMF), two strains of P-solubilizing fungi (PSF) (Aspergillus niger [An] and Penicillium brevicompactum [Pb]), the possible combinations of the latter fungi and an uninoculated control were used. CAMF inoculums should contain 60 spore/g and colonization rate should be 79% [202]. This consortium contain following species: Acaulospora morrowiae, A. spinosa, A. scrobiculata, Gigaspora rosea, Scutellospora pellucid, Glomus macrocarpum, Funneliformis mosseae, F. geosporum, and Rhizophagus aggregatus. Outcome: When these treatments were given to the coffee plant, it was found that mycorrhizal colonization is more in CAMF and Pb + CAMF treatments (35%–51.33%), whereas in control (uninoculated), it was 0% [202]. In case of available phosphorus in the soil, all the treatments having CAMF and its combination with An and Pb have more phosphorus content (3.39 to 5.77 mg/kg) compared to control (0.0879 mg/kg). But this content is more in CPSF compared to rest all (3.8 mg/kg more). Coinoculation of phosphate solubilizing fungus and Glomus etunicatum add more

446  Biofertilizers phosphorus (2.9 mg/kg) to the cashew plants [203]. Coinoculation of arbuscular mycorrhizal fungus and Aspergillus tubingensis increases phosphorus content (3.1 mg/kg) in bamboo plants [204]. CAMF, An + CAMF, CPSF + CAMF, Pb + CAMF, and CPSF have higher foliar phosphorus (increased by 110%), plant height [202, 205] root length [202, 203], foliar area, and dry weight [202] compared to individually Pb or An individually and control. All the treatments showed higher phosphatase activity compared to control [202]. These inoculations enhance growth of the plant because of phosphate solubilization and also because of release of hormones, vitamins, or amino acids.

15.5.5 Glutamicibacter halophytocola KLBMP 5180 on Tomato Seedlings Characteristics: One of the major stress conditions is salinity [206]. It reduces germination of seed, growth of seedling, and hence the yield. In high-salt condition, plant’s ability to absorb nutrients decreases [207]. Therefore, use of microbes to improve tolerance of crop for stress is an immediate necessity. This is an eco-friendly approach for salt stress problem. Glutamicibacter halophytocola KLBMP 5180 is a natural halotolerant strain isolated from halophyte Limonium sinense [208]. It is capable of promoting growth of plant under salt stress conditions [209–211]. Outcome: The results were evaluated for 2% NaCl salinity. Inoculation with KLBMP 5180 promotes growth of tomato seedling in saline and non-saline condition, whereas there was no growth in control (uninoculated) under saline condition. G. halophytocola KLBMP 5180 can promote the growth of Limonium sinense, from where it is isolated, at 10% salt condition [212]. This strain is a new member of genus Glutamicibacter [208]. It also increases the fresh root weight by 28.6% and 26.5% with and without NaCl stress, respectively. It helps to increase proline concentration in leaves and stem by 110% and 86.7%, respectively. It also helps to enhance antioxidant enzyme concentration. It increases stem superoxide dismutase enzyme activity by 9.4% under saline concentration [212–214]. It also helps to regulate Na+ concentration under stress situation. After salt stress situation, Na+ concentration increases in the plant, and with the help of KLBMP 5180, this content is reduced by 96.5% and 23.5%, respectively, in leaves and stem. It also maintains K+ concentration higher in stem in 200 mM NaCl condition [215–218]. The ratio of k+/Na+ decreases under stress situation but KLBMP 5180 increases it in stem and leaves by 170.5% and 33.3%, respectively. This ratio is important in protecting from salt stress toxicity. Inoculation by KLBMP 5180 also enhances Ca2+ concentration

Biofertilizers: Features and Applications  447 by 0.3-fold in leaves in salt conditions [214, 219]. This biofertilizer also increases proline content, which is an osmoprotectant that protects the cells from damage due to oxidation [214].

15.6 Screening of Microbes for Biofertilizer Biofertilizer is useful to agriculture because it made available nutrients to the plants which are otherwise unavailable to them. It helps to solubilize macro- and micronutrient and produce phytohormones to participate directly in plant growth. So for biofertilizer preparation, microbes having such potentials need to be screened from the population of microbes. In vitro screening follows specific protocol to fractionate desired microbes from whole occupants.

15.6.1 Screening for Phosphate Solubilization For screening of microbes for phosphate solubilization, microbes are grown on Pikovskaya agar medium supplemented with either Ca3(PO4)2 or FePO4 or CaHPO4. Formation of halo zone around grown colonies considered as phosphate solubilization positive. Phosphate solubilizing index and efficiency is calculated by the following formula: solubilization index (SI) = colony diameter + halo zone diameter / colony diameter, solubilization efficiency (SE) = halo zone diameter / colony diameter × 100 [14, 117, 188, 220– 224]. Another media for phosphate solubilization are National Botanical Research Institute’s phosphate (NBRIP) growth medium (glucose 10.0 g/L, (NH4)2SO4 0.15 g/L, KCl 0.2 g/L, MgCl2⋅6H2O 0.5 g/L, MgSO4⋅7H2O 0.5 g/L, pH 7.0) supplemented with tri-calcium phosphate (TCP, 5.0g/L). After growth of culture on this medium upto 3 days, it is transferred to NBRIP agar medium with tri-calcium phosphate. Halo zone is the indication of positive test result [13, 225–227]. Another method to measure efficiency is by adding vanadomolybdate reagent (ammonium molybdate 5% + ammonium vandate 0.25% + nitric acid with water 1:3) [124].

15.6.2 Screening for Potassium Solubilizing For potassium solubilization, strains are inoculated in Aleksandrove media [CaCO3: 0.10 g/L; Glucose: 5.0 g/L; MgSO47H2O: 0.50 g/L; FeCl3: 0.006 g/L; Ca3(PO4)2: 2.0 g/L; Insoluble Mica powder (insoluble potassium source): 3.0 g/L; and Agar: 20.0 g/L]. Halo zones around colonies are indication of positive response [114].

448  Biofertilizers

15.6.3 Screening for Nitrogen-Fixing Burk’s media [MgSO4 (0.2 g/L), K2HPO4 (0.8 g/L), KH2PO4 (0.2 g/L), CaSO4 (0.13 g/L), FeCl3 (0.00145 g/L), NaMoO3 (0.000253 g/L), and sucrose (20 g/L), and Agar (18 g/L)] are the media for selection of nitrogen-fixing microbe. This media does not contain any external nitrogen source; therefore, to grow microbe has to fix nitrogen from air [221, 222]. Efficiency of nitrogen fixation can be measured by Kjeldahl method. In this method, in 2 ml of supernatant (from where culture grown), 1 ml of K2Cr2O7 and 20 ml of sulfuric acid are added and heated for 1 min and then 200 ml of water is added and 4 to 5 drops of ferroin indicator. Titration against 0.5N FeSO­4 gives the result of total carbon released from total volume of ferrous sulfate used [124].

15.6.4 Screening for Zinc Solubilization For screening of microbes having capacity for zinc solubilization, microbes are grown in zinc oxide/zinc phosphate amended Tris-minimal salt agar (Tris-MSA). Halo zone around colonies is the indication of zinc solubilization [224, 226].

15.6.5 Screening for Ammonia Production For ammonia production determination, microbial strains are inoculated in peptone broth and incubated at 30°C for 72 h. After incubation, Nessler’s reagents are added, and change in color is observed [224, 225].

15.6.6 Screening for Hydrogen Cyanide (HCN) Production To check for HCN production, organism is grown on agar plates having 0.44% glycine. There after filter paper soaked in 2% sodium carbonate and 0.5 ml of picric acid solution has to be placed and sealed on agar plate. Change of color of filter paper from yellow to brown is the indication of positive result [222, 225].

15.6.7 Screening for Siderophores To evaluate for siderophore production bacteria is grown in chrome azurol sulfonate (CAS) agar plate. Orange halo zone is the indication of siderophore production [13, 15, 188, 222, 225, 226].

Biofertilizers: Features and Applications  449

15.6.8 Screening for Auxin Production Dworkin and Foster (DF) minimal salt broth or YEMA medium or Luria Bertani (LB) broth supplemented with 1.0 g/L of tryptophan is used for selection of strains having potential for auxin production. Broth inoculated with specific strain incubated for 72 h. After incubation, broth is centrifuged and 1 ml of Salkowski reagent (50 ml of 35% sulfuric acid/perchloric acid + 1 ml of 0.5 moles of FeCl3) is added to the supernatant. Analysis by spectrophotometry at 600 nm and plotting at standard curve gives the result [12, 13, 188, 223, 224, 226, 228], or adding 2 drops of orthophosphoric acid and then adding 4 ml of Salkowski reagent gives pink color after keeping 24 h in dark if the organism produces IAA [168]. Cells can also be grown in B medium having 20 g/L of tryptone and 0.2 g/L of NH4Cl and keep in dark for 48 h. After centrifuge to 2 ml of supernatant, 8 ml of Salkowski reagent is added, and pink color shows positive result [122, 225].

15.6.9 Screening for Gibberellic Acid Production For screening of gibberellic acid production, culture can grow in nutrient broth (NB) media. After that, it is centrifuged and in supernatant 2 ml of acetate zinc solution is added. Then, after 2-min potassium ferrocyanide solution is added and centrifuged. In 5 ml of supernatant, 5 ml of 30% hydrochloric acid is added and incubated for 75 h at 27°C. Absorbance at 254 nm in spectrophotometer and plotting the graph against standard curve gives the result [168].

15.6.10 Screening for Production of Chitinase For chitinase production screening, strain is inoculated in minimal salt agar medium having colloidal chitin. Plates stained with Congo red (0.1%) giving appearance of halo zone around the colonies are indication of positive result [224, 229].

15.7 Limitations of Biofertilizers One of the major problems related to biofertilizer is its adoptability. According to one of the surveys conducted in Indonesia related to acceptance of use of biofertlizers among farmers showed that attributes of farmers like farming experience, age, and education affects the acceptance of

450  Biofertilizers biofertilizers for use. The same survey showed that productive age group (15–64 years old) of farmers is more responsive. Education level of farmers is also very important for the process of acceptance. The farmers that are more educated can easily accept any new technology compared to less educated farmers [230]. This is because education opens the mindset to adopt any new thing and gives vision to see and understand any new change. Another big problem related to the use of biofertilizer is its response. Because biofertilizers are made up of microorganisms; therefore, its functionality depends on environmental conditions. The most important factor for organism’s viability is water [5, 231]. Therefore, water deficiency is the major limiting factor for the response efficiency of biofertilizer. So, in arid and semiarid areas, the use of biofertilizer is restricted because no plant growth and yield become visible. Another limitation of biofertilizer is its instability of vitality and efficiency. Due to this, its usage became restricted among farmers [5]. To overcome this problem, some techniques were developed named plasma. Plasma is a new technique that helps to increase its vitality and efficiency, making it popular for its use among farmers [7]. Other limitations of biofertilizers are poor shelf life, shortage of useful bacterial strains, and require high dose for large coverage area. This problem of high dose can get reduced if slow release fertilizer technique be used. Urea-formaldehyde encapsulation is the slow release fertilizer developed using polymeric matrix and biofertilizer. Two encapsulated biofertilizers developed were polymeric urea-formaldehyde matrix (PUFM) with Chlorella sp. (CHLO) and PUFM and Nannochloropsissp (NANNO). PUFM + CHLO releases the nutrient at a slowest rate compared to conventional application procedure [232].

15.8 Success of Biofertilizer One of the successes of using biofertilizer is that it increases the productivity of crop plant more compared to the chemical fertilizers. The survey conducted on soybean in Indonesia has showed that when biofertilizer is used along with liquid fertilizer and organic fertilizer, it increases the productivity and profitability of soybean farming more [230]. Biofertilizers also make plants to overcome stress conditions. Some examples are as follows: Pseudomonas aeruginosa PW09, which is an endophyte in cucumber, helps it to fight NaCl stress as well as Sclerotium rolfsii stress when added to its seedlings [5, 233]. Pseudomonas putida SI and Pseudomonas aeruginosa Cgr help chickpea to tolerate NaCl and

Biofertilizers: Features and Applications  451 Sclerotinias clerotiorum stress [5, 234]. Burkholderia phytofirmans PsJN helps plant to reduce drought stress of wheat crop [5, 235]. Some microbes can help tolerate stress situation of salt along with increasing growth parameters. Germination of seed under salt condition is a challenge for the plant. But with the assistance of biofertilizer plant can overcome this situation. Studies in this respect and also in productivity parameters were performed by [8]. Various bacteria are used for the treatment. These are Stenotrophpmonas maltophilia P (phosphate solubilizing bacteria) TV14B, Bacillus sp. P (phosphate solubilizing bacteria) TV119E, Bacillus atrophaeus N (nitrogen-fixing bacteria) TV83D, Cellulomonas turbata N (nitrogen-fixing bacteria) TV54A, Kluyvera cryocrescens NP (nitrogen fixing + phosphate solubilizing bacteria) TV113C, and B. atrophaeus + Bacillus sp. (binary combination) TV83D + TV119E. Germination rate of wheat was found as 95.33% in B. atrophaeus + Bacillus-GC, 84.33% for K. cryocrescens, 83.67% for Bacillus-GC, 79.33% C. turbata, and 78.67% for S. maltophilia. Germination rate in presence of 0, 50, 75, 100, and 125 mM salt concentration was found to be 94.29%, 87.38%, 81.91%, 79.05%, and 68.33%, respectively [8, 236]. Response with respect to growth parameters under 0, 50, 75, 100, and 125 mM salt concentration was found to be 99.52%, 99.76%, 100%, 100%, and 100%, respectively. Plant height under stress situation with the help of microbes was found to be 25.38, 25.26, 24.37, 24.24, and 24.44 cm, respectively [8, 237]. Similarly, for root length obtained was 24.38, 25.59, 24.61, 24.26, and 23.57 cm, respectively. For plant wet weight and dry weight, the result was 0.268, 0.258, 0.221, 0.227, and 0.254 g, respectively, and 0.020, 0.024, 0.024, 0.026, and 0.027 g, respectively [8, 238]. These organisms can fix nitrogen under these salt conditions. For Stenotrophpmonas maltophilia P, it is 0.055, 0.048, 0.050, 0.037, and 0.050, respectively; for Bacillus-GC group NP, it is 0.055, 0.053, 0.049, 0.049, and 0.053, respectively; for Bacillus atrophaeus N, it is 0.052, 0.054, 0.051, 0.050, and 0.055, respectively; for Cellulomonas turbata N, it is 0.049, 0.053, 0.049, 0.053, and 0.052, respectively; for Kluyvera cryocrescens NP, it is 0.054, 0.050, 0.051, 0.051, and 0.045, respectively; and for B Bacillus atrophaeus + Bacillus-GC group NP, it is 0.051, 0.051, 0.050, 0.057, and 0.052, respectively [8, 239]. Similarly, these are useful for phosphorus addition to the soil. Therefore, use of microbe can help reduce the pressure of environmental conditions on plants. Because these are eco-friendly, these can able to maintain the integrity of the soil and hence the whole ecosystem. Metal contamination, like Fe, Mn, Cu, Zn, Pb, Cr, Ni, Cd, and Co, also creates stress on plants. Cadmium is toxic to the plant because it inhibits many enzymes and hinders growth [240–243]. It disturbs auxin level in

452  Biofertilizers plants by inhibiting PIN1 protein activity. To overcome cadmium stress, cadmium-tolerant IAA producing bacteria or fungi is needed. Two organisms were identified that has IAA production capacity and are cadmium tolerant. These are Lysinibacillus varians and Pseudomonas putida, both can produce 20 ppm of IAA, responsible for root development [244]. They also help in increase of seed germination of mustard seed under Cd stress condition by 1.21-fold (L. varians) and 1.26-fold (P. putida). Shoot and root length decreases under Cd stress but, with the help of these microbes, it increases by 1.73- and 1.62-fold (L. varians) and by 2.08- and 2.12-fold (P. putida), respectively. L. varians and P. putida also increase leaf area by 2.29- and 2.18-fold, respectively, under Cd stress condition [15, 243]. Other heavy metal-tolerant rhizobacteria are Strenotrophomonas acidaminiphila [245], Klebsiella pneumonia MCC 3091 [242], and Pseudomonas aeruginosa KUCd1 [246]. Another application of biofertilizer is its promotion of seed germination of crop plants. For examples, addition of biofertilizer (Pseudomonas fluorescens, Enterobacter hormaechei, and Pseudomonas migulae) promotes foxtail millet seed germination and seedling growth under drought condition [5, 247]. Burkholderia sp. L2 and Bacillus sp. A30 could increase tomato seed germination [5, 248]. Non-rhizobial endophytes can act as biofertilizer and can help in nodule formation in leguminous plants. The bacterial genera that help in nodule formation are mainly from α-proteobacteria and β-proteobacteria [249]. Paenibacillus polymyxa ANM59 and Paenibacillus sp. ANM76 are two such species which have been isolated from nodule of chickpea and found to be involved in nodule formation. They help to promote growth of the plant by producing growth regulator IAA, by solubilizing phosphorus and chitin, by various enzymatic activities, and by producing ammonia [250– 254]. Many other endophytes also produce IAA like Bacillus subtilis LK14, Pseudomonas aeruginosa LK17, and Sphingomonas LK18 [255]. IAA helps in plant growth because it helps to fight plant against stress situation [256]. It also enhances plant bacterium symbiosis and improves root architecture [224, 257]. Biofertilizer helps to reduce the cost of extra overage on the farmers. To grow crops on acidic soil, farmers need to add significant amount of lime to the soil [258]. This extra overage burdens with loads of cost [259]. Using biofertilizer along with chemical fertilizer in the form of method called fertilizer micro-dosing can reduce the need of excess amount of lime, thereby reducing the expenses [260–263]. Micro-dosing of fertilizer increases the maize yield with comparatively low cost that is a benefit to small scale farmers [264].

Biofertilizers: Features and Applications  453 Biofertilizers can also able to control plant pathogens when incorporated into the soil. Fusarium wilt is a disease of banana plant which can be controlled by the application of biofertilizer [265, 266]. It is very hard to control because it can remain for decades as chlamydospores in soil. Soil acidity is one of the factors that increase the frequency of occurrence of this disease [267, 268]. So, biofertilizer along with acid soil ameliorants (ASAs) can prove to be the only way to control fusarium wilt [268, 269]. A novel biofertilizer generated using Bacillus velezensis H-6, isolated from banana rhizosphere. The novel biofertilizer (BIO) is mixture of organism Bacillus velezensis H-6 and organic substrate (cattle manure compost + peanut bran 9:1 w/w). This novel BIO increases the soil pH, adds organic matter to soil, manipulates soil bacteria and actinomycetes, and controls pathogen causing fusarium wilt [270].

15.9 Debottlenecking Biofertilizer has immense potential for crop improvement if it can be used in a proper way. The potentiality of biofertilizer can be amended by using it along with super absorbant polymer (SAP). SAP increases the efficiency and viability of biofertilizer. SAP increases biofertilizer efficiency because SAP helps in water absorption, which otherwise a major limiting factor for its function [231, 271]. The use of biofertilizer (Paenibacillus beijingensis BJ-18) and SAP shortens the germination time of wheat when compared to control (no biofertilizer and SAP) and SAP treatment by 4 and 2 days, respectively. SAP when used along with biofertilizer also helps to increase the germination rate of seed. The data showed that SAP and Paenibacillus beijingensis BJ-18 biofertilizer increases the germination rate of seed by 47.3% in wheat. Another biofertilizer, Bacillus sp. L-56, when used with SAP reduces the germination time of cucumber by 2–4 days and increases the germination percentage of seed by 35.7% [5]. Biofertilizer along with SAP increases the viability of inoculants by increasing the population density of microbes in the rhizospheric soil. Biofertilizer and SAP together are also helpful in improving growth parameters of crop plant. For example, SAP + BJ-18 enhances shoot length by 28.2%, root length by 42.3%, shoot FW by 86.9%, root FW by 83.4%, shoot DW by 104.9%, and root DW by 79.7% of wheat seedlings. SAP + L-56 enhances shoot length by 47.8%, root length by 50.8%, shoot FW by 71.9%, root FW by 175.7%, shoot DW by 68.9%, and root DW by 76.9% for cucumber crop plant [5].

454  Biofertilizers This combination also helps to reduce proline content and soluble sugar content of same crop plant. It also increases chlorophyll content of plants [272, 273]. It also helps to increase the enzymatic activity of soil, which, in turn, helps to maintain the nutrient cycle of atmosphere due to which ecosystem biotic and abiotic factor balance is maintained well. It was also found that this combination of SAP with biofertilizer also helps to down-regulate some drought stress related genes. Another approach toward betterment of biofertilizer is its mixing with biochar. Biochar is the product formed when organic matter is degraded thermally in absence of oxygen [274, 275]. It helps to increase soil property like nutrient content, microbial activity, water absorption capacity, and nutrient cycling [276–280]. It helps to improve the overall structure of soil and make it suitable for plant growth [281]. Biochar formation also helps to reduce air pollution because organic waste if not converted to biochar release greenhouse gases with due course of time [282, 283]. When various particle sizes of biochar are mixed with biofertilizer, it gives better result with respect to fertility, nutrient content, and organic matter content. The particle size < 2mm of biochar with biofertilizer is effective in increasing organic content of soil. This particle size with biofertilizer is also effective in extracting Fe, Zn, Mn, and Cu by 37%, 32%, 38%, and 96%, respectively [284]. Biofertilizer Rhizobium leguminoserum and biochar with particle size < 2mm increase number of branches per plant in lentils. It also helps in nitrogen cycling of soil [285]. One of the studies on application of biochar with biofertilizer showed that the efficiency of this combination depends on the genotype of the plant on which it is to be applied [286]. When rice husk biochar and Bacillus pumilus applied to two different genotypes of rice, Fukuhibiki and the newly bred line, LTAT-29, it was found that in LTAT-29, grain yield increases but not in Fukuhibiki. Therefore, genotype of plant is an important criterion to keep in mind before designing biofertilizer with biochar [287]. In acidic soil, growing maize is a challenge to the farmers. To obtain 4 tons/ha of maize, grain yield from soil having 54% acid saturation 4.5 tons/ha of dolomite lime is required. The solution to this problem is use of fertilizer micro-dosing. The biofertilizer Bacillus megaterium along with micro-dosing of NPK 2:3:2 (34) increases maize yield by 54.7% and 48.1%, respectively, in season 1 and season 2 [288, 289]. This percentage is 64.6% and 13.6% when NPK alone used. Bacillus megaterium also helped to absorbs phosphorus and can fix nitrogen to the soil [264, 290, 291]. Biofertilizers’ efficiency can also be increased by combining two different antagonistic organisms. The synergistic effect of antagonistic bacteria is observed in rice crop plant. MO plus biofertilizer when combined with

Biofertilizers: Features and Applications  455 Paenibacillus polymyxa gives better response in various parameters. In case of pioneer varieties of rice, the height of crop is more when MO plus biofertilizer is added along with Paenibacillus polymyxa (7.07 cm) than MO plus (4.62 cm) and Paenybacillus (4.07 cm) individually at 10% concentration. This combination also helps to increase the root length (7.39 cm) with 10% concentration as compared to MO plus (1.95 cm) and Paenybacillus (4.00 cm) individually [168]. Biofertilizer can be also used in combination of recommended fertilizer and green manure. The increased effectiveness of biofertilizer mixed with organic matter was reported in sugarcane, rice, and zea mays. The efficiency of biofertilizer increases when it is mixed with green manure of Crotalaria juncea [292, 293]. The combination of biofertilizer + 100% recommended fertilizer + C. juncea is effective in absorbing nitrogen (41.16 g/ plant) which is 62.56% more than control (100% recommended fertilizer) in case of sugarcane. In case of phosphorus uptake, the most effective combination was found to be 75% recommended fertilizer + biofertilizer + C. juncea. It absorbs 10.87 g/plant of phosphorus which is 1.40 times more compared to control. In case of potassium uptake, 50% recommended fertilizer + biofertilizer + C. juncea is more effective, it absorbs 92.94% more than control. These combinations are also found to be effective for sugarcane growth. Length of stalk is more (343 cm) when 100% recommended fertilizer + biofertilizer + C. juncea was used. In addition, 100% recommended fertilizer and biofertilizer increase the density of sugarcane (17.55 stalk/m), which is 12.06% more than control. Number of internodes is found to be more when 75% recommended fertilizer and biofertilizer were used [294]. Biofertilizer along with organic fertilizer also gives better response compared to its individual use. Trichoderma helps to improve soil microbial diversity and solubilize phosphate but, when used with organic fertilizer, induce stronger positive effects [295, 296]. It was also evident in strawberry plant that combination of recommended fertilizer along with biofertilizer (Azospirillum/Azotobacter) shows enhanced growth with respect to leaves/plant (25.33%), leaf area (44.15% more), etc. [297]. To remove one of the limitations of biofertilizer of instability of vitality and efficiency, the technique developed and used was micro Dielectric Barrier Discharge (DBD) plasma. This technique was applied on Bacillus subtilis CB-R05. This is a new technique that uses plasma, that is, ionized gas [298]. Depending upon its usage, it can activate or deactivate biological substances. In this technique, plasma was generated using burst pulses of high voltage. The energy of plasma can be controlled by changing the duty ratio of burst pulses. Plasma is of two types N2 plasma and air plasma. This treatment proved to be useful in increasing bacterial number and

456  Biofertilizers efficiency. Bacteria treated with plasma when used in rice crop, it showed enhanced growth and yield, tolerance to fungal diseases, higher IAA level, higher salicyclic acid level, improved vitality, colonization, phytohormone level, and tolerance to diseases. The levels of oleamide, cyclohexanone, and phosphoric acid were significantly increased, whereas significant decrease in the levels of succinic acid, glycerol, oleanitrile, and stearic acid was observed in plasma treated bacteria [7]. Microbes can grow as biofilms on the surface of biotic or abiotic materials [299]. These biofilms provide nutrients and water. Therefore, biofilms support bacteria better compared than planktonic form, and hence, biofertilizer in the form of biofilms can serve better and can give better response [300]. Bacillus and Pseudomonas species can serve better when used as a biofilms on greenhouse tomatoes [301, 302]. Pseudomonas species can increase some parameters of growth like height and root dry weight when applied as biofilm. Similarly, Bacillus species can give positive result for root length of tomatoes. It proves that this method can be a better option if it can be optimized [303].

15.10 Optimization of Biofertilizer 15.10.1 Optimization of Phosphate Solubilization Microbe: Pantoea agglomerans ZB Phosphate sources: Ca3(PO4)2 (TCP), Hydroxyapatite (HP), CaHPO4, AlPO4, FePO4 along with rock phosphates (RPs). Optimization: For the optimization of pH, temperature, and rotation speed, 1-ml ZB inoculum was added to 100 ml of liquid medium with 5.0 g/L TCP. For temperature optimization, pH was fixed at 7.0, and cultures were kept at 10°C, 15°C, 20°C, 25°C, 28°C, 29°C, 30°C, 31°C, 32°C, 34°C, 36°C, 38°C, 40°C, and 45°C on a rotary shaker at 200 r/min. For optimization of pH, temperature is kept at 30°C and pH at 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 on a rotary shaker at 200 r/min for optimization of rotation speed, temperature is kept at 30°C, pH at 7.0, and shaker at different rotation speeds of 100, 125, 150, 175, 200, and 225 r/min for optimization of carbon source. Different carbon (10.0g/L) sources used were glucose, xylose, sucrose, maltose, galactose, fructose, and mannitol. For optimization of nitrogen source (0.15g/L), different nitrogen sources used were (NH4)2SO4, KNO3, yeast extract, peptone, and urea. For optimum concentration of carbon source, different concentrations of glucose were tested: 5.0, 7.5,

Biofertilizers: Features and Applications  457 10, 12.5, 15, 20, 25, 30, and 40 g/L. For optimum concentration of nitrogen sources, different concentrations of ammonium sulfate were tested: 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, and 0.40  g/L. To test the concentration of phosphorus source, TCP was used in various concentrations: 1.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 40.0, and 50.0 g/L. Different phosphorus sources were also tested at 5.0g/L. Effect of coinoculation with planktonic cell and symplasmata on bio‑solubilization was also tested. The influence of pre‑cultivation on bio‑solubilization was also tested. For pre-cultivation, insoluble phosphates were added into the pre-grown cultures after 24 h of incubation [227]. Preparation of biofertilizer: Microbe was inoculated to the soil in the form of biofertilizer prepared from semi-solid fermentation with spent mushroom substrate (SMS) compost [227]. In this process of biofertilizer preparation, ZB was inoculated at late exponential stage (5%) into 30 g of medium which was autoclaved (5-g corn flour + 25-g sun dried SMS) and moisture content was maintained at 65%–70% [304, 305]. This was maintained for 6 days till the concentration of cells reached upto 5 × 108 cells/g biofertilizer. This biofertilizer then can be applied to the soil (1%). Outcome: Optimum temperature for phosphate solubilization (629.74 mg/L P­­­2O5) is 30°C. Optimum pH was found to be 7.0 for solubilizing phosphorus (642.34 mg/L P2O5). Best rotation speed is 175 r/min (675.23 mg/L P2O5). Maximum solubilization of phosphorus (525.76 mg/L P2O5) obtained with glucose as a carbon source, and its optimum concentration is 12.5 to 15 g/L [227, 306]. Ammonium sulfate was found to be best for phosphorus solubilization (629.74  mg/L P2O5); in some studies, ammonium nitrate was found to be the potential nitrogen source which proves that nitrogen in the form of ammonium is the best nitrogen source [225], and suitable concentration is 0.25g/L. TCP concentration gives best result at 1.0  and 15  g/L of TCP. Coinoculation with symplasma has negative effect on bacterial growth, whereas with plankton, cells have positive effect. Inoculation with planktonic cells has more dissolved phosphorus content (21.89% more) [227, 307]. Precultivation has an effect on phosphorus solubilization. For different phosphorus sources, different carbon source preincubation is better. Like for TCP, preincubation with glucose gives better result [227, 308]. Phosphate solubilizing bacteria can able to solubilize phosphorus because they secrete organic acid that lowers the pH of the soil and helps in phosphate uptake [309, 310]. Low pH supplies protons and chelates the cations especially calcium ions that remain bound with phosphate [311, 312].

458  Biofertilizers

15.11 Concomitant of Biofertilizer Biofertilizer has proved to be one of the useful discoveries for humankind because it helps to maintain the sustainability of environment. But every useful thing also has its aftermath. One of such with biofertilizer is development of antibiotic resistant genes in microorganisms of aquatic ecosystem. Biofertilizers that are formed from biological treatment of animal manure transfer plasmid-based resistant genes horizontally to microorganisms. Fluoroquinolone resistant developed in vibrio parahaemolyticus due to application of biofertilizer in aquatic environment. This makes the sea food unsafe for consumption and is a threat to human health [313, 314]. Therefore, control of these resistant genes has to be considered so that this factor should not become next limiting agent in the use of biofertilizer.

15.12 New Approach A new way by which biofertilizers can be produced is by making use of prawns and crab shells. These are rich in chitin, protein, and inorganic compounds like calcium carbonate. Using crab shell to make biofertilizer makes it eco-friendly. Crab shell is the waste product of sea crab meat production. Because crab shell contains chitin and chitosan, it helps to promote plant growth. Biofertilizers produced from crab shell can be used in aquatic ecosystem. Watercress showed increase shoot length, root growth, and wet weight by crab shell biofertilizer [315]. Amalgamation of nano-biotechnology with fertilizer is a new way to improve the existing biofertilizer. This is the union of nanomaterial and biofertilizer to generate novel nano-biofertilizer. This combination gives advantage of more soil moisture absorption and microbe vitality due to covering of nanomaterial. Nano-biofertilizer is an advanced version of biofertilizer that helps in more effective and efficient sustainable agricultural development. It is formed by reducing biofertilizer to nanosize (1–100 nm) by using some nanomaterial coating [316]. It provides some useful features: eco-friendly, renewability, cost effective, less time of production, required in fewer amounts, and enhanced nutrient use efficiency [316, 317]. Due to coating of nanomaterial, the nutrients are released in slow pace and hence made available to the plants for a longer time [318]. It has the potential to solve the limitation of biofertilizer and make it a better product for sustainable growth and development. It solves the problem of shelf life, low efficiency, high cost, and non-renewability because in its formation

Biofertilizers: Features and Applications  459 nutrient and plant growth promoting bacteria is coated inside nanomaterial. Nanomaterials used for coating are chitosan, zeolite, and polymer [319]. Nano-biofertilizer can enhance plant yield because it increases nutrient absorption and their distribution in plant and accumulates photosynthetic components. Biofertilizer coated with gold or silver nanoparticle is very much effective for crop plants. A study from Iran found that Biozar® as nano-biofertilizer increased the crop growth, seed yield, and yield components of the wheat crop [320, 321]. Another study from Iran found that Zea mays grain yield increases by nano-Zn and biofertilizer combination [321, 322]. One study is from Ludhiana which showed increased leaf area (19.6%), root biomass (42.6%), net photosynthetic productivity (15.8%), and sucrose content 1.03% of sugar beet plants [321, 323].

15.13 Conclusion and Future Prospects Biofertilizer is a potent tool for reducing the pollution pressure and for sustainable environmental growth. It helps in plant growth directly or indirectly. For preparation of biofertilizer, proper pH, temperature, carrier molecule, etc., is required. Most suitable pH for biofertilizer is 6–7, temperature is 30–40°C, and carrier molecule is peat. Using combination of biofertilizers and biofertilizers with chemical fertilizers provides more support for the plant and the soil. These combinations provide better response and output in terms of maintaining soil fertility and yield of plant because they gives more nutrient and organic matter to the soil. For the better response of crop plant, it is important to apply biofertilizer in correct amount, correct combinations, as well as in correct time interval. This understanding of biofertilizer is very important for its more beneficial results [324, 325]. Not only bacteria but also fungus, combination of fungus, combination of bacteria and fungus is also very important in increasing plant growth, adding nutrient to the soil, increasing fertility of the soil and maintaining the natural microbial population of the soil. Therefore, it is the need to find and search more and more natural innate microbial populations that has the capacity of increasing crop yield and growth along with maintenance of soil structure and integrity. Nature and texture of soil is also very important in determining the type of biofertilizer to be used. It is important to study the soil type before choosing biofertilizer. Soil is acidic or alkaline determine which microbe to be used for making biofertilizer because microbe can work in specific pH, temperature and other growth conditions. Therefore, to get the full advantage of biofertilizer its optimization is very important [227]. Due to establishments of

460  Biofertilizers more and more industries now-a-day metal contamination is also increasing day by day. These toxic heavy metals hinder plant growth by blocking their mechanisms of development. Biofertilizers that are heavy metal tolerant help plant to get through these conditions. So it is essential to search for more and more metal-tolerant microorganisms that can act as a plant growth promoting microbes [243]. Biofertilizers has some limitations but can be solved by various methods like by using SAP, urea-formaldehyde encapsulation, biochar, and nano-biofertilizer. Nano-biofertilizer which is a new and emerging technology to provide better results in sustainable development is still not explored completely. Only few studies have been conducted in this respect. In future focus should also be more toward the development and optimization of more and more nano-biofertilizer. Keeping its negative effects in mind the way to solve that problem should also be developed. As combination of biofertilizer with other biofertilizer/ green manure/chemical fertilizer increases the efficiency of biofertilizers, in the similar way combinations of all these with nano-biotechnology should be tried to get more promising eco-friendly product for agriculture [321]. In future more such combination also with organic fertilizer should be tried to get more efficient and suitable way for agricultural evolution. It was found that coinoculation of microbes gives better results compared to when applied individually. Therefore, more such concoction must be tried in immediate future to get more efficient product and to save our environment from deterioration. Apart from using rhizobacteria as a biofertilizer, search of rhizofungus also is a need for imperishable ballooning of agronomy because fungus can help plant to face and subjugate challenging circumstances. Along with rhizobacteria, phyllobacteria also can be fruitful in solving many problems related to agricultural niche. So much research is needed to find and optimize phyllosphere microorganisms. Metabolites synthesized by microbes get affected by surroundings and therefore sometimes cannot able to give their best in field conditions. To improve this situation, genome sequencing and bioinformatics is needed. With the help of genome mining and genetic engineering, biosynthetic and action potential can be increased. Hence, future research in this direction should be devoted toward the development of more effective and highly promising biofertilizer [326].

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16 Fabrication Approaches for Biofertilizers Andrew N. Amenaghawon1*, Chinedu L. Anyalewechi1 and Heri Septya Kusuma2† 1

Department of Chemical Engineering, Faculty of Engineering, University of Benin, Benin City, Nigeria 2 Department of Chemical Engineering, Faculty of Industrial Technology, Universitas Pembangunan Nasional “Veteran” Yogyakarta, Indonesia

Abstract

The use of biofertilizers as a viable substitute for chemical fertilizers is increasingly gaining attention as a very important source of nutrient supply to plants. Biofertilizers are eco-friendly and economical with potential for wider agricultural applications. This is due to the several benefits accruable from the use of biofertilizers in terms of increased growth and productivity of plants, elimination of adverse health and environmental problems arising from the use of chemical fertilizers, and the added protection against various plant diseases. This book chapter provides an in-depth understanding of biofertilizers and the various approaches available for their preparation. The different types of biofertilizers and their respective roles are covered in considerable detail. The types of inoculant carriers for biofertilizers formulation, carrier forms, and the various application modes of biofertilizers are extensively discussed. The challenges and limitations affecting the application of biofertilizers are also highlighted. The chapter ends with some future prospects and recommendations needed for further improvements in the development, preparation, and application of biofertilizers in order to achieve green, cleaner, and sustainable food production. Keywords:  Biofertilizers, nutrients, inoculant, bacteria, solubilization, carrier, formulation, nitrogen fixation

*Corresponding author: [email protected] † Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (491–516) © 2021 Scrivener Publishing LLC

491

492  Biofertilizers

16.1 Introduction The production output as well as quality of agricultural yields obtained is dependent on the quality of the soil. Drought, salinization and the continuous exploitation of arable land without adequate nutrient replenishment are the main factors responsible for poor soil quality [1]. Generally, the reduction in crop productivity is often caused by poor maintenance of the top soil, hence the need to properly maintain the soil quality. The commonly and widely used approach in maintaining soil quality and ensuring adequate supply of nutrients to the soil for increased crop yields is via the application of chemical fertilizers. However, the utilization of biofertilizers as a viable alternative to ­synthetic/chemical fertilizers is widely gaining importance in sustainable agriculture. This is due to the adverse health and environmental issues posed by the application of chemical fertilizers in agriculture, such as water and air pollution, soil deterioration, greenhouse effect, and the need for eco-friendly and cost-efficient solutions for the production of food capable of meeting the needs of our growing human population. Biofertilizers are natural, eco-friendly, affordable, and viable solutions that can be used for agricultural activities regardless of the scale of production. These biofertilizers in addition to providing nutrients to the soil also help in maintaining the soil structure and the biodiversity of farmlands. The nutrient enhancement of biofertilizers in the soil is achieved mainly via nitrogen fixation, solubilization of phosphorus, and synthesis of substances that promote plant growth.

16.2 Biofertilizers Biofertilizers also referred to as microbial inoculants are substances designed to meet the nutritional needs of plants via microbial means. These substances contain living microorganisms that promote plant growth when applied to soil, seeds, or plants. Biofertilizers are basically carrier-based microbial formulations which contain beneficial microorganisms in a viable state that enhance the growth of plants by increasing nutrient availability. Microbial inoculants that are commonly used include nitrogen, phosphorus, and plant growth-promoting rhizobacteria (PGPR). Biofertilizers are preparations comprising of strains of microorganisms, organic products, as well as dead tissues of plants which enrich the soil and plants with desired nutrients. The application of biofertilizers serves to enhance soil fertility, thereby increasing crop yield [2]. By increasing the

Fabrication Approaches for Biofertilizers  493 microbial population of the rhizosphere, biofertilizers are able to convert nutrients in erstwhile unavailable forms to available plant usable form. The supply of nutrients in soluble form in the soil for plant uptake is triggered by microbial populations. The following are some benefits derivable from the use of biofertilizers [3]: • Increased plant growth and crop yield arising from improvement in soil fertility • Reduction in environmental pollution resulting from the production and application of chemical fertilizers • Protection of plants from various pathogens present in the soil • Cost-effectiveness and economic attractiveness • Improvement in soil fertility, overall soil health and conditioning Biofertilizers are used to enhance the microbial processes which serves to boost the supply of nutrients that are available for plant use. Biofertilizers help to improve the fertility of soils through the fixation of nitrogen in the atmosphere, solubilization of insoluble phosphates, as well as by producing plant growth-promoting substances in the soil [4]. Biofertilizers are ecofriendly in nature, cost effective, and their continued use promotes soil fertility [5].

16.3 Types of Biofertilizers There are various types of biofertilizers which will be discussed under the following categories: nitrogen-fixing biofertilizers, phosphate-­ solubilizing biofertilizers, phosphate-mobilizing biofertilizer, potassium biofertilizer, blue-green algae (BGA), and plant growth-promoting biofertilizers.

16.3.1 Nitrogen-Fixing Biofertilizers Nitrogen is important for the growth and productivity of plants. It is readily available and abundant in the atmosphere. However, atmospheric nitrogen cannot be used by plants. In order for the nitrogen to be available for plant use, it has to be converted to plant usable form. This is achieved by converting the atmospheric nitrogen into ammonia which is easily accessible by plants. This conversion process is known as biological nitrogen

494  Biofertilizers fixation (BNF) [6]. During the BNF process, nitrogen-fixing microorganisms convert the atmospheric nitrogen to ammonia by means of a catalytic enzyme (nitrogenase) [7]. Common examples of nitrogen-fixing microorganisms are Rhizobium, Azospirillum, Azotobacter, Bradyrhizobium, etc. These microorganisms can be further classified as free living, symbiotic, or asymbiotic/associative.

16.3.1.1 Rhizobium Rhizobium belongs to the Rhizobiaceae family. They are symbiotic in nature and form root nodules in plants. Rhizobium is capable of fixing atmospheric nitrogen in symbiotic association with legumes (e.g., soybean, pea, lentil, berseem, black gram, and groundnut) and non-leguminous plant (e.g., parasponia). Rhizobium has nitrogen-fixing ability of about 50–100 kg/ha with legumes only [8]. Rhizobium enters the root hairs, multiplies, and inhabits the roots of legumes forming tumor-like growths known as root nodules in which the production of ammonia occurs. Specific species of rhizobium are required for different legumes to achieve successful nodulation. Hence, the availability of compatible strain for a particular legume determines the extend of effective nodule formation by rhizobium. Although various legumes can be modulated by varying strains of rhizobia, improvement in growth only occurs when nodules are formed by effective strains of rhizobia. Consequently, for maximum nitrogen fixation, the matching of microsymbionts is of paramount importance and should be carefully performed [8]. More so, the population of rhizobium in the soil is dependent on the availability of leguminous crops in the field. Therefore, as the legumes become unavailable, the rhizobium population diminishes.

16.3.1.2 Azospirillum Azospirillum belongs to the Spirilaceae family. They are heterotrophic, gram-negative, aerobic, and associative non-nodule forming nitrogenfixing bacteria [9]. Azospirillum can fix about 20–40 kg/ha of nitrogen and is capable of producing growth regulating substances [8]. There are several species of Azospirillum which include A. lipoferum, A. amazonense, A. brasilense, A. halopraeferens, and A. irakense [10]. However, the most important and beneficial species of Azospirillum are A. lipoferum and A. brasilense [11]. The interaction between Azospirillum and several plants, especially plants which have C4 dicarboxylic pathway (Hatch-Slack pathway) of photosynthesis, is that of associative symbiosis. This is because of the

Fabrication Approaches for Biofertilizers  495 growth and nitrogen fixation of Azospirillum on the organic salts of malic and aspartic acid [8]. Azospirillum is very suitable for growing crops like sorghum, rice, millet, maize, sugarcane, and wheat [12, 13]. Azospirillum produces growth promoters such as cytokinin, indole acetic acid (IAA), and gibberellins which stimulate root development and enhances the plant nutrient uptake of nitrogen, phosphorus, and potassium [12]. Trabelsi and Mhamdi [14] reported significant increase on root development and exudation from the use of Azospirillum inoculant.

16.3.1.3 Azotobacter Azotobacter belongs to the Azotobacteriaceae family. They are free-living, non-symbiotic, aerobic, and photoautotrophic bacteria that are mostly found in neutral and alkaline soils. The most commonly found species in arable soils is Azotobacter chroococcum. Other species of Azotobacter include Azotobacter beijerinckii, Azotobacter insignis, Azotobacter macrocytogenes, and Azotobacter vinelandii [11]. Azotobacter produces vitamin B complex and phytohormones such as naphthalene acetic acid (NAA) and gibberellins, as well as other substances, which promote plant growth and plant mineral uptake, but inhibits the growth of certain root pathogens [15, 16]. Nitrogen fixation of about 15–93 kg/ha has been reported using Azotobacter on roots of Paspslum notatum plant [17]. The Azotobacter indicum strain have been reported as been able to produce a variety of antifungal antibiotics which can prevent various fungi pathogens from growing in the root region, thus resulting in considerable decline in the mortality rate of seedlings [18].

16.3.2 Phosphorus-Solubilizing Biofertilizers Phosphorus is an essential element needed for plant growth and development, constituting about 0.2% of plant dry weight [3]. Phosphorus is present in large amounts in the soil. However, most of it is present in forms which are unavailable to plants. Hence, phosphorus is regarded as the second major limiting plant nutrient after nitrogen [19]. Phosphate rock minerals are usually highly insoluble making it difficult to adequately provide phosphorus for plant uptake. Some of the noticeable bacteria strains with capability to solubilize insoluble inorganic phosphorus include Bacillus, Pseudomonas, Rhizobium, Micrococcus, Acetobacter, Achromobacter, Agrobacterium, Flavobacterium, Burkholderia, and Erwinia [20]. These phosphorus solubilizing bacteria can be used to make phosphorus more available by converting it to its more available forms in the

496  Biofertilizers soil [21]. They can combine with other soil fungi like Aspergillus and Penicillium to facilitate the breakup of complex phosphates in the soil by producing acids that lower the pH of the surroundings. Increased crops yield of about 70% have been reported using phosphate-solubilizing microorganisms [3]. The sugar yield and juice quality of sugarcane was reported to have increased by 12.6% when phosphorus solubilizing bacteria, Bacillus megaterium var. phosphaticum, was applied to an inexpensive rock phosphate [22]. The phosphorus requirement was observed to have reduced by 25%, resulting in additional 50% reduction of the costly superphosphate usage [22]. Biofertilizers containing phosphate-­ solubilizing microorganisms can be used in the control of nematode disease [23]. The application of Bacillus megaterium have been reported to have resulted in about 50% reduction in the penetration of M. chitwoodi and Pratylenchus penetrans into potato roots [24]. Padgan and Sikora [25] from their studies reported a reduction of about 40% in nematode penetration and gall formation for rice plants treated with Bacillus megaterium. Microorganisms that have phosphate-solubilizing capability as well as capacity for phosphate fixing in soil can also facilitate growth of plant via nitrogen fixing in soil.

16.3.3 Phosphate-Mobilizing Biofertilizer (Mycorrhizae) Mycorrhizae are very popular and important phosphorus mobilizers. Phosphorus mobilizers facilitate the acquisition of soluble phosphorus from locations in the soil that are inaccessible to plants roots by making them available to plant roots. Mycorrhizae denote a symbiotic association between fungi and the roots of plants. This symbiosis enables both sides to meet their nutritional needs [26]. Fungi are ubiquitous in nature and are present in the roots of many Gymnosperms, Angiosperms, Thallophytes, and Pteridophytes [27]. The fungi derive carbohydrates from the photosynthates of the host plant and provide the needed nutrients, water, hormones, and protection from root pathogens. Phosphorus mobilizers are useful in increasing nutrient uptake and plant growth. This uptake of nutrients is made possible by means of the fine absorbing hyphae of the fungi. The fungal endophyte is able to stimulate growth, enhance reproduction, as well as provide the host plant protection from both biotic and abiotic stresses [28]. Mycorrhizae plants have been reported to have greater tolerance to stresses such as toxicity from heavy metals, soil salinity, high soil temperature, poor soil pH, as well as stresses resulting from the transplanting of plants [29].

Fabrication Approaches for Biofertilizers  497 The arbuscular mycorrhizae (AM) fungi are commonly regarded as the most abundant fungi present in agricultural soils, comprising of about 5%–50% of the biomass of soil microorganisms [30]. There are seven genera of fungi that produce Arbuscular mycorrhizal symbiosis with plants, namely, Acaulospora, Gigaspora, Entrophospora, Glomus, Gigaspora, Archaeospora, Scutellospora, and Paraglomus. The AM fungi also contribute to the water economy of plants. Through their association, the hydraulic conductivity of the plant roots in the soil at lower water regions is improved; thus, promoting the uptake of water by the plants [8]. Certain mechanisms have been proposed by which AM fungi can help to facilitate the activation of plant defense systems. They include higher cell wall lignification, modifications in the exudation patterns and that of the ­mycorrhizo-sphere, and competition for colonization space and infection sites [31].

16.3.4 Potassium Biofertilizer Potassium biofertilizer is an important class of biofertilizers that contribute significantly to the growth and development of plants. Potassium biofertilizers can be derived from waste materials such as mica after undergoing modifications using chemical or biological techniques. By means of composting technique, unavailable potassium (K) in the waste mica can be converted into plant-accessible forms. This is due to the acidic environment present during composting of the waste mica. On the other hand, some rhizobacteria can produce potassium in accessible form in the soil. For example, silicate bacteria have been reported as having the capability to dissolve potassium, silicon, and aluminum from insoluble minerals. Bacillus, Aspergillus, and Clostridium microorganisms have been reported as very suitable for potassium solubilization in different crops [32]. Potassium helps in the activation of enzymes and in the transport of sugars and starch. It also aids in the uptake of nitrogen, promotes photosynthesis, as well as maintain the turgor pressure of the cell. Potassium is vital to the synthesis of protein. Other benefits of potassium include higher resistance to diseases, thereby enabling the plant to withstand stress better and higher quality of crops [2]. The application of potassium to the soil is usually done externally in the form of potassic fertilizers.

16.3.5 Growth-Promoting Biofertilizers These are biofertilizers with capacity for enhancing growth of plants. They are a type of rhizobacteria found naturally in soil within the vicinity of

498  Biofertilizers plant roots where the form specific symbiotic relationships with plants that aid the nutrient availability and safety of plants. These bacteria types comprise of the majority of phosphorus-solubilizing as well as nitrogen-fixing bacteria that can serve as biofertilizers with capability to enhance plant growth through the synthesis of growth promoting chemicals [33]. These rhizobacteria help to ensure the availability of vital macro and micronutrients like nitrogen, phosphorous, potassium, iron, and copper. They also help in enhancing the growth of other beneficial bacteria and fungi [34]. There are two main routes by which they may promote growth in plants, viz., directly or indirectly. The direct route is by enhancing nutrients availability or via modification of plant hormones. The indirect route is through reducing pathogen inhibition to plant growth [20, 35]. Growth enhancing rhizobacteria have various positive effects on plants such as improved growth and crop yields, better seed germination, higher chlorophyll contents, and improved nodule formation in legumes [36, 37]. In addition to providing suitable conditions for nutrients uptake, they also provide protection to plants against certain abiotic and biotic stresses including the ability to defend themselves from the intrusion of various pathogens [38]. Azotobacter, Arthrobacter, Bacillus, Alcaligenes, Azospirillum, Pseudomonas, Burkholderia, Serratia, Klebsiella, and Enterobacter are some of the known plant growth-promoting rhizobacteria (PGPR) that help in soil enrichment by changing complex matter into simple and usable forms. These PGPR can be further classified into bio-stimulants, biofertlizers, and bio-protectants.

16.3.6 Blue-Green Algae (Cyanobacteria) These are living organisms also known as cyanobacteria and are freeliving nitrogen-fixing microorganisms. They are phototrophic and belong to eight different families. Cyanobacteria comprise of unicellular and colonial species. Some of the commonly used cyanobacteria for field applications are Anabaena, Aulosira, Cylindrospermum, Nostoc, Plectonema, Scytonema, and Tolypothrix [2, 3]. BGA produce several growth promoting substances such as nicotinic acid, folic acids, pantothenic acid, amino acids, IAA, sugars, and polysaccharides [39]. The nitrogen-fixing ability of BGA varies with the agro-climatic conditions. Nitrogen fixation of about 25%–30% N/ha/season in rice fields has been reported using BGA [40]. Besides the fixation of nitrogen in the soil, BGA also enriches the soil with several secondary metabolites, hormones, and extracellular carbohydrates. BGA help to improve the structure and health of the soil. They also increases soil porosity and water retention and provides some remediation

Fabrication Approaches for Biofertilizers  499 for soils degraded by excessive use of chemical fertilizers and those affected by salts [41].

16.4 Preparation Approaches for Biofertilizers 16.4.1 Inoculant Formulation The formulation of biofertilizers is usually performed such that although the living microbial cells are in a viable state; they are able to increase soil fertility, growth, and productivity of the plant. Biofertilizers formulation is basically a multi-stage process involving the combination of different strains with specific additives that serves to preserve the cells during storage [42]. The formulation method used in the preparation of microbial inoculant is central to its viability as a biofertilizer. Basically, the preparation approach involves the incorporation of viable microbial cells into an appropriate carrier with the addition of additives that provide stabilization and protection to the inoculants during storage, transportation, and application. It is important for the formulation to be such that its handling and application are easy to carry out. This will ensure effective delivery of the formulation to the target by protecting the bacteria from harsh environmental factors thereby maintaining the microbial activity of the soil. Achieving desirable formulations of biofertilizers is mainly dependent on two factors, namely, the microbial strain and the preparation method of the inoculant. Generally, the potential success of an inoculant is hinged on the formulation approach used. Studies have shown that the development of biofertilizers formations that are very good greatly increases its activity upon introduction to the target plants [43]. However, the optimization of an inoculant is usually not dependent on the microbial strain used because of the similarities in the physiological properties of most strains derived from the same bacteria species. Hence, a procedure developed using a particular strain can be easily adapted to another strain of the same species [33]. Some of the desirable qualities required in a good formulation are as follows: the formulation should have the capacity for nutrients addition; it should be prepared from readily available low cost raw materials; ecofriendly (non-pollutant, nontoxic, and biodegradable); adjustable pH; flexible and gradual release of bacteria into the soil; easy application that is compatible with standard seedling equipment; and stability and extended shelf life under harsh conditions [20, 42, 44].

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16.4.2 Carriers for Biofertilizer Preparation Carriers are the delivery vehicles used in conveying the formulated microbial inoculants (biofertilizers) from industrial fermenters to rhizosphere in the field. They play an important role in ensuring the delivery of the required cell population in viable state [45]. These carriers provide the biofertilizers temporal protective environment in the soil. This protection may be physical, i.e., by means of a protective surface of pore space, or nutritional, i.e., via the provision of a suitable substrate [43]. The carriers can be made from inorganic, organic, or synthetic materials. A good carrier should exhibit the following properties: high water retention ability; support growth and survival of bacteria; affordable and readily available in powder and granular form; nearly neutral or easily adjustable pH; amenable to nutrient augmentation; easy to handle during mixing, curing, and packaging operations; no heat of wetting; almost sterile or easy to sterilize; chemically and physically uniform; and with good adhesion to seeds for seed coated carriers. It should be nontoxic to the inoculated biofertilizers, nonpolluting, and easily biodegradable, suitable for all rhizobia, and also devoid of lump forming materials [3, 33, 46–49]. However, a single carrier that possesses all these properties and can be used as a universal carrier for all biofertilizers hardly exists. Nevertheless, a good carrier should have most of these qualities.

16.4.2.1 Sterilized Carriers Inoculants prepared using sterilized carriers leads to increased microbial growth as well as increase in the shelf life. More so, the use of pre-sterilized carriers eliminates or greatly reduces the presence of contaminants and provides an alternative for extending culture preparations by diluting the broth without sacrificing the final quality of the inoculant [50]. However, there are some drawbacks associated with the use of sterilized carriers. These include increase in cost of production, installation of a sterilizing unit with the required capacity to meet the production demands, increase in labor and requirement of aseptic procedures during the introduction of the culture to the pre-sterilized carrier package. Some of these packages may be contaminated, and detecting which package have been contaminated is usually difficult. Whereas, these limitations may be negligible and not too obvious for small-scale productions, they however pose serious challenges during manufacturing on a large scale [47]. There are various sterilization methods that have been used. Examples are these sterilization

Fabrication Approaches for Biofertilizers  501 methods are gamma irradiation, autoclaving, microwave radiation, and fumigation using ethylene oxide or methyl bromide.

16.4.3 Carrier Form The carrier form is mainly dependent on the mode of application to be used. It is also dependent on the planting equipment to be used and the cost involved. The typical carrier forms used are granules, beads, or powder forms (for solid based carriers) and liquid form. Most inoculants used for seed applications before planting are usually in powdered forms. Powdered forms of inoculant carriers can be used in seed coating. It can also be suspended in a liquid to form a slurry, which can either be directly applied to soil. Alternatively, the seeds/plants can be dipped in the slurry to coat the seeds/plants before the planting process. Powder materials with particle sizes that can pass through a 0.25-mm sieve have been recommended by [51, 52] on the other hand recommended as a requirement that 50% of the particles size should pass through a 0.075-mm mesh screen. Better seed adhesion has been achieved using finer particle size. Inoculant in granular form can be directly applied into soil with the seed. Particle sizes that are within the range of 0.35–1.18 mm are granular, thus ensuring easy absorption, curing, and uniform flow for the culture. The granular form in comparison with the seed application method provides significantly more rhizobia during application, for application rates of 5–30 kg/ha, depending on the width of the row. The seed application method delivers only 210 g/ ha of inoculant on the total seeds planted [47]. A major drawback of granular inoculant is the high cost involved. However, it gives better results when applied under various conditions especially under conditions with environmental stresses after planting and greatly improves plant nodulation.

16.5 Methods of Biofertilizer Formulation The various types of biofertilizer formulations can be classified based on the type of carrier used which forms a larger part of inoculant that provides nourishment and protection to the microorganisms from environmental stresses form the formulation to application.

16.5.1 Solid-Based Carrier Bioformulation Initial formulations of biofertilizers were made using only solid-based carrier. Solid-based carriers bioformulation refers to inoculants prepared

502  Biofertilizers using solid carriers. In this process, the inoculum is mixed to a solid carrier made of an inert material which serves as a vehicle for transporting the microbes from the laboratory to the field. There are several examples of solid carriers which have been considered as carriers for inoculants. They include peat, perlite, mineral soil, charcoal, inorganic clay, zeolite, talc, vermiculite, sand, and plant waste materials. These solid carriers can be further grouped into the following broad categories [47]: i.

Soils: peat, coal, peat with additives, coal with additives, clays, inorganic soils, etc. ii. Plant waste materials: compost obtained from bagasse, rice husk, farmyard manure, sawdust, corncob, coir dust, cellulose, etc. iii. Inert materials: vermiculite, vermiculite with additives, perlite, calcium sulphate, ground rock phosphate, polyacrylamide gel, and encapsulated alginate beads. iv. Lyophilized microbial cultures or oil dried bacteria, which is the incorporated into a solid carrier. However, only peat, which is the most widely used solid carrier for the preparation of biofertilizers, will be discussed.

16.5.1.1 Peat Formulations Peat is easily the most widely used carrier because it is dependable and has recorded several successful applications over the years [47]. Peat is essentially a complex and undefined material comprising of different origins that differ in their capacity to support cell growth and survival [44]. Peat formulations consist of partially decayed plant materials that have accumulated over the years. Peat, when used as a carrier provides a protective environment that is rich in nutrients which aids the growth of various microorganisms with ability grow and form microcolony particle surfaces and cervices as well [45]. Some of the desirable properties of peat are its enormous surface area which facilitates the thriving of the inoculant, high water retention capacity, ease of use, and general acceptability. However, lower tolerance of the bacteria to physical stress during storage, inconsistency in quality, high cost, and non-availability in several countries (mostly in the tropics) are some of its drawback [33, 47, 53].

Fabrication Approaches for Biofertilizers  503

16.5.2 Liquid Inoculants Formulation These are basically aqueous, oil, or polymer-based products. Liquid formulations in addition to having the required microorganisms and nutrients also consist of special cell protectants and additives that supports the survival of the cells during storage as well as after application to seeds/ soil [3]. Liquid inoculants comprise of a medium which contains carbon, nitrogen, and vitamins that promotes the growth of microorganisms as well as other additives that serve as cell protectant. These added additives help in stabilizing the product, protect the inoculant from environment stresses upon application to seed and soil, provide better seed adhesion, help in the binding or inactivation of soluble toxins coated on the seeds, provide prevention from osmolysis, as well as enhance the survival of rhizobia during storage [49]. The presence of cell protectants in liquid inoculum in addition to maintaining the high microbial numbers helps to facilitate the formation of resting cells (e.g., cysts and spores) that provide greater resistance to abiotic stresses, which consequently leads to higher survival rates of the bacteria [49]. The nature and concentration of additives have been reported to affect the performance of the inoculum [54]. The selection of an additive should be based on its ability to provide protection to bacteria cells during storage and on application to seeds from various harmful environmental stresses that includes high temperature, drought, and toxic conditions of seeds and seed chemical. Polymers, which have high molecular weights, good water solubility and water activity, limited heat transfer, good rheological properties, and non-toxic and complex chemical nature, are very suitable for use as additives [55]. Examples of polymers commonly used as additives include polyvinylpyrrolidone (PVP), polyethylene glycol, polyvinyl alcohol, trehalose, sodium alginate, Fe-EDTA, gum Arabic, tapioca flour (cassava starch), glycerol, and methyl cellulose [56]. Advantages of liquid inoculant include easy handling and application to seeds; compatibility with modern agriculture machineries for its application; size reduction, as less amount of inoculant is required, ease of production, packaging and storage with longer shelf life; higher cell counts; higher performance in soil; elimination of contamination problems; and cost efficiency, as it does not require processing and sterilization of solid carrier material [49].

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16.5.3 Polymer-Based Formulation The increasing interest in the application of biofertilizers as plant protectants has led to further research studies aimed at improving the stability and shelf life of biofertilizers. These studies have resulted in the development of ­polymer-based carriers. In this method, the polymer immobilizes the microbial cells in the matrix and then gradually release them. These formulations have the advantage of longer shelf life even when kept at room temperature due to the protection they provide against environmental stresses. Polymer-based formulations also ensure the preparation of products with consistent quality due to the standardized production process. However, storing the inoculant at temperature of 4 °C is preferable and helps to maintain the viability of the encapsulated cells for a prolonged period of time. They inoculants can also be augmented with other nutrients to increase the survivability of the bacteria during application [44].

16.5.3.1 Alginate Formulations Alginate has been established as a suitable polymer for encapsulating microorganisms. Beyond that, it is naturally occurring and is made up of bacteria and brown algae-derived L-guluronic and β-1,4-linked D-mannuronic acids. The reaction between alginate and a multivalent cation (e.g., Ca2+) results in the formation of a gel made up of a compact three-dimensional lattice with pore sizes ranging from 0.005–0.2 mm in diameter [57]. Beads are formed when the alginate solution is added to the cation solution. These alginate beads typically have diameters in the range of 2–3 mm. However, microbeads with sizes ranging between 50 and 200 µm that are capable of entrapping about 108 to 109 CFU g−1 have also been considered [58]. Alginate-based formulations are increasingly used due to their desirable properties such as gradual release of microorganisms into a soil, nontoxicity, and biodegradability [59]. However, a major drawback on the use of alginate is its high cost. Under field conditions, wheat plants have been successfully inoculated with Pseudomonas fluorescens and Azospirillum brasilense using with alginate as the agent of encapsulation [60]. Longer survival rates comparable to results obtained using other carrier-based inoculants have also been reported for alginate encapsulated bacteria during field inoculation [61].

16.5.4 Fluidized Bed Dried Formulation The fluidized bed dryer (FBD) formulation approach for the preparation of biofertilizers potentially eliminates the problems of biofertilizers

Fabrication Approaches for Biofertilizers  505 contamination and their short shelf life. By reducing the moisture content of carrier-based inoculants, the contaminants can be reduced. Longer shelf life and higher inoculant activity at field conditions can be achieved by improving the production process for dried inoculants. Generally, the FBD utilizes an upward flow of air stream to suspend the introduced material against gravity, thereby creating a fluidized condition. A major advantage of using the FBD for the drying of bioinoculant is that reduction in the drying temperature. Products drying is possible in temperatures as low as 37 °C–38 °C and at even lower temperatures, allowing the drying of more temperature sensitive organisms [62, 63]. However, despite the promising potentials of FBD, more research studies are still needed on the usage of fluid bed dryer for the production of biofertilizers. Other advantages of FBD include absence of contamination, adjustable drying temperature, limited decline in number of cells, and the combination of several ingredients prior to drying [62, 63].

16.5.5 Particles From Gas Saturated Solutions (PGSS) Method The particles from gas saturated solutions (PGSS) process is a novel method that involves the application of supercritical fluid properties. The PGSS process, which is performed at low temperatures, makes use of carbon dioxide as supercritical fluid. This process has the potential to produce formulations with no negative effects on the viability of the microorganisms as well reduce the production costs [44]. The end product of the PGSS process is the formation of particles that are nearly spherical in shape that are in powdered-like forms which can be suspended in water [64]. This method has been evaluated for the encapsulation of virus formulations [65]. Other successful applications of the PGSS method have been performed for different solids and liquids [64, 66].

16.5.6 Bionanoformulations Bionanofromulation is another novel method with potentials for use for the production of biofertilizers. Bionanotechnologies can be deployed in the development of carrier-based microbial inoculants [67, 68]. The desirable properties of nanomaterials such as physical stability, high surface area, cost effectiveness, and ease of fabrication are triggering their application in biofertilizer production. The utilization of these nanoformulations has the capability to improve biofertilizer stability as well as that of biostimulators in terms of desiccation, heat, and UV inactivation [44].

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16.6 Application Modes for Biofertilizers There are several methods by which biofertilizers can be introduced to the soil. Some of the application methods include [33]: a. Sprinkling method, which involves wetting the seeds with little amount of water before mixing with peat powder b. Inoculating of seeds with powder formulations c. Mixing of seeds with dry biofertilizers in the seed hopper d. Seed treatment e. Slurry method, which involves the suspension of the biofertilizers in water before adding and mixing with seeds f. Spraying a peat-in-water suspension in the furrow created while planting g. Seed pelleting h. Seedling root dip i. Soil application j. Coating a slurry mixture of biofertilizers and adhesive However, methods d, h, and i are the prominent methods for applying microbial biofertilizers and will be briefly discussed.

16.6.1 Seed Treatment This is the most commonly used technique for applying various types of inoculants. It is a very effective and economical mode of application [69]. The seed treatment method basically involves uniformly coating a mixture of seeds in a slurry, which is then dried in a shade and planted within 24 h. However, for liquid biofertilizers, the seed coating can be done via the use of a bucket or plastic bag depending on the quantity of seeds involved [70]. The bucket can be used for large quantity of seeds while the plastic bag can be used for small quantity of seeds. The seed treatment allows the use of different bacteria combinations without any negative effect, as well as ensuring adequate delivery of the amount of bacterium on the respective seeds needed to achieve desirable results [71].

16.6.2 Seedling Root Dipping The seedling root dipping involves dipping of the seedling roots in a water suspension made up of biofertilizers for a particular period of time before transplanting. The treatment time required for the dipping of the seedling

Fabrication Approaches for Biofertilizers  507 roots before transplanting vary from crop to crop with paddy crops requiring a much longer dipping period (about 8–12 h) than vegetable crops (about 20–30 min) [72]. The seedling root dipping mode of application is mostly used for plants like vegetables, grapes, cereals, tobacco, banana, fruits, sugarcane, and trees.

16.6.3 Soil Application This method involves the direct application of the biofertilizers to the soil. The application can be done distinctly or in combination with other biofertilizers. For instance, phosphate-solubilizing biofertilizer in addition to cow dung and rock phosphate have been combined together and stored overnight in shade with it moisture content kept at 50% before applying to the soil [73]. Soil applied inoculants have the following advantages: elimination of seed mixing, minimization of direct contact with treated seeds, ability to increase the delivery rates, thereby providing more rhizobia per unit area, and better ability to withstand low moisture conditions when compared with that of powder form [74]. Biofertilizers that have been applied via the soil application method include Rhizobium (for either trees or leguminous plants) and Azotobacter (for coffee, tea, leaves, coconuts, spice, gum, rubber, nuts, seeds, flowers, and fruits) [75].

16.7 Factors Affecting the Preparation of Biofertilizers A major factor that affects the production of biofertilizers is the cost required for the production. The market price for biofertilizers is expected to be at par or even below that of conventional fertilizers in order to ensure market sustainability [44]. Also, the inoculant formulation is also an important factor to consider during the preparation of biofertilizers. According to Sahu and Brahmaprakash [49], the following points should be considered during the production of biofertilizers: • The inoculant produced should be user friendly in terms of handling and application. • The delivery of the inoculant formulation to the target sites should be performed in an efficient manner and form. • The biofertilizer prepared should be capable of providing protection for the agent against various environmental threats.

508  Biofertilizers • The inoculant formation should be able to promote or maintain the soil’s microbial activity. • The stability of the formulated inoculum is vital during the stages of production, distribution, storage, and transportation. • The biofertilizers produced should be able to improve soil properties as well as resist pH changes during storage.

16.8 Beneficial Effects of Biofertilizers The use of biofertilizers presents several benefits considering the problems arising from the use of chemical fertilizers. Some of these problems include leaching, destruction of microorganisms and other organisms of economic importance, contamination of water bodies, susceptibility of crops to diseases, and reduction in soil fertility [76]. However, the utilization of biofertilizers can help to eliminate these challenges. Biofertilizers have the capability to satisfy the nutrient requirements of plants, in addition to limiting the need for inorganic fertilizers. Biofertilizers can be used to facilitate plant growth and improve crop yields by means of bioinoculants. These bioinoculants contain different strains of living microorganisms. These microorganisms are capable of converting and mobilizing nutrients from unavailable form into usable form in the rhizosphere. They can also facilitate the conversion of complex organic compounds to smaller compounds that serve as nutrients to improve the fertility of the soil, thus increasing crop yields, while maintaining the ecology of the soil [20, 77]. Biofertilizers have positive effects on several plants in terms of growth and yield. Malusà et al. [77] observed that microorganisms that facilitate the growth of plants can be effectively used in improving plant nutrition when used in synergy with organic and inorganic fertilizers. Bhardwaj et al. [78] reported higher crop yields (about 10%–40%) from the use of biofertilizers resulting from an increase in the protein content, vitamins, amino acid, and nitrogen fixation. The eco-friendly, non-toxic, easy to apply, and cost-effective nature of biofertilizers have continued to make them attractive as potential alternatives to chemical fertilizers. Biofertilizers are also able to convert nutrients that are naturally available in the atmosphere or soil into plant usable form as well as serve as supplements to agrochemicals. Other benefits accruable from the use of biofertilizers in agriculture include increased vigor in seedlings and adult plants, excellent source of micronutrients and microchemicals, secretion of growth hormones and source of organic matter,

Fabrication Approaches for Biofertilizers  509 reduction in harvesting time as improvement in post-harvesting of crops, and cleanup of heavy metals and crude oil contaminated soils [76].

16.9 Challenges and Limitations of Biofertilizers A major limitation of biofertilizers is that of inconsistency in terms of their performance. For optimal utilization of biofertilizers in agriculture, further improvements are needed to ensure consistency of their performance. There also exists a gap in the formulation and application of inoculants. This is mostly due to the inability of the manufacturers of these inoculants (most based in developed countries) to take into consideration the unique challenges encountered during the application of the inoculants in developing countries which are mostly their target market. Another limitation of biofertilizers is their application in certain soil conditions such as in semi-arid conditions. When applied to semi-arid conditions, the survival rate of the introduced bacteria is very minimal due to the prevailing adverse conditions which include but not limited to drought, high salinity, insufficient irrigation, and soil erosion. It is also possible for the bacteria introduced into the soil to be unable to sufficiently populate the rhizosphere due to competition from native strains. Moreso, limited data exists on the factors that control competition among bacteria strains, especially under field conditions [3]. Other factors limiting the application of biofertilizers are inadequate understanding of the inoculation process by extension personnel and farmers, production of inoculants with poor quality, and low nutrient density, ineffective inoculants delivery/supply system, poor sensitization and enlightenment among farmers on biofertilizer usage and benefits, and lack of concerted effort in terms of policy to ensure the utilization of biofertilizers [70, 77].

16.10 Future Prospects As the demand for green, cleaner, and sustainable methods for agricultural production increases, the utilization of biofertilizers will continue to be an integral part of such drive. This will consequently lead to the development of improved biofertilizer formulations. With the increasing popularity and the successful applications of these biofertilizers in various countries, it is safe to assume that in years to come, the continuous and widespread utilization of biofertilizers will provide several useful insights for the complete improvement of the agricultural sector [79].

510  Biofertilizers The development of biofertilizers for large-scale commercial application from laboratory and greenhouse experiments will require detailed studies and development of novel methods for the formulation, storage, and application of the bioinoculants. Extensive awareness and enlightenment of farmers and other stakeholders on the utilization and long-term benefits of biofertilizers instead of using chemical fertilizers, as well as the inherent dangers to life and environment involved with the prolonged use of chemical fertilizers. A paradigm change in the public mindset will be required to dispel the misconceptions about bacteria as only agents of diseases for general acceptability. Whereas, current biofertilizers are often made from carefully selected non-transformed bacteria strains with certain desirable traits, the development of genetically engineered bacteria strains that are highly effective and efficient in enhancing plant growth will be necessary. However, relevant regulatory bodies will need to thoroughly ascertain that such inventions do not pose any hazard or risk to the public and environment. Also, quality control system will suffice to monitor the production of inoculants and their applications. Stringent regulations/act for quality control of biofertilizers and their applications should be established. Research studies on the adaptability of microorganisms in biofertilizers under adverse environmental soil conditions should be carried out in conjunction with agronomic and economic evaluation of biofertilizers for different agricultural applications.

16.11 Conclusion The several benefits derivable from use of biofertilizers in terms of growth and productivity of plants and the added protection against some plant diseases make them a very desirable and viable source of nutrient supply to plants that is eco-friendly and economical, with potential for wider agricultural applications. Determining the most favorable plant-microorganism interaction is very crucial in augmenting productivity. More studies need to be carried out to understand the actual mechanism of biofertilizers, in addition to identifying the various strains of biofertilizers and their properties. As advancement in biotechnology continues, more robust and efficient biofertilizer formulations will be developed in order to achieve clean, green, and sustainable agriculture. The development of improved, easy to use formulations of inoculants that are cost effective, stable, and affordable will no doubt translate into wider usage, application, and general acceptability of biofertilizers.

Fabrication Approaches for Biofertilizers  511

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512  Biofertilizers 17. Youssef, M., Eissa, M., Biofertilizers and their role in management of plant parasitic nematodes. A review. E3 J. Biotechnol. Pharm. Res., 5, 1–6, 2014. 18. Martin, X.M., Sumathi, C.S., Kannan, V.R., Influence of agrochemicals and Azotobacter sp. application on soil fertility in relation to maize growth under nursery conditions. EurAsian J. Biosci., 5, 19–28, 2011. 19. Schachtman, D.P., Reid, R.J., Ayling, S.M., Phosphorus Uptake by Plants: From Soil to Cell. Plant Physiol., 1998. 20. Mahanty, T., Bhattacharjee, S., Goswami, M., Bhattacharyya, P., et al., Biofertilizers: a potential approach for sustainable agriculture development. Environ. Sci. Pollut. Res., 24, 3315–3335, 2017. 21. Richardson, A.E., Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Funct. Plant Biol., 28, 897, 2001. 22. Sundara, B., Natarajan, V., Hari, K., Influence of phosphorus solubilizing bacteria on the changes in soil available phosphorus and sugarcane and sugar yields. F. Crop. Res., 77, 43–49, 2002. 23. Khan, M.R., Khan, S.M., Mohiddin, F.A., Askary, T.H., Effect of certain phosphate-solubilizing bacteria on root-knot nematode disease of mungbean, in: First International Meeting on Microbial Phosphate Solubilization, Springer Netherlands, Dordrecht, pp. 341–346, 2007. 24. Al-Rehiayani, S., Hafez, S.L., Thornton, M., Sundararaj, P., Effects of Pratylenchus neglectus, Bacillus megaterium, and oil radish or rapeseed green manure on reproductive potential of Meloidogyne chitwoodi on potato. Nematropica, 1999. 25. Padgham, J.L., Sikora, R.A., Biological control potential and modes of action of Bacillus megaterium against Meloidogyne graminicola on rice. Crop Prot., 26, 971–977, 2007. 26. Barea, J.M., Vesicular-Arbuscular Mycorrhizae as Modifiers of Soil Fertility, in: Advances in Soil Science, Springer, New York, NY, pp. 1–40, 1991. 27. Mosse, B., Stribley, D.P., LeTacon, F., Ecology of Mycorrhizae and Mycorrhizal Fungi, in: Advances in Microbial Ecology, Springer, Boston, MA, pp. 137–210, 1981. 28. Singh, L.P., Gill, S.S., Tuteja, N., Unraveling the role of fungal symbionts in plant abiotic stress tolerance. Plant Signal. Behav., 6, 175–191, 2011. 29. Vasanthakrishna, M., Bagyaraj, D.J., Selection of efficient VA mycorrhizal fungi for inoculating Casuarina equisetifolia. Arid Soil Res. Rehabil., 7, 377– 380, 1993. 30. Olsson, P., Thingstrup, I., Jakobsen, I., Bååth, E., Estimation of the biomass of arbuscular mycorrhizal fungi in a linseed field. Soil Biol. Biochem., 31, 1879–1887, 1999. 31. Kasiamdari, R.S., Smith, S.E., Smith, F.A., Scott, E.S., Influence of the mycorrhizal fungus, Glomus coronatum, and soil phosphorus on infection and disease caused by binucleate Rhizoctonia and Rhizoctonia solani on mung bean (Vigna radiata). Plant Soil, 238, 235–244, 2002.

Fabrication Approaches for Biofertilizers  513 32. Badr, M.A., Shafei, A.M., El-Deen, S.H.S., The Dissolution of K and P-bearing Minerals by Silicate Dissolving Bacteria and Their Effect on Sorghum Growth. Res. J. Agric. Biol. Sci., 2, 5–11, 2006. 33. Bashan, Y., Inoculants of plant growth-promoting bacteria for use in agriculture. Biotechnol. Adv., 16, 729–770, 1998. 34. Ahmad, F., Ahmad, I., Khan, M.S., Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol. Res., 163, 173–181, 2008. 35. Bevivino, A., Characterization of a free-living maize-rhizosphere population of Burkholderia cepacia: effect of seed treatment on disease suppression and growth promotion of maize. FEMS Microbiol. Ecol., 27, 225–237, 1998. 36. Mishra, M., Kumar, U., Mishra, P.K., Prakash, V., Efficiency of Plant Growth Promoting Rhizobacteria for the Enhancement of Cicer arietinum L. Growth and Germination under Salinity. Adv. Biol. Res. (Rennes)., 4, 92–96, 2010. 37. Holzinger, A., Nagendra-Prasad, D., Huys, G., Plant protection potential and ultrastructure of Bacillus subtilis strain 3A25. Crop Prot., 30, 739–744, 2011. 38. Saravanakumar, D., Vijayakumar, C., Kumar, N., Samiyappan, R., PGPRinduced defense responses in the tea plant against blister blight disease. Crop Prot., 26, 556–565, 2007. 39. Parasuraman, P., Pattnaik, S., Busi, S., Plant-Microbe Interactions in Ecosystems Functioning and Sustainability, in: New and Future Developments in Microbial Biotechnology and Bioengineering, 2019. 40. Venkataraman, G.S., Blue-green algae for rice production: a manual for its promotion, 1981. 41. Kaushik, B.D., Subhashini, D., Amelioration of salt-affected soils with bluegreen algae. II. Improvement in soil properties., in: Proc. Ind. Natl. Sci. Acad., pp. 380–389, 1985. 42. Herrmann, L., Lesueur, D., Challenges of formulation and quality of biofertilizers for successful inoculation. Appl. Microbiol. Biotechnol., 2013. 43. Arora, N.K., Khare, E., Maheshwari, D.K., Plant Growth Promoting Rhizobacteria: Constraints in Bioformulation, Commercialization, and Future Strategies, in: pp. 97–116, 2010. 44. Malusá, E., Sas-Paszt, L., Ciesielska, J., Technologies for Beneficial Microorganisms Inocula Used as Biofertilizers. Sci. World J., 2012, 1–12, 2012. 45. Bashan, Y., De-Bashan, L.E., Prabhu, S.R., Hernandez, J.-P., Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013). Plant Soil, 378, 1–33, 2014. 46. Kaljeet, S., Keyeo, F., Amir, H.G., Influence of Carrier Materials and Storage Temperature on Survivability of Rhizobial Inoculant. Asian J. Plant Sci., 10, 331–337, 2011. 47. Smith, R.S., Legume inoculant formulation and application. Can. J. Microbiol., 38, 485–492, 1992.

514  Biofertilizers 48. Smith, R.S., Inoculant Formulations and Applications to Meet Changing Needs, in: Nitrogen Fixation: Fundamentals and Applications, Springer, Dordrecht., pp. 653–657, 1995. 49. Sahu, P.K., Brahmaprakash, G.P., Microbial Inoculants in Sustainable Agricultural Productivity, in: Singh, D.P., Singh, H.B., Prabha, R. (Eds.), Springer India, New Delhi, pp. 179–198, 2016. 50. Somasegaran, P., Halliday, J., Dilution of Liquid Rhizobium Cultures To Increase Production Capacity of Inoculant Plants. Appl. Environ. Microbiol., 44, 330–333, 1982. 51. Burton, J.C., Rhizobium culture and use, in: Microbial Technology, pp. 1–33, 1967. 52. Strijdom, B., Deschodt, C.C., Carriers of rhizobia and the effects of prior treatment on the survival of rhizobia. Symbiotic Nitrogen Fixat. Plants, 7, 151–168, 1976. 53. van Elsas, J.D., Heijnen, C.E., Methods for the introduction of bacteria into soil: A review. Biol. Fertil. Soils, 1990. 54. Patil, N., Gaikwad, P., Shinde, S., Sonawane, H., et al., Liquid formulations of Acetobacter diazotrophicus L1 and Herbaspirillum seropedicae J24 and their field trials on wheat. Int. J. Environ. Sci., 3, 1116–1129, 2012. 55. Mugnier, J., Jung, G., Survival of Bacteria and Fungi in Relation to Water Activity and the Solvent Properties of Water in Biopolymer Gels. Appl. Environ. Microbiol., 50, 108–114, 1985. 56. Singleton, P., Keyser, H., Sande, E., Development and evaluation of liquid inoculants, in: Inoculants and Nitrogen Fixation of Legumes in Vietnam, pp. 52–56, 2002. 57. Smidsrod, O., Skjakbrk, G., Alginate as immobilization matrix for cells. Trends Biotechnol., 8, 71–78, 1990. 58. Bashan, Y., Hernandez, J.-P., Leyva, L., Bacilio, M., Alginate microbeads as inoculant carriers for plant growth-promoting bacteria. Biol. Fertil. Soils, 35, 359–368, 2002. 59. Kitamikado, M., Yamaguchi, K., Tseng, C.-H., Okabe, B., Method Designed To Detect Alginate-Degrading Bacteria. Appl. Environ. Microbiol., 56, 2939– 2940, 1990. 60. Fages, J., An Industrial View of Azospirillum Inoculants - Formulation and Application Technology. Symbiosis, 13, 15–26, 1992. 61. Bashan, Y., Gonzalez, L.E., Long-term survival of the plant-growthpromoting bacteria Azospirillum brasilense and Pseudomonas fluorescens in dry alginate inoculant. Appl. Microbiol. Biotechnol., 1999. 62. Sahu, P.K., Gupta, A., Singh, M., Mehrotra, P., et al., Bioformulation and fluid bed drying: A new approach towards an improved biofertilizer formulation, in: Eco-Friendly Agro-Biological Techniques for Enhancing Crop Productivity, 2018. 63. Sahu, P.K., Lavanya, G., Gupta, A., Brahmaprakash, G.P., Fluid bed dried microbial consortium for enhanced plant growth: A step towards next generation bio formulation. Vegetos, 2016.

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17 Biofertilizers From Waste Rafaela Basso Sartori, Ihana Aguiar Severo, Álisson Santos de Oliveira, Paola Lasta, Leila Queiroz Zepka and Eduardo Jacob-Lopes* Bioprocess Intensification Group, Federal University of Santa Maria (UFSM), Santa Maria, RS, Brazil

Abstract

The intensive utilization of chemical fertilizers over the past few years has led to serious environmental complications besides to negatively influencing human and animal health. It is suggested that these products are responsible for up to 10% of global greenhouse gas emissions, in addition to severely worsening soil quality and crop productivity. Recently, biofertilizers appear as the main substitutes for chemical fertilizers through a growing awareness of the use of sustainable products and which generate less risk than synthetic products. Biofertilizers comprise living microorganisms that, upon application, provide almost all the nutrients necessary for the growth of the cultures, minimizing the environmental impact of land use. Formulations based on photosynthetic microorganisms, such as cyanobacteria and microalgae, are of particular interest due to the valuable biomass production. Furthermore, many studies and researches have concentrated on the development and commercialization of waste-based biofertilizers. The use of organic materials as a basis for biofertilizers production is an environmentally friendly approach in the integrated management and waste use, as they are cheap and renewable sources of nutrients for sustainable use. In this sense, this chapter aims to address an overview of biofertilizers through waste recycling, main sources, appropriate treatment processes, emerging technologies, and applications. Keywords:  Organic waste, chemical fertilizers, waste treatment, bioremediation, nutrient recovery, microalgae/cyanobacteria, sustainable agriculture

*Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (517–540) © 2021 Scrivener Publishing LLC

517

518  Biofertilizers

17.1 Introduction The arrival of the green revolution around 1960, intensive agricultural practices emerged in order to meet the high demand in grain production and in parallel the resistance of crops to diseases and pests. Despite the improvement in the yield of food crops, the high use of chemical fertilizers ended up leading to many environmental problems, such as severe nutrient disequilibrium, groundwater pollution, salinization, and decreased long-term soil fertility [1, 2]. It is also known that these products contribute severely for the greenhouse gas emissions in addition to requiring high energy demand. Therefore, the need to use more sustainable products has become inevitable to overcome these limitations [3]. Currently, biofertilizers are the best alternative to synthetic fertilizers due to their environmentally friendly, cost-effective, ease of application, and non-toxicity [4]. Biofertilizers are microbial products that can improve the availability and transfer of nutrients in the soil, colonize the interior of plants, and promote the growth and productivity of the most diverse crops [5, 6]. They are composed of live microorganisms and a transporter material in which fungi, bacteria, cyanobacteria, and microalgae are the most used [1]. There are several formulations for the production of biofertilizers; however, those based on organic waste have called a lot of attention due to the excellent ability to increase soil fertility. Organic waste, mainly derived from agriculture, municipal, and industrial sources, is composed of a significant amount of biological material and important nutrients which facilitate their use in relation to chemical fertilizers [7]. The use of biofertilizers based on waste recycling is considered today the backbone of sustainable agriculture, because it adopts modern recycling techniques, reduces the environmental impact by diverting the negative carbon footprint and greenhouse gases, and circumvent the high cost of landfills [8]. However, the use of these bioalternatives is still relatively low when compared to the use of synthetic products, conquering only between 1% and 2% of the world market. In summary, there is still a great need to find new approaches and sustainable solutions for the most diverse environmental areas in additional bioeconomy [9]. Researchers all over the world are getting involved in the development of more straightforward to use bioproducts that solve the harmful problems of chemicals and reach more cultures, as well as biofertilizers from waste.

Biofertilizers From Waste  519

17.2 Waste Sources The rapid advance in industrial development has resulted in a significant intensification in the generation of solid, liquid, and gaseous waste. The problems related to waste are mainly linked to the rise in production, to the variety of discarded materials, and the difficulty in finding areas for its deposit or disposal in the correct way [10]. Waste management has become a significant challenge for most countries around the world. According to Vincentin et al. [11], waste generation in developed countries is much higher when compared to developing countries. In fact, the increase in these residues is due to the wide variety of sources related to the most diverse sectors and segments, which have increased in recent years. Figure 17.1 shows the main sources of organic waste generated today that contain a wide range of chemical, physical, and biological pollutants and which, after proper treatment, can become potentials high added-value products, such as biofertilizers. Among the main sources in the generation of organic waste, those from agriculture are current highlights due to the great need to discover new research and development solutions. These residues present a great source of pollution, pathogens, odours, and greenhouse gases since they are produced most often by manure, farm and silage residues, oil processing, veterinary drugs, pesticides, and fertilizers enriched with carbon and nutrients like nitrogen and phosphorus [12]. Among the main crops responsible for most of this agricultural waste is the rice production, wheat, cotton, and Primary production Animal feed industry

Food industry

Animal feed (fodder)

Food

Animal products

Agricultural waste: plant production

Wood/Paper industry

Fermentation industry

Pharmaceutical industry

Slaughterhouses Tanneries

Agricultural waste: animal production

Municipal waste: wastewater

Industrial waste

Waste-processing for application as biofertilizers, or other value-added products

Figure 17.1  Different sources of wastes. Adapted from Insam et al. [8].

520  Biofertilizers corn. However, livestock waste is also another significant source of pollution since it is often discharged directly into estuaries and rivers without any pretreatment [13]. These residues are reported as one of the most important in greenhouse gas emissions, contributing about 20% of total methane emissions in the world [14]. Annually, each municipality produces a significant amount of wastewater, which contains a high number of nutrients and organic materials [15]. It is estimated that cleaning products, such as detergents, generate one of the principal residual loads in municipal treatment plants, contributing about 0.03 tons. In contrast, human waste makes around 0.06 tons/capita/year [16]. Other large residual volumes come from the most diverse industrial sectors, such as the pharmaceutical industry, paper, and wood production, and fermentation products. The characteristics of industrial waste are high chemical oxygen demand (COD), biological oxygen demand (BOD), and a strong smell resulting from the presence of organic fillers and volatile compounds. However, today many countries require industries to use appropriate technologies to reduce the content of these residual contributors [17, 18]. Despite having some common characteristics, each waste has its own average compositions, which can change depending on the time and operating conditions. Organic matter, for example, is the primary contaminant found in any waste. COD includes total degradable compounds, while BOD includes biodegradable parts. In addition, nitrogen is present mainly in the form of ammonia, while phosphorus is generally found in the form of phosphate. Table 17.1 presents the average composition of the waste sources mentioned. Table 17.1  Average composition of some waste sources. Waste

COD (g/L)

BOD (g/L)

N (g/L)

P (g/L)

TSS (g/L)

Turbidity (NTU)

Manure

16

12

9

0.5

3

9,000

Digestate

9

7

8

0.4

10

30,000

Breweries

4

3.8

0.03

0.01

1

3,000

Dairy

0.95

0.21

0.12

0.028

0.32

nr

Sewage

0.7

0.5

0.065

0.011

0.3

900

Centrate

0.3

0.2

0.5

0.012

1

3,000

COD, chemical oxygen demand; BOD, biological oxygen demand; N, nitrogen; P,  phosphorus; NTU, nephelometric turbidity unity; nr, not reported. Adapted from Fernández et al. [19].

Biofertilizers From Waste  521

17.3 Technologies for Waste Treatment 17.3.1 Conventional Technologies The biofertilizers production coupled with waste treatment has been widely publicized; however, for each type of composition, the treatment process must be properly designed and operated. In this sense, Figure 17.2 presents a model of the conventional waste treatment process carried out by some industries. Based on the scheme in Figure 17.2, the first stages are summarized in an initial treatment in which the removal of suspended sediments and floating solids occurs [20]. After this phase, the processes become more intensive and are based on the conversion of organic materials by microbial decomposition. Here, many types of configurations are presented. However, the treatment process by activated sludge is highlighted for having several advantages, mainly due to its low operating cost [21]. Tertiary treatment, in turn, is an additional treatment that can reduce the amount of nitrogen and phosphorus in the waste [22]. Nitrogen removal occurs mainly through the nitrification-denitrification process. In contrast, for phosphorus removal, in addition to biological processes, the

A

B

C

D

E

Waste

F

G

H Clean waste disposal

Input Output

Figure 17.2  Scheme of a conventional waste treatment process. (A) Pre-treatment waste tank; (B) primary sedimentation (C) biological treatment; (D) secondary sedimentation; (E) tertiary treatment; (F) concentration; (G) anaerobic digestion; (H) dewatering/ disposal. Adapted from Fernández et al. [19].

522  Biofertilizers use of chemical processes and fertigation has also been used recently to reduce the compounds [21]. Finally, the analysis of current treatment processes highlights the high cost of chemicals, energy, and the elevated demand for manpower, techniques, and practices related to the current increase in treatment plants. Besides, the excessive production of pollutants in the atmospheres must be assessed, as some transformation processes can further burden the system. Based on this, a new concept for developing and optimizing of emerging technologies must be evaluated to solve current problems.

17.3.2 Emerging Technologies Based on the premise that the excessive use of chemical fertilizers hurts the soil, unbalances and reduces elements such as N, P, and K, pollutes groundwater, and increases salinity, some questions arise: How to reduce the requirements for chemical fertilizers? What technologies can contribute to the sustainable development of actual agriculture? These queries have been of great interest to many scientific researchers. Today, there are several types of biofertilizers, but productions reasoned on photosynthetic microorganisms are of particular interest due to the valuable biomass production. Cyanobacteria and microalgae have been used as emerging technologies over conventional ones for application in modern agriculture. Other microorganisms, including fungi and bacteria, can also be utilized to produce biofertilizers. Alternatively, these distinct classes of microorganisms have broadened their scope of application. They can act together, that is, in the development of a consortium between microalgae/bacteria or cyanobacteria/bacteria or their respective biofilms [23]. Thus, both microalgae and cyanobacteria as bacteria can assimilate considerable amounts of nutrients from waste (COD, N, and P) phototrophically, heterotrophically, or mixotrophically and convert them into many products of commercial interest [24]. Microalgae, for example, constitute a broad group of organisms, preferably photosynthetic, which use the light energy to bioconvert nutrients into organic matter, producing biomass. Its distinct structural, physiological, and morphological characteristics expand the possibilities of use in different processes and to obtain products such as food, pharmaceuticals, biofuels, bioenergy, animal feed, and, especially, agricultural as a natural fertilizer [25]. These microorganisms have (i) simple cultivation requirements, (ii) high cell growth rates and productivities, (iii) develop quickly and efficiently, (iv) inhabit environments of

Biofertilizers From Waste  523 extreme conditions, and (v) do not require arable land or freshwater, growing under the supply of different types of wastes (as explained above—see Section 17.2) [26].

17.3.2.1 Nutrients Recovery From Wastes by Microalgae In terms of wastes reuse, microalgae and cyanobacteria can recover and recycle the nutrients contained in those to achieve higher biomass productivity and then target your final application [27]. As an example, it is possible to produce about 1 and 10 kg per m³ of dry biomass-based waste sewage and manure, respectively. This means that microalgae have been successfully used as a nutrient cycling technology from wastes [28]. However, when considering the use of microalgae for the waste bioremediation, what should be analyzed is that this type of technological route is purely based on a consortium of the attached microalgal/cyanobacterial versus bacteria. The development of microbial consortia consists of a mixed population of species of microorganisms that, in synergism, are potentially applied in waste biodegradation. They extend the diversity of enzymatic activities in the medium, related to xenobiotic substances, thereby obtaining a broad spectrum and higher rates of degradation. Some studies have shown that the consortium can achieve high removal rates (>80%) of COD, NH3, and P in laboratory-scale systems [29]. Due to the non-sterile conditions, the association that will predominate in the cultivation systems it’s going to be one that occurs typically depending on the waste chemical composition, environmental parameters, bioreactor design, and operational conditions [30]. Figure 17.3 shows that, regardless of the microbial consortium composition, a pattern model is followed for the process. According to Figure 17.3, microalgae use light energy during photosynthesis to assimilate inorganic nutrients N and P (typical concentrations of these compounds are less than 0.5 and 0.03 mg/L for total N and P, respectively). They produce biomass and release O2, which is then required by bacteria to oxidize organic compounds into inorganic. This occurs via assimilation, anaerobic ammonia oxidation, nitrification and denitrification of nitrogen, and phosphorus uptake. In this summary scheme, the bacteria release the CO2 which is finally consumed via microalgae. The natural balance in the consortium between the attached microalgae-bacteria defines the operational conditions in the bioreactor [29, 31]. Although the consortium seems simple, the biochemical reactions involved in microalgal and bacterial metabolisms are quite complex, as shown in Table 17.2.

524  Biofertilizers Light energy Biomass

Organic and inorganic matter

O2

Microalgae

Waste Consortium

N P

NO3PO43Bacteria

CO2

Cleanwaste

Figure 17.3  Simplified schematic representation of the main biological phenomena during the attached microalgae-bacteria consortium.

Table 17.2  Metabolisms involved in waste treatment processes from the microalgae-bacteria consortium. Microorganism Microalgae

Bacteria

Reaction

Metabolism + 4

−3 4

CO2 + H2O + NH + PO → biomass + O2

Phototrophic

NO3− + 4H2O → NH+4 + 7OH−

Nitrate reduction

COD + O2 + nutrients → biomass + CO2

Heterotrophic

NH+4 + 2O2 → NO3− + 2H+ + H2O

Nitrification

8NO3− + 5CH3COOH → 8HCO3− + 6H2O + 2CO2 + 4 N2

Denitrification

Adapted from Acién et al. [28].

The main reactions involved in microalgae-based metabolism include biomass production via photosynthesis and the release of hydroxyl ions by the assimilation of nitrate and reduction to ammonia. On the other hand, in heterotrophic metabolism, microalgae assimilate smaller organic molecules, such as short-chain carbohydrates. Bacteria have aerobic metabolism for growth, nitrification, denitrification, and biomass production decay. Also, other reactions (e.g., N2 ammonification) can occur [28].

Biofertilizers From Waste  525 Regardless of metabolic reactions, it must be taken into consideration that the performance of nutrient recovery processes from wastes by microalgae and cyanobacteria can be directly related to several factors including light intensity, pH value, temperature, and inoculum and nutrients concentration (C/N and N/P ratios). These, in turn, will differ according to the algal strains used, type of bioreactor (open or closed systems), and operational mode (batch, fed-batch or continuous) [32]. The key factor of these processes is based on light availability as a function of location, photoperiod, and solar radiation, which also depends on the cultivation system depth. Many studies prove the importance of this parameter in the global performance of microalgae-based processes and also for recovering nutrients from waste [33–36]. For example, the smaller the bioreactor depth, the higher the light irradiation in the system, which will favor the nutrients assimilation and culture growth. It should be noted that this implies a reduced volume of bioreactor and, consequently, low waste loads can be treated. The critical point is to establish the system depth for cultivating microorganisms accurately. However, this should balance two variables: the quality biomass production, where it is suggested that the culture depth is less than 0.2 m, and the treatment of high waste loads, with a depth greater than 0.3 m. On the other hand, if the cultures are exposed to great depths, the light incidence may be sufficient enough for the O2 production and organic matter oxidation by bacteria, or insufficient for the microalgae to assimilate the produced CO2 and, therefore, the nitrification/denitrification phenomena would occur in excess [28]. In parallel to light, cell growth and biomass production are also affected by photosynthetic efficiency. This factor depends on the temperature and the appropriate pH of the medium. Some studies prove that the pH changes during the lighting period and when CO2 is not supplied, reaching values close to 9.0. This is due to the CO2 and NO3 assimilation by microalgae. However, pH value decreases later because the bacteria release CO2 and H+ ions. Thus, it is crucial to know the parameters that affect these systems [37]. Finally, in addition to the importance of knowing the parameters that affect nutrient recovery systems from wastes by microalgae and cyanobacteria, the composition of the waste can also affect their assimilation. The removal capacity of the nutrients contained in the wastes can be increased via optimization and modeling of the microorganisms’ productivity. Besides, the technologies used during the processing stages of these bioprocesses must be understood.

526  Biofertilizers

17.3.2.2 Overall Process Operations The cycling nutrients process from waste by microalgae consists of many unit operations. The main processing steps include waste pre-treatment, nutrients recovery, biomass production in the bioreactor, harvesting, waste treatment for recirculation or final disposal, and obtaining microalgal biomass for conversion into the desired target product. These steps are briefly outlined in Figure 17.4 through a process flow diagram. The first two stages, which consist of waste pre-treatment and nutrients recovery, are resembling those applied in conventional waste treatment facilities. In this sense, it is necessary to perform the filtering of total solids aiming to reduce the turbidity of the residue to improve its quality. Besides, if during the biomass harvest, the solids withdrawal is sufficient, the waste treatment is discarded. Likewise, considering that in microalgae-based processes, pathogenic microorganisms are found in low concentrations, extra disinfection steps (e.g., ultraviolet radiation and O3) are eliminated before reusing or dumping the clean waste back into the environment. The cultivation step in the bioreactor and harvesting are considered as the most crucial in microalgae-based processes for obtaining biomass.

A

B

C

Waste

D

Biomass

E

Input Output

Clean waste disposal

Figure 17.4  Process flow diagram of the main steps in waste recovery for microalgae biomass production. (A) Pre-treatment waste tank; (B) nutrient recovery tank; (C) bioreactor; (D) centrifugal pump; (E) recirculation/disposal tank.

Biofertilizers From Waste  527 There are numerous researches in the literature demonstrating the various reactor designs and operational conditions for the microalgae cultivation, such as open and closed systems. However, for wastes recovery, open systems are the most frequently studied. Open bioreactors have been around since the 1960s. Currently, different models are investigated, such as circular ponds, shallow lagoons, and ponds, mixed ponds, and inclined systems, but raceway ponds are traditionally used for commercial purposes [26]. They are characterized by large culture reservoirs (~30m³), with low depth (around 0.2 to 0.5 m) and operated in average cell residence times of 8 days; they are moved with the aid of a paddlewheel to maintain the mixture flow of the microalgal broth and regularly expose the cells to light energy. Raceway ponds are cheaper and simpler to build, as well as easy to scale [38, 39]. Today, systems up to 5,000 m² are used at large scale. In fact, there are few shortcomings associated with its design; however, as the general objective of any cultivation system is to achieve higher productivity, this aspect is still a critical point in this type of system. Although the raceway ponds performance for waste treatment has been proven, several further improvements have been proposed. The central claims are in terms of mass transfer coefficient and fluid dynamics [40, 41]. After cultivation ceases, harvesting is performed to recover the cells diluted in the broth. Generally, cell concentration is low, around 0.5 g/L in raceway ponds, and cell size is tiny (2–20 μm). Therefore, current harvesting technologies are challenging in terms of recovering as much biomass as possible. Its concentration can be done through centrifugation, filtration, floatation, sedimentation, and electrical process [42, 43]. Centrifuges are more widely used for microalgal broth dewatering, with a recovery rate of up to 95%. Nevertheless, they are incredibly energy-intensive precisely because of the water volume to be removed. Existing harvesting processes have been reported to account for a third of the total biomass production cost [44]. Given this scenario, for the nutrients recovery from wastes, technologies with reduced energy demand (below 0.1 kWh/m3) must be inserted in the microalgae biomass processing [28]. Finally, regardless of the upstream and downstream processing steps, the application of microalgae-based processes to treat different residues coupled with the biomass production for as inputs of market interest is a rapidly growing field. Worldwide, many companies are working on manufacturing biomass as an agricultural commodity. Biofertilizers are promising candidates for this purpose. Its applicability in modern agriculture will be discussed below.

528  Biofertilizers

17.4 Main Applications of Microalgae Biofertilizers The main requirement for agrarian progress is to apply a resistant source of nutrients to agriculture, without harming the environment and without damaging the country’s economy. With a focus on the production of sustainable agriculture, biofertilizers are gaining prominence, as they can benefit the fertility of crops, in an economically and environmentally viable way, and also decrease the aggravating from the actual fertilizers [45–47]. Microalgae biorefinery is a current commercial need and has vast potential in bio-resource for different industries, treating its waste and producing bioproducts making these residues promising to be applied as biofertilizers in a symbiotic system to improve the soil structure and contribute to the replacement of chemical fertilizers [48, 49]. These microorganisms are already applied in agriculture, and they have a high capacity to scatter nutrients from the soil, improving the macro and micronutrients and quality soil. Besides, they can produce hormones, carbohydrates, antimicrobials, and control the pests and diseases, as can be seen in Figure 17.5 [49, 50].

17.4.1 Fertility and Soil Quality 17.4.1.1 Nitrogen Fixation In the nitrogen fixation process, fixing microorganisms convert nitrogen into ammonia. As the amount of ammonium salt is low in the soil, this fixation becomes extremely important for maintaining the nitrogen cycle

Fertility and soil quality Nitrogen fixation Carbon sequestration

Biofertilizer application

Soil organic matter Soil improvement Recovery soil Plant growth, disease and pest control Disease and pest control Plant colonization Hormone production

Figure 17.5  Potential applications of microalgae biofertilizers.

Biofertilizers From Waste  529 in the ecosystem. Fixation occurs only for certain types of microorganisms, the best-known being bacteria and microalgae, which have so-called heterocysts, a specific nitrogenase system for these species, so they end up not competing with plants for the demand for N and end up helping availability of the soil [51–53]. Studies indicate that the applicability of microalgae can reduce 25% to 40% of chemical nitrogen fertilizers. According to Pereira et al. [54], the application of microalgae how biological nitrogen fixers in rice crops can decrease the utilization of chemical fertilizers by up to 50% without affecting grain quality and production yield. Osman et al. [55] also concluded by applying cyanobacteria as biofertilizers in pea plantations, that the use of cyanobacteria reduces the use of fertilizers by up to 50% and still improves the nutritional value of peas. The application of microalgae biofertilizers, in addition to having an economic impact by reducing the values of chemical fertilizers, also increased crop productivity not only in rice but also in several other crops [56]. Jha and Prasad [57] concluded by inoculating microalgal strains in rice plantations that the increase in straw and grains was significant, and reduced the use of chemical fertilizers by 25%. However, environmental care is necessary due to the possibility of a process called leaching, a process in which nutrients run out of channels in the fields. Although, according to Mager and Thomas [58], they reported that microalgae are exopolysaccharide products responsible for the formation of biological crusts in the soil, helping to immobilize access to N is much smaller than to the leaching caused by commonly used synthetic fertilizers. Therefore, the utilization of microalgae can generate low output costs, to reducing pollution, avoiding leaching [49].

17.4.1.2 Carbon Sequestration The use of chemical fertilizers increases the carbon dioxide and nitrogen emissions, which are also emitted from the soil when they are applied. The employment of microalgae becomes essential since they are organic matter sources and are directly linked to the assimilation of atmospheric carbon dioxide through photosynthesis, thus being able to significantly increase the organic carbon content of the soil [53, 59]. Improved carbon fixture in the ground can subserve the reparation of damaged environmental; for this reason, further studies of these fixation mechanisms are needed. According to Yan-Gui et al. [60], the establishment in biological scrabs in the ground showed to be promising, a compound of microalgae, microorganisms, and fungus in different proportions,

530  Biofertilizers restoring ground nutrients and balancing the ecosystem, serving as an important carbon reservoir in the soil due to high photosynthetic capacity. In a proposed study, Yilmaz and Sönmez [61] evaluated the potential of several types of biofertilizers in the organic carbon of the soil, and the outcome demonstrated that concerning the control, the changes of the ground with applying microalgae biofertilizers increased the organic carbon of the soil. Demonstrating then that microalgal biofertilizers are an advance for improvement in the absorption of nutrients, contributing positively to the environment.

17.4.1.3 Soil Organic Matter, Improvement, and Recovery The conservation of an appropriate standard of organic materials and the structure and the appropriate composition of the soil is fundamental for sustainable farming. Furthermore, to the contribution in fixing the nitrogenous carbon, microalgae contribute to the progress of the grounds organic material, and they have the function of organic matter production and solubilization of nutrients in the ground, essential for the growing and evolution of plants [62]. These microorganisms are responsible for the high volume of organic carbon, increasing the expansion of flora and fauna. The organic content of the soil rises through the deterioration of microalgae biomass. Studies on the application of microalgae in the soil and in different cultures exhibited an increase in the biochemical properties and general organic material [52, 62]. Algae, more specifically cyanobacteria, have a mineralization function, so much for the output of organic acids but also by siderophores, these compounds made by microorganisms that act in the chelation of ferric iron when there is an iron deficit making them accessible to plants [64]. Studies reported on the improvement of nutrients in plants and seeds using microalgae in the biofortification of basic and food crops also report that the application of microalgae leads to changes in the frame and expands microbic species implied in processes of the solubilization (of macro and micronutrients) and mineralization [65–69]. Studies reported on the enrichment of micronutrients, particularly Fe, Mn, Cu, and Zn in plants and grains using microalgae in the biofortification of basic and food crops, also report that the application of microalgae leads to changes in the structure and expands microbial species involved in the processes of mineralization and solubilization of nutrients [65–69]. The soil can be damaged by various weather conditions, which end up affecting fertility and productivity in agriculture. Microalgae produce

Biofertilizers From Waste  531 so-called exopolysaccharides (EPS), and this is a mechanism that improves soil microbial activities, providing organic carbon and forming bioflocs and biofilms, which are important for the rhizosphere [70, 71]. According to Trejo et al. [72], microalgae can act in soil recovery since they were applied in the treatment of three-stage sewage, and after treating increased the organic matter significantly, mainly carbon. Abed [73] also reported that microalgae are related to the correction of areas contaminated by oil. Tripathi et al. [74] used microalgae as a biofertilizer in mixed soil with fly ash, and these improved the stress capacity of rice crops to fly ash and prevented the accumulation of heavy metals in plants, acting positively on their growth. Thus, microalgae can operate as inoculants, biofertilizers applied, and in the recuperation of contaminated grounds.

17.4.2 Promotion of Plant Growth, Disease, and Pest Control 17.4.2.1 Plant Colonization and Hormone Production As already mentioned in the previous topics, microalgae have several benefits for the soil and can also be applied as plant colonizers. Several species have symbiotics with different plants, and it has been reported that microalgae are colonizers of epidermal cells, intercellular spaces, cortex, sub-stomatal chambers, and form intracellular loops [75, 76]. According to Bidyrani et al. [77], cyanobacteria can colonize various parts of plants and can also act in defense, nitrogen fixation, and nutritional status and development. By applying microalgae inoculants, a symbiotic yield of chickpeas was also obtained, presenting benefits in the soil content, increasing soil fertility and improving the harvest [78, 79]. Seeking better progress of plants, phytohormones are of great importance; in agriculture, there is have an increment in the supplementation of plant hormones to assist in the growth and benefitting control of weeds [80]. Microalgae have intracellular hormones, and this is also capable of producing and/or excreting these in the medium set for growth or in the environment [81]. According to Hussein and Hassner [82], the hormones that are secreted by these microorganisms for the development of plants in sterile fields show the levels of cytokine and help to have positive action with the growth of plants, and this interaction occurs, through microalgae interaction/roots. It was also reported in a study that the levels of auxin in wheat, using microalgae as a phytohormone supplement, increased the symbiotic system, demonstrating that there is a positive transfer between microalgae/ wheat [83]. There are not many pilots-scales studies that use algae as

532  Biofertilizers hormones, but it becomes necessary more to study it until it becomes an environmentally positive option to apply.

17.4.2.2 Disease and Pest Control Microalgae can be appointed as articulators of defense mechanisms in plants, mainly antioxidant activities. In rice, microalgae significantly increased the action of plant protective enzymes, both in the source and in the aerial part of plants [84]. Babu et al. [85] said that the use of microalgae contributed in a beneficial way to the immunity of plants, analyzing enzymatic activities. Investigating the real potential of microalgae, for use as a biofertilizer, shows a variety of applications and the differential of each species. The application of these demonstrate significant growth in the defense response of plants, such as increased RNA, and also in the enzymatic activities of nutrient assimilating enzymes [86]. Another function of microalgae is which can be applied to reduce pests present in plants (e.g., nematodes, who are toxin producers). According to Khan et al. [87], an application of cyanobacteria in the soil reduced by up 97.5% the number of nematodes in tomato seedlings, thus managing to enlarge production. The use of these microorganisms is more effective before changes in the anti-nematocidal action, promoting the growth of plants as well [88]. By using biofertilizer microalgae, there was a reduction in the number of mosquitoes in the field, contributing to better development when they are compared with the utilization of chemical fertilizers [89]. Metal extracts of microalgae were also described as antifungal, insecticidal, nematicidal, and herbicidal [90]. Further study is still needed to ascertain strategies in applications of these microorganisms as biofertilizers and or their combined use, so that we can successfully apply them in conventional agro-industrial practices, and their action has a successful integration with routine agricultural practices.

17.5 Conclusions and Recommendations Various governments about the world are supporting the inclusion of sustainable projects and developments to avoid the harmful problems caused by fertilizers and chemical products. Many countries are introducing policies and programs in order to promote organic agriculture and finally

Biofertilizers From Waste  533 overcome environmental pollution. Recently, market research and strategy reports informed that the biofertilizer segment will grow in the coming years, and the market participation of these products could reach USD 3 billion. Recent developments on the use of green microalgae and cyanobacteria as a biofertilizer have shown to be promising potential. Microalgae biofertilizers offer several benefits in terms of cost-effectiveness, pathogen biocontrol, and non-toxicity, besides, and they have a proven capacity in the recovery of soil nutrients. However, the success in the production of microalgae biofertilizers is totally dependent on the cost and expenses caused by biomass production. In this sense, the use of organic residuals for the generation of biofertilizers is a valuable strategy, as it has economic and environmental advantages. Nutrient recovery based on microalgae is a safe technology that can be used for different waste. The junction of the production of these microorganisms to the waste treatment has a great impact on reducing the treatment cost and in increasing the processes sustainability by reducing the need for energy and greenhouse gas emissions. The main defiance today is to present these processes that are economically viable in an industrial-­ scale implementation. Finally, the challenges associated with the microalgae biofertilizers generated by residues still need complete studies and evaluation on a demonstration-scale before they are commercialized. Research on the increase of high-value properties and their applications on agriculture are emerging areas that need attention. Development of genetically modified microorganisms and the utilization of new molecular techniques are providing new knowledge about the paths involved in the interactions with soil and plants and are useful to authenticate the application of biofertilizers and increase their commercialization. Also, there is still a profound need for awareness of environmentally friendly products and further research to potentialize your investment in the future.

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534  Biofertilizers 3. Saritha, M., Tollamadugu, N.V.K.V.P., The Status of Research and Application of Biofertilizers and Biopesticides: Global Scenario, in: Recent Developments in Applied Microbiology and Biochemistry, V. Buddolla (Ed.) pp. 195–207, Academic Press, 2019. 4. Nayak, M., Swain, D.K., Sen, R., Strategic valorization of de-oiled microalgal biomass waste as biofertilizer for sustainable and improved agriculture of rice (Oryza sativa L.) crop. Sci. Total Environ., 682, 475–484, Elsevier, 2019. 5. Mazid, M., Khan, T.A., Mohammad, F., Potential of NO and H2O2 as signalling molecules in tolerance to abiotic stress in plants. J. Ind. Res. Technol., 1, 56–68, Hatam Publishers, 2011. 6. Arriola, K.G., Queiroz, O.C., Romero, J.J., Casper, D., Muniz, E., Hamie, J., Adesogan, A.T., Effect of microbial inoculants on the quality and aerobic stability of bermudagrass round-bale haylage. J. Dairy Sci., 98, 478–485, Elsevier, 2015. 7. Dahiya, S.A., Kumar, N.J., Sravan, S., Chatterjee, S., Sarkar, O., Mohan, S.V., Food waste biorefinery: Sustainable strategy for circular bioeconomy. Bioresour. Technol., 248, 2–12, Elsevier, 2018. 8. Insam, H., Gómez-Brandón, M., Ascher-Jenull, J., Recycling of Organic Wastes to Soil and Its Effect on Soil Organic Carbon Status, in: The Future of Soil Carbon, C. Garcia, P. Nannipieri, T. Hernandez (Eds.), pp. 195–214, Elsevier, Academic Press, 2018. 9. Höfer, R., History of the Sustainability Concept-Renaissance of Renewable Resources, in: Sustainable Solutions for Modern Economies, R. Höfer (Ed.), pp. 1–11, RSC Publ., Cambridge, 2009. 10. Querino, L.A.L., Pereira, J.P.G., Geração de resíduos sólidos: a percepção da população de São Sebastião de lagoa de roça, Paraíba, Revista Monografias Ambientais – REMOA/UFSM, 15, 404–415, Santa Maria, 2016. 11. Vincentin, R., Fdz-Polanco, F., Fdz-Polanco, M., Energy Integration in Wastewater Treatment Plants by Anaerobic Digestion of Urban Waste: A Process Design and Simulation Study. Int. J. Chem. Eng., 1–11, 2019. 12. Ramírez-García, R., Gohil, N., Singh, V., Recent Advances, Challenges, and Opportunities in Bioremediation of Hazardous Materials, in: Phytomanagement of Polluted Sites, V.C. Pandey, K. Bauddh (Eds.), pp. 517– 568, Elsevier Inc., 2019. 13. Sany, S.B.T., Tajfard, M et al, The West Coast of Peninsular Malaysia, in: World Seas: an Environmental Evaluation (Second Edition), C. Sheppard (Ed.), pp. 437–, Academic Press, 2019. 14. Sorathiya, L.M., Fulsounder, A.B., Tyagi, K.K., Patel, M.D., Singh, R.R., Ecofriendly and modern methods of livestock waste recycling for enhancing farm profitability. Int. J. Recycl. Org. Waste Agric., Springer, 2014. 15. Guo, C.H., Stabnikov, V., Ivanov, V., The removal of nitrogen and phosphorus from reject water of municipal wastewater treatment plant using ferric and nitrate bioreductions. Bioresour. Technol., 101, 3992–3999, Elsevier, 2010.

Biofertilizers From Waste  535 16. Karunanithi, R., Szogi, A et al, Phosphorus recovery from wastes, in: Environmental Materials and Waste. Resource Recovery and Pollution Prevention, M.N.V Prasad, K. Shih (Eds.) pp. 687–705, Elsevier Inc., 2016. 17. Parsons, S.A., Smith, J.A., Phosphorus Removal and Recovery from Municipal Wastewaters. Elements 4, 109–112, 2008. 18. Molinuevo, B., García, M.C., Karakashev, D., Angelidaki, I., Anammox for ammonia removal from pig manure effluents: effect of organic matter content on process performance. Bioresour. Technol., 100, 2171–2175, Elsevier, 2009. 19. Fernández*, F.G.A., Gómez-Serrano, C., Fernández-Sevilla, J.M., Recovery of Nutrients From Wastewaters Using Microalgae. Front. Sustain. Food Syst., 2018.  20. Nemerow, N.L., Industrial Waste Treatment, Contemporary Practice and Vision for the Future, pp. 53–77, Elsevier, 2007. 21. Santos, A.M., Santos, A.M et al, Nutrient cycling in wastewater treatment plants by microalgae-based processes, in: Industrial waste management, assessment and environmental issues, S.N. Barton (Ed.), pp. 41–64, Nova Science Pub. Inc., 2016. 22. Cabanelas, I.T.D., Arbib, Z., Chinalia, F.A., Souza, C.O., Perales, J.A., Almeida, P.F., Druzian, J.I., Nascimento, I.A., From waste to energy: Microalgae production in wastewater and glycerol.  Appl. Energy.,  109, 283–290, Elsevier, 2013. 23. Rana, K.L., Kour, D et al, Agriculturally important microbial biofilms: Biodiversity, ecological significances, and biotechnological applications, in: New and Future Developments in Microbial Biotechnology and Bioengineering Microbial Biofilms: Current Research and Future Trends, M.K. Yadav, B.P. Singh (Eds.), pp. 221–265, Elsevier, 2020. 24. Andrade, D.S., Telles, T.S., Castro, G.H.L., The Brazilian microalgae production chain and alternatives for its consolidation. J. Clean. Prod., 250, 119526, Elsevier, 2020. 25. Koutra, E., Economou, C.N., Tsafrakidou, P., Kornaros, M., Bio-Based Products from Microalgae Cultivated in Digestates. Trends Biotechnol., 36, 819–833, Cel Press, 2018. 26. Borowitzka, M.A., Biology of Microalgae, in: Microalgae in Health and Disease Prevention, I. Levine, J. Fleurence (Eds.), Academic Press, Elsevier, pp. 23–72, 2018. 27. Tekerlekopoulou, A.G., Economou, Ch. N et al, Wastewater treatment and water reuse in the food industry, in: The interaction of Food Industry and Environment, C. Galanakis (Ed.), pp. 245–280, Academic Press, 2020. 28. Acién, F.G., Gómez-Serrano, C., Morales-Amaral, M.M., Fernández-Sevilla, J.M., Molina-Grima, E., Wastewater treatment using microalgae: how realistic a contribution might it be to significant urban wastewater treatment? Appl. Microbiol. Biotechnol., 100, 9013–9022, Springer, 2016.

536  Biofertilizers 29. Liu, J., Wu, Y., Wu, C., Muylaert, K., Vyverman, W., Yu, H.-Q., Muñoz, R., Rittmann, B., Advanced nutrient removal from surface water by a consortium of attached microalgae and bacteria: A review. Bioresour. Technol., 241, 1127–1137, 2017. 30. Nagarajan, D., Lee, D.J., Chen, C.Y., Chang, J.S., Resource recovery from wastewaters using microalgae-based approaches: A circular bioeconomy perspective. Bioresour. Technol., 302, 122817, Elsevier, 2020. 31. Muñoz, R., Guieysse, B., Algal-bacterial processes for the treatment of hazardous contaminants: A review. Water Res., 40, 2799–2815, Elsevier, 2006. 32. Koutra, E., Economou, C.N., Tsafrakidou, P., Kornaros, M., Bio-Based Products from Microalgae Cultivated in Digestates. Trends Biotechnol., 36, 819–833, CelPress, 2018. 33. Fernández, I., Acién F.G., Guzmán, J.L., Berenguel, M., Mendoza, J.L., Dynamic model of an industrial raceway reactor for microalgae production. Algal Res., 17, 67–78, Elsevier, 2016. 34. Jebali, A., Acién, F.G., Sayadi, S., Molina-Grima, E., Utilization of centrate from urban wastewater plants for the production of Scenedesmus sp. in a raceway-simulating reactor. J. Environ. Manage., 211, 112–124, Elsevier, 2018a. 35. Jebali, A., Acién, F.G., Rodriguez Barradas, E., Olguín, E.J., Sayadi S., Molina Grima, E., Pilot-scale outdoor production of Scenedesmus sp. in raceways using flue gases and centrate from anaerobic digestion as the sole culture medium. Bioresour. Technol., 262, 1–8, Elsevier, 2018b. 36. Barceló-Villalobos, M., Fernández-del Olmo, P., Guzmán, J.L., FernándezSevilla, J.M., Acién Fernández, F.G, Evaluation of photosynthetic light integration by microalgae in a pilot-scale raceway reactor. Bioresour. Technol., 280, 404–411, Elsevier, 2019. 37. Posadas, E., Morales, M.M., Gomez, C., Acién, F.G., Muñoz, R., Influence of pH and CO2 source on the performance of microalgae-based secondary domestic wastewater treatment in outdoors pilot raceways. Chem. Eng. J., 265, 239–248, 2015. 38. Pawar, S., Effectiveness mapping of open raceway pond and tubular photobioreactors for sustainable production of microalgae biofuel. Renew. Sust. Energ. Rev., 62, 640–653, 2016. 39. Li, K., Liu, Q., Fang, F., Luo, R., Lu, Q., Zhou, W., Hou, S., Cheng, P., Liu, J., Addy, M., Chen, P., Chen, D., Ruan, R., Microalgae-based wastewater treatment for nutrients recovery: A review. Bioresour. Technol., 291, 121934, Elsevier, 2019. 40. Mendoza, J.L., Granados, M.R., de Godos, I., Acién, F.G., Molina, E., Banks, C., Heaven, S., Fluid-dynamic characterization of real-scale raceway reactors for microalgae production. Biomass Bioenerg., 54, 267–275, Elsevier, 2013. 41. Kumar, K., Mishra, S.K., Shrivastav, A., Park, M.S., Yang, J., Recent trends in the mass cultivation of algae in raceway ponds. Renew. Sustain. Energy Rev., 51, 875–885, Elsevier, 2015.

Biofertilizers From Waste  537 42. Kadir, W.N.A., Lam, M.K., Uemura, Y., Lim, J.W., Lee, K.T., Harvesting and pre-treatment of microalgae cultivated in wastewater for biodiesel production: A review. Energ. Convers. Manage., 171, 1416–1429, Scopus, 2018. 43. Tang, D.Y.Y., Khoo, K.S., Chew, K.W., Tao, Y., Ho, S.H., Show, P.L., Potential utilization of bioproducts from microalgae for the quality enhancement of natural products. Bioresour. Technol., Elsevier, 2020. 44. Suparmaniam, U., Lam, M.K., Uemura, Y., Lim, J.W., Lee, K.T., Shuit, S.H., Insights into the microalgae cultivation technology and harvesting process for biofuel production: A review. Renew. Sust. Energ. Rev., 115, 109361, Elsevier, 2019. 45. Singh, D.P., Prabha, R., Yandigeri, M.S., Arora, D.K., Cyanobacteria-mediated phenylpropanoids and phytohormones in rice (Oryza sativa) enhance plant growth and stress tolerance. Anton. Leeuw., 100 (4), 557–568, 2011a. 46. Singh, J.S., Pandey, V.C., Singh, D.P., Efficient soil microorganisms: A new dimension for sustainable agriculture and environmental development. Agric. Ecosyst. Environ. 140 (3-4), 339–353, Elsevier, 2011b. 47. Khan, S.A., Sharma, G.K., Malla, F.A., Kumar, A., Rashmi, Gupta, N., Microalgae based biofertilizers: A biorefinery approach to phycoremediate wastewater and harvest biodiesel and manure. J. Clean. Prod., 211, 1412– 1419, Elsevier, 2019. 48. Thilagar, G., Bagyaraj, D.J., Rao, M.S., Selected microbial consortia developed for chilly reduces application of chemical fertilizers by 50% under field conditions. Sci. Hortic. 198, 27–35, Elsevier, 2016. 49. Renuka, N., Guldhe, A., Prasanna, R., Singh, P., Bux, F., Microalgae as multi-functional options in modern agriculture: current trends, prospects and challenges. Biotechnol. Adv., 36, 1255–1273, Elsevier, 2018. 50. Guo, S., Wang, P et al, Microalgae as Biofertilizer in Modern Agriculture, in: Microalgae Biotechnology for Food, Health and High Value Products, Md.A. Alam, J.-L. Xu, Z. Wang (Eds.), pp. 397–411, Springer Nature Singapore, 2020. 51. Babu, S., Prasanna, R., Bidyarani, N., Singh, R., Analysing the colonisation of inoculated cyanobacteria in wheat plants using biochemical and molecular tools. J. Appl. Phycol., 27 (1), 327–338, Springer, 2015. 52. Renuka, N.,  Prasanna,  R., Sood, A.,  Ahluwalia, A.S.,  Bansal,  R., Babu,  S., Singh, R., Shivay, Y.S., Nain, L., Exploring the efficacy of wastewater-grown microalgal biomass as a biofertilizer for wheat.  Environ. Sci. Pollut. Res. Int., 23 (7), 6608–6620, 2016. 53. Guo, S., Wang, P et al., Microalgae as Biofertilizer in Modern Agriculture, in: Microalgae Biotechnology for Food, Health and High Value Products, M.A. Alam, J.-L. Xu, Z. Wang (Eds.), pp. 397–411, Springer, 2020. 54. Pereira, I., Ortega, R., Barrientos, L.X, Moya, M., Reyes, G., Kramm, V., Development of a biofertilizer based on filamentous nitrogen-fixing cyanobacteria for rice crops in Chile. J. Appl. Phycol., 21(1), 135–144, Springer, 2009.

538  Biofertilizers 55. Osman, M.E.H., El-Sheekh, M.M., El-Naggar, A.H., Gheda, S.F., Effect of two species of cyanobacteria as biofertilizers on some metabolic activities, growth, and yield of pea plant. Biol. Fertil. Soils, 46(8), 861–875, Springer, 2010. 56. Prasanna,  R., Joshi,  M., Rana, A., Shivay, Y.S.,  Nain, L., Influence of co-­ inoculation of bacteria-cyanobacteria on crop yield and C–N sequestration in soil under rice. World J. Microbiol. Biotechnol., 28 (3), 1223–1235, Springer, 2012. 57. Jha, M.N., Prasad, A.N., Efficacy of New Inexpensive Cyanobacterial Biofertilizer Including its Shelf-life. World J. Microb. Biot., 22(1), 73–79, Springer, 2006. 58. Mager, D.M., Thomas, A.D., Extracellular polysaccharides from cyanobacterial soil crusts: A review of their role in dryland soil processes. J. Arid Environ., 75 (2), 91–97, Elsevier, 2011. 59. Leloup, M., Nicolau, R., Pallier, V., Yéprémian, C., Feuillade-Cathalifaud, G., Organic matter produced by algae and cyanobacteria: Quantitative and qualitative characterization. J. Environ. Sci., 25 (6), 1089–1097, Elsevier, 2013. 60. Yan-Gui, S., Xin-Rong, L., Ying-Wu, C., Zhi-Shan, Z., Yan, L., Carbon fixation of cyanobacterial–algal crusts after desert fixation and its implication to soil organic carbon accumulation in desert. Land Degrad. Dev.,  24  (4), 342–349, 2011. 61. Yilmaz,  E., Sönmez, M., The role of organic/bio–fertilizer amendment on aggregate stability and organic carbon content in different aggregate scales. Soil Tillage Res., 168, 118–124, Elsevier, 2017. 62. Uysal,  O., Uysal, F.O., Ekinci, K., Evaluation of Microalgae as Microbial Fertilizer. Eur. J. Sustain. Dev., 4 (2), 77–82, 2015. 63. Prasanna, R., Jaiswal, P., Singh, Y.V., Singh, P.K., Influence of biofertilizers and organic amendments on nitrogenase activity and phototrophic biomass of soil under wheat. Acta Agron. Hung., 56 (2), 149–159, 2008. 64. Ahmed, E., Holmström, S.J., Siderophores in environmental research: roles and applications. Microb. Biotechnol., 7 (3), 196–208, 2014. 65. Prasanna, R., Bidyarani, N., Babu, S., Hossain, F., Shivay, Y.S., Nain, L., Moral, M.T., Cyanobacterial inoculation elicits plant defense response and enhanced Zn mobilization in maize hybrids. Cogent Food Agric., 1 (1), 998507, 2015. 66. Rana,  A., Joshi, M., Prasanna, R.,  Shivay, Y.S., Nain, L., Biofortification of wheat through inoculation of plant growth promoting rhizobacteria and cyanobacteria. Eur. J. Soil Biol., 50, 118–126, Elsevier, 2012. 67. Manjunath, M., Kanchan, A., Ranjan, K., Venkatachalam, S., Prasanna, R., Ramakrishnan, B., Hossain, F.,  Nain, L., Shivay, Y.S.,  Rai,  A.B., Singh, B., Beneficial cyanobacteria and eubacteria synergistically enhance bioavailability of soil nutrients and yield of okra. Heliyon, 2 (2), Elsevier, 2016. 68. Ranjan, K., Priya,  H., Ramakrishnan, B., Prasanna,  R.,  Venkatachalam, S.,  Thapa,  S., Tiwari, R., Nain,  L., Singh,  R., Shivay, Y.S., Cyanobacterial

Biofertilizers From Waste  539 inoculation modifies the rhizosphere microbiome of rice planted to a tropical alluvial soil. Appl. Soil Ecol., 108, 195–203, Elsevier, 2016. 69. Renuka, N., Prasanna, R., Sood, A., Bansal, R., Bidyarani, N., Singh, R., Shivay, Y.S., Nain, L., Ahluwalia., A.S., Wastewater grown microalgal biomass as inoculants for improving micronutrient availability in wheat. Rhizosphere, 3, 150–159, Elsevier, 2017. 70. Weiss, T.L., Roth, R., Goodson, C., Vitha, S., Black, I., Azadi, P., Rusch, J., Holzenburg, A., Devarenne, T.P., Goodenough, U., Colony Organization in the Green Alga  Botryococcus Braunii  (Race B) Is Specified by a Complex Extracellular Matrix. Eukaryot. Cell, 11 (12), 1424–1440, 2012. 71. Xiao, R., Zheng, Y., Overview of microalgal extracellular polymeric substances (EPS) and their applications. Biotechnol. Adv.,  34  (7), 1225–1244, Elsevier, 2016. 72. Trejo, A., De-Bashan, L. E., Hartmann, A., Hernandez, J.P., Rothballer, M., Schmid, M., Bashan, Y., Recycling waste debris of immobilized microalgae and plant growth-promoting bacteria from wastewater treatment as a resource to improve fertility of eroded desert soil. Environ. Exp. Bot., 75, 65–73, Elsevier, 2012. 73. Abed, R.M.M., Interaction between cyanobacteria and aerobic heterotrophic bacteria in the degradation of hydrocarbons. Int. Biodeter. Biodegr., 64(1), 58–64, Elsevier, 2010. 74. Tripathi, R.D., Dwivedi, S., Shukla, M.K., Mishra, S., Srivastava, S., Singh, R., Rai, U. N., Gupta, D.K., Role of blue green algae biofertilizer in ameliorating the nitrogen demand and fly-ash stress to the growth and yield of rice (Oryza sativa L.) plants. Chemosphere, 70(10), 1919–1929, Elsevier, 2008. 75. Gantar,  M., Kerby,  N.O., Rowell, P., Colonization of wheat (Triticum vulgare L.) by N2-fixing cyanobacteria: III. The role of a hormogonia-promoting factor. New Phytol., 124 (3), 505–513, 1993. 76. Krings,  M., Hass, H., Kerp, H., Taylor, T.N., Agerer, R., Dotzler., N., Endophytic cyanobacteria in a 400-million-yr-old land plant: A scenario for the origin of a symbiosis? Rev. Palaeobot. Palynol., 153, 62–69, Elsevier, 2009. 77. Bidyarani, N., Prasanna, R., Chawla,  G., Babu, S., Singh, R., Deciphering the factors associated with the colonization of rice plants by cyanobacteria. J. Basic Microbiol., 55 (4), 407–419, 2015. 78. Ramakrishnan, B., Kaur, S., Prasanna, R., Ranjan, K., Kanchan, A., Hossain, F.,  Shivay, Y.S., Nain, L., Microbial inoculation of seeds characteristically shapes the rhizosphere microbiome in desi and kabuli chickpea types. J. Soils Sediments, 17, 2040–2053, Springer, 2017. 79 Prasanna,  R., Ramakrishnan, B., Simranjit, K., Ranjan, K.,  Kanchan, A., Hossain, F., Nain, L., Cyanobacterial and rhizobial inoculation modulate the plant physiological attributes and nodule microbial communities of chickpea. Arch. Microbiol., 199 (9), 1311–1323, Springer, 2017. 80. Epp, J.B.,  Alexander, A.L.,  Balko, T.W., Buysse, A.M., Brewster, W.K.,  Bryan,  K., Daeuble, J.F., Fields, S.C., Gast,  R.E., Green,  R.A., Irvine,

540  Biofertilizers N.M., Lo, W.C., Lowe, C.T., Renga, J.M., Richburg, J. S., Ruiz, J.M., Satchivi, N.M., Schmitzer, P.R., Siddall, T.L., Webster, J.D., Weimer, M.R., Whiteker, G.T.,  Yerkes, C.N., The discovery of Arylex active and Rinskor active: two novel auxin herbicides. Bioorg. Med. Chem., 24 (3), 362–371, Elsevier, 2016. 81. Lu, Y., Xu, J., Phytohormones in microalgae: a new opportunity for microalgal biotechnology? Trends Plant Sci., 20 (5), 273–282, CelPress, 2015. 82. Hussain, A., Hasnain, S., Phytostimulation and biofertilization in wheat by cyanobacteria. J. Ind. Microbiol. Biot., 38 (1), 85–92, Springer, 2011. 83. Mazhar, S.,  Cohen, J.D., Hasnain, S., Auxin producing non-heterocystous Cyanobacteria and their impact on the growth and endogenous auxin homeostasis of wheat. J. Basic Microbiol., 53, 996–1003, 2013. 84. Priya, H., Prasanna, R., Ramakrishnan, B., Bidyarani, N., Babu, S., Thapa, S., Renuka, N., Influence of cyanobacterial inoculation on the culturable microbiome and growth of rice. Microbiol. Res., 171, 78–89, Elsevier, 2015. 85. Babu, S., Bidyarani, N.,  Chopra, P.,  Monga, D.,  Kumar, R.,  Prasanna, R., Kranthi, S., Saxena, A., Evaluating microbe-plant interactions and varietal differences for enhancing biocontrol efficacy in root rot challenged cotton crop. Eur. J. Plant Pathol., 142, 345–362, Springer, 2015. 86. Grzesik, M.,  Romanowska-Duda, Z.,  Kalaji, H.M., Effectiveness of cyanobacteria and green algae in enhancing the photosynthetic performance and growth of willow (Salix viminalis L.) plants under limited synthetic fertilizers application. Photosynthetica, 1-12, Springer, 2017. 87. Khan, Z., Park, S.D., Shin, S.Y., Bae, S.G., Yeon, I.K., Seo, Y.J., Management of Meloidogyne incognita on tomato by root-dip treatment in culture filtrate of the blue-green alga,  Microcoleus vaginatus. Bioresour. Technol.,  96  (12), 1338–1341, Elsevier, 2005. 88. Khan, Z., Kim,  Y.H., Kim, S.G., Kim, H.W., Observations on the suppression of root-knot nematode (Meloidogyne arenaria) on tomato by incorporation of cyanobacterial powder (Oscillatoria chlorina) into potting field soil. Bioresour. Technol., 98 (1), 69–73, Elsevier, 2007. 89. Victor, T.J., Reuben, R., Effects of organic and inorganic fertilisers on mosquito populations in rice fields of southern India. Med. Vet. Entomol., 14 (4), 361–368, 2000. 90. Biondi,  N., Piccardi,  R., Margheri, M.C., Rodolfi, L., Smith,  G.D., Tredici, M.R., Evalation of Nostoc strain ATCC 53789 as a potential source of natural pesticides. Appl. Environ. Microbiol., 70 (6), 3313–3320, 2004.

18 Biofertilizers Industry Profiles in Market Kashish Gupta

*

Noida International University, Greater Noida, India

Abstract

Continuous rising population and increasing urban and rural incomes are driving the popularity of agriculture market. Data consensus report also validates that about 50% exclusive share is directly contributed by the fertilizer in the agriculture industry. Considering the whooping industry requirements, various governments and other organizations are actively involved in promoting research and development in this area. Motivation behind writing this chapter is to make the people aware of the status quo of biofertilizer and also to motivate contribution toward achieving targets of food demand in an eco-friendly and sustainable manner. Keywords:  Green fertilizers, biofertilizer market, rhizosphere, bioavailability, liquid fertilizers

18.1 Biofertilizers and Biofertilizer Technology Biofertilizers are also popularly called as microbial inoculants or bioformulations but the term has also varied number of synonym such as manure, green manure, organic supplemented or intercrop, and chemical fertilizer [1–3]. Biofertilizers are rightly defined in terms of the Vessey (2003) as the substance that contains living MOs which are applied to seeds, leaves, roots, or as soil treatment, permitting the colonization of the rhizosphere and thus promoting growth either by enhancing the supply or bioavailability of the major nutrients to the host plant. For the first time, this term was used in 1978, referring to a cluster of microorganisms, now known as PGPR [4, 5] which are basically the free-living soil, rhizosphere/ Email: [email protected]

*

Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (541–560) © 2021 Scrivener Publishing LLC

541

542  Biofertilizers rhizoplane, and phyllosphere bacteria that flourish under some conditions and show immense benefits to the plants [4–6]. Biofertilizer has promising present and future in the integrated nutrient management system, which widely reduces the adverse environmental impact by fertilizer inputs [7, 40]. In this line of action, “Nitragin” (Rhizobium sp.) was the first commercial biofertilizer by USA (1896) and others are “Azotogen” (Azetobacter chroococcum) and “phosphobactin” (Bacillus megatarerium cv phospahticum) by Russia (1900s). As far as India is concerned, we got our first biofertilizer, rhizobium biofertilizer, back in 1934 (Joshi) and its commercial production was initiated by ICAR Indian Agricultural Research Institute (IARI), New Delhi, and Agricultural College and Research Institute, Coimbatore (1956).

18.1.1 Benefits of Different Biofertilizers 1) As Supplement With Chemical Fertilizer: Biofertilizer has the potential to heighten the productivity in short span of time, but due to its limitation, its casual use is not preferred. It is used along with chemical fertilizer, rather supplemented it with biofertilizers that are supplement to chemical fertilizers [8]. 2) High Crop Production: It increases the crop yield by 4%–5%, on an average, can fix biological nitrogen in the soil, is readily available to the host plant, can improve soil properties, and can sustain soil fertility. It is an effective nutrient resource and maintains and sustains the yield and quality. Use of biofertilizer is gaining momentum due to its nutritional value, less environmental impacts, and being a renewable source. Biofertilizers enhance the soil biota and favor plant health by increasing organic content of the soil [9]. 3) Economic Reasons: Biofertilizers are comparatively less expensive to chemical ones and can largely reduce the cost of cultivation. It provides plant nutrient at low cost and is useful especially for cereal crops. Microbial inoculant–based biofertilizers are used to give economic input toward crop productivity and to lower the dosage requirement [10, 11]. 4) Restoration of Soil Structure: Biofertilizers show immense potential toward protection from drought and soil diseases.

Biofertilizers Industry Profiles in Market  543 It is an effective and eco-friendly alternative against the chemical fertilizer. Biofertilizers also show capacity toward resistance against abiotic stress for the host plant. It serves as a wonderful alternative to fulfill the nutrient requirement of crops [12].

18.2 Limitations in Usage of Biofertilizers 1) Lack of sufficient information regarding the biofertilizers scenario and scope in the market. Moreover, the crop growers and farmers are not well aware and rather ready to accept it as a part of crop plantation integral part. 2) For successful and effective integration of biofertilizers in farmers’ field, it is still restricted to certain crops and also locations [13]. Generally, the microorganism activity is host-specific and site-specific applications. 3) Biofertilizers, being live microorganisms inoculants, usually die off or loss their activity, when temperature fluctuates. Low shelf life is its major market constraints. Marketing of biofertilizers is cumbersome since biofertilizer agroproducts have restricted shelf life (ca. 6 months) [14, 15]. It has also been reported that standards for packing, labeling, and pricing are not followed, leading to chaos, quality degradation, and loss to farmers [16, 17]. Subsidization system of the government is unsystematic and indirectly responsible for promotion of the discrimination and manipulation. Furthermore, different state governments sometimes provide subsidies up to 50% of the sales [18]. Although biofertilizer has multiple advantages in sustainable agriculture, still it suffers from constraints at various levels, production unit, farmers’ field, and distribution unit, thus making it less popular in India. 4) Non-availability of suitable carrier for biofertilizer is decreasing the market. Carrier acts as the medium that loads effectively the microbial inoculants used in the biofertilizer. Good quality carrier directly ensures the production of a quality biofertilizer. Non-availability of good quality carrier peat in India has also led to the development of alternative sources for carrier materials like lignite and charcoal, which

544  Biofertilizers are mostly used unsterilized [19]. Biofertilizers sold in markets suffer from serious contamination and low count of MOs due to the unskilled working personnel for its manufacturing process. In general, producers, due to lack of proper training, are not able to pay attention to use host-specific strains for a particular biofertilizer production, leading to its lower potency on application [20, 21]. Substandard biofertilizer products are supplied by spurious manufactures, leading to less acceptability of new product.

18.3 Biofertilizer Market Segments Market break for any biofertilizer is based on the following criteria: a) Based on Region: Indian agricultural market can be segmented as North India, South India, East India, and West India regions. b) Based on Types: Majorly, three types of biofertilizers are popularized in Indian market such as nitrogen fixing, phosphate fixing, and potash mobilizing. Nitrogen fixing biofertilizers are being considered as the superior alternatives among the available industrial nitrogen fertilizers. Marketing of ­nitrogen-based fertilizers is huge, especially for crops like rice, oats, and grains. Rice is the staple crop in various Asian countries such as India, China, Japan, and Indonesia due to which nitrogen fertilizer consumption, and production is enormous. Tables 18.1 and 18.2 clearly depict the status quo of production and consumption of different fertilizers in the country. c) Based on Crop: Oilseeds, vegetable, and grain crops are the most grown crops. Requirement of fertilizer for cereal crops is highest among all crops. Biofertilizers’ market based on crop includes fruits and vegetables, cereals, plantations pulses, and oilseeds and others. Cereals and grain need the biofertilizers in ample amount as compared to fruits and vegetables. Second major intake of biofertilizers is for the vegetables and fruits, which further enhances the scope for biofertilizers. d) Based on Microorganism: Rhizobium sps., Acetobacter sps., Phosphate solubilizing, and immobilizing bacteria.

Biofertilizers Industry Profiles in Market  545 Table 18.1  Table depicting all Indian production of N- and P2 O5-based fertilizers. ~Through N based fertilizers

~Through straight P2 O5

~Total (tonnes)

S. no.

Year

1

Plan VIII(1992–1997)

7,993

2,385

19,264

2

Plan IX(1998–2002)

10,612

3,460

32,200

3

Plan X(2003–2007)

11,045

4,042

33,747

4

Plan XI(2008–2012)

11,050

4,047

36,100

5

Plan XII(2013–2017) 2018 2019

32,785 13,423 13,337

3,498 4,098 3,938

39,505 41,560 41,564

Table 18.2  Depicting all Indian consumption of N-, P2 O5-, and K2O-based fertilizers. ~Through N based fertilizers

~Through straight P2 O5

~ Through K2O

~Total (tonnes)

S. no.

Year

1

Plan VIII(19921997)

9,369

2,863

1,100

13,314

2

Plan IX(19982002)

11,215

4,284

1,523

17,022

3

Plan X(20032007)

11,952

4,702

2,001

15,644

4

Plan XI(20082012)

15,985

7,051

3,134

25,965

5

Plan XII(20132017) 2018 2019

16,925 13,423 13,337

6,413 6,854 6,910

2,480 2,779 2,680

25,660 26,650 27,228

546  Biofertilizers Depending on the type of group, compatible biofertilizer is applied. e) Based on Form: Liquid biofertilizer and powder biofertilizer. Powder biofertilizer was initially used but now discontinued due to certain limitations. Liquid biofertilizer has carved its prominent niche not only in Indian but also in global market [22–24].

18.4 Biofertilizers Market Drivers in India Major biofertilizer market drivers can be due to rising environmental awareness and increasing demand for organic and demand for improvising soil fertility. On the contrary side, limiting factors being listed for the barriers in exponential growth of biofertilizers market are regarded as insufficiency in knowledge about the benefits and due to which lower acceptance in adoption rate by farmers. As far as the current scenario of fertilizer market is concerned, demand of fertilizers is not satisfied by the current level of production. Various policies are being framed by government and agriculturally economy-based countries to promote the awareness and implement schemes in the industry. a) Sustainability: Although the green revolution has brought immense gains to the agriculture output productivity, but sadly, it lacked in the sustainability aspect of agriculture growth. This revolution has led to the uncontrolled usage of chemical fertilizers, and pesticides gave rise to new ecological problems and burden along with high productivity of crops such as serious decline in soil quality, fertility, and disruption of the natural microbial flora. Biofertilizers are like the panacea in such cases which can definitely provide the efficient use of the various resources (factors) for sustainable and high yield. b) Increasing Population Growth Rate: Food demand is directly proportional to the increasing population. Biofertilizers can be the appropriate solution for feeding such huge population especially when agriculture industry is largely facing problems due to environmental stress [25]. Realizing the useful prospects and aspects of biofertilizers, implementation along with modern agricultural practices becomes essential. Secondly, growing awareness of the organic India

Biofertilizers Industry Profiles in Market  547 has made the users aware and enlightened. Farmers and common men are accepting and adopting the production and usage of biofertilizers on wide scale. Robust demand from the health and economic perspective has led to its high growth. Developing country and high amount of arable land in India make it the hot spot for the investors whether national or international because of high availability of arable land. Government of India is making tremendous efforts both at the state and central levels to extend the use of biofertilizers for its use in agriculture industry. Growth rate of both consumption and production is quite satisfactory in India; still, very low percentage increase in demand is seen as supported by Tables 18.1 and 18.2.

18.5 Present Scenario of Biofertilizer Market There is no doubt that chemical fertilizer are responsible for a definite positive role in the as far as Indian agriculture is concerned. Increase in fertilizer consumption has largely occurred during the past decade [25]. Need of sustainability has promoted the use of biofertilizers to a new Indian agriculture paradigm and practices such as biofertilizers, biopesticides, organic farming, low input agriculture, and sustainable agriculture. Integrated farming practices are largely marked as the indicators not only of developed nation but also of developing nation [24]. India has gross cropped area of over and above 190 million hectares, suiting the prospective demand of the biofertilizer of about 627,000 million tonnes (MT) [26]. In India, the first commercial biofertilizer was started in 1956 by eminent scientist N.V. Joshi. Since the ninth 5-year plan, environmentally sustainable aspect in agriculture is added. Popular scheme of national project on research, development, and use of biofertilizer (NPDB) was largely implemented by the Government of India, for both production and consumption of biofertilizers. At national level, National Biofertiliser Development Centre at Ghaziabad, UP, for delivering the training programs in biofertilizer development is being promoted. The Natural Input Complete Utilization (NIKU) Bioresearch laboratory was established in Pune (1996). Under the seventh 5-year plan schemes, one national and seven regional centers were established. Various grants, 20 lakh per unit of 150 tonnes per year, were released for

548  Biofertilizers the setting up of biofertilizer plant. Nearly 83+ biofertilizer production units have been installed since the inception of this plan. Sanctioning of these units was done by Department of Biofertilizers (9 units) and 74 units by Department of Agriculture and Cooperation. About 39 units were moted by the different private companies, entrepreneurs, and organizations. Estimated biofertilizer production capacity increased to about the 18,500 tonnes per year in the country. The tenth 5-year plan had clearly focused on the usage of biological agents for biocontrol, organic manures, and biofertilizers. Although, due to green revolution, initially, chemical fertilizer consumption showed hike but later after observing and releasing its impact, its consumption became less acceptable. Efforts made by National Centre for Organic Farming (NCOF) for the commercialization of the biofertilizer had led to the rhizobium biofertilizer demand of about 0.43 MT. Later, it is estimated that the demand for biofertilizers for various seed and root treatment is about 0.426 MT based on gross cultivated area. Government is promoting the biofertilizers under several schemes fabricated by National Mission for Sustainable Agriculture (NMSA)/ Paramparagat Krishi Vikas Yojana (PKVY), National Food Security Mission (NFSM), National Mission on Oilseeds and Oil Palm (NMOOP), and Indian Council of Agricultural Sciences (ICAR). ICAR also installed network projects on organic farming to provide location specific package for over 18 different crops and cropping systems. This project was aimed at targeting over 20 centers located in 16 different states. Another network project based on the soil diversity-biofertilizer was executed for improvising and producing efficient strains for biofertilizer specific for different soil types and the crops. Improvised shelf life for liquid biofertilizer technology was also one of the fruits of this project. The council also worked on the developing technologies for improvised organic manures such as phospho-compost and bio-enriched compost. ICAR provides training program and also front-line demonstration (FLD) to farmers in support of biofertilizer and know-how for applying the biofertilizer to seed, soil, and soil under its AI-NOF, all India network programs on organic farming. ICAR project “Network Project on Soil Biodiversity-Biofertilizers” has produced the improved and efficient strains of biofertilizers for various crops and soil types. ICAR study has validated that biofertilizers have the capacity to improve the crop yield top nearly 10%–25% and, as supplement with chemical fertilizer, show increment by 20%–25%. From Bihar, Bihar Agriculture University and KVKs including KVK Gopalganj are also engaged in research study on biofertilizers. Government of India has motivated and promoted the usage of biofertilizers over chemical

Biofertilizers Industry Profiles in Market  549 fertilizers in various state governments including in district Gopalganj of Bihar under the various innovative schemes/programs, viz., PKVY, NMOOP, Mission Organic Value Chain Development for North Eastern Region (MOVCDNER), and NFSM. Promotion of soil health under various soil test schemes under the integrated nutrient management schemes, soil health card program, and integrated pest management has also been undertaken. Indian fertilizer market showed prized worth INR 4,675 billion in 2017. As we look forward, the market predicted to show projected growth to reach INR 9,987 billion by 2023, showing CAGR of around 13% during 2018–2023. Biofertilizers are now an integral part of various agricultural practices in India. However, its promotion has some limitations. This aspect will be discussed in the later section of the chapter.

18.6 Key Players of Biofertilizers in Indian Market National Fertilizers Limited (NFL) had made its name not only in Indian market but in global market. NFL manufactures and does marketing of mainly three types of biofertilizers, phosphate solubilizing bacteria Rhizobium and Azetobacter sps. by the brand name of Kisan. NFL marketing of biofertilizer is targeted to states Uttar Pradesh, Madhya Pradesh, Uttarakhand, Jammu and Kashmir, Maharashtra, Himachal Pradesh, Chattisgarh, Bihar, Jharkhand, Rajasthan, and Punjab and Haryana. Madras Fertilizers Limited (MFL) was the joint venture GOI and AMOCO India. MFL is actively engaged in the manufacturing biofertilizers products, e.g., Vijay organic products. Gujarat State Fertilizers and Chemicals Ltd (GSFC) are active from past 45 years for providing diverse products mix ranging from more than 24 brands of fertilizers to chemicals, petrochemicals, industrial gases, plastics, fibers, and other products, e.g., Sardar brand. RCF is a leading chemical and fertilizer manufacturing company. It has further reorganized to five new companies, viz., Hindustan Fertilizer Corporation Limited (HFC), Fertilizer Corporation of India (FCI), NFL, Projects and Development India Limited (PDIL), and Rashtriya Chemicals and Fertilizers Limited (RCF). RCF does production of not only biofertilizers but also micronutrients and 100% water soluble, e.g., Biola, Microla, and Ujjwala. Other key biofertilizer manufacturers in market are Root Care Glomus Ambica Biotech, MP, JOSH super/ plus Glomus intraradices Rajan Laboratories, Tamil Nadu, Bioproducts

550  Biofertilizers

Figure 18.1  Different biofertilizer product in Indian market.

(Gujrat) Samrat Biotech Ltd, and Karnataka (Kisan Power) [26, 27], as shown in Figure 18.1.

18.7 Problems in Promotion of Biofertilizer Marketing of biofertilizer is quite cumbersome task since it is not the primary input like the seed and other variables. Limited acceptance of biofertilizer by farmers is as follows: Extensive measures are undertaken by the government to convince and give awareness to the farmers of the essential benefits of the biofertilizers. Moreover, the acceptance of biofertilizers is limited to Rhizobium, Azotobacter, and phosphate solubilizing bacteria. To create awareness among farmers, various seminars on biofertilizers and micronutrients are regularly being organized by the Government of India, convincing farmers about the need of biofertilizers in crop yield. In order to popularize the concept of biofertilizer was popularized, field day farmers visit are arranged to agricultural universities that are arranged and sponsored by the government.

Biofertilizers Industry Profiles in Market  551 Table 18.3  Type of biofertilizers used for different types of crops (data.govt.in). S. no.

Bacteria used

Crops used

Contribution

1

Rhizobium strains

Legumes like pulses, groundnut, soybean, black bersem, lucern

10%–35% yield increase, 50–200 kg N/ha. Fodders give better results. Leaves residual N in the soil.

2

Azotobacter

Soil treatment for non- legume crops including dry land crops. Mustard, sunflower, banana, sugarcane, grapes, papaya, water melon, tomato, ladyfinger, coconut, spices, fruits, flowers, plantation crops

10%–15% yield increase - adds 20– 25 kg N/ha Also controls certain diseases

3

Azospirillum

Non-legumes like maize, barley, oats, sorghum, millet, sugarcane, rice, wheat etc.

10%–20% yield increase. Fodders give higher/ enriches fodder response. Produces growth promoting substances. It can be applied to legumes as co-inoculant. (Continued)

552  Biofertilizers Table 18.3  Type of biofertilizers used for different types of crops (data.govt.in). (Continued) S. no.

Bacteria used

Crops used

Contribution

4

Phosphate Solubilizers and mobilizers

Bacillus megaterium var. phosphaticum, Bacillus circulans, Pseudomonas striata. Soil application for all crops. Penicillium sp., Asper­ gillus awamori

5%–30% yield increase. Can be mixed with rock phosphate.

Phosphate mobilizers Mycorrhizae (VAM) Glomus sp., Gigaspora sp(Arbuscular)

Many trees, some crops, and some ornamental plants

30%–50% yield increase, enhances uptake of P. Zn, S, and water. Usually inoculated to seedlings.

Laccaria sp(Exto­ mycoorhiza) Rhizoctonia solani (orchid) 5

Biofertilizers for Micro nutrients

Bacillus sp.

Increases the availability of the essential nutrients in the rhizosphere zone. Leaf moisture retention increases.

6

Plant Growth Promoting Rhizobacteria Pseudomonas fluorescens

Protein rich crops, e.g., soybean Energy crops willows and poplars

15% and above increase yield Mineral nutrition and growth, aggressive colonization, plant growth stimulization, and biocontrol.

Source: Biofertilizers - types and their application, KrishiSewa.

Biofertilizers Industry Profiles in Market  553

18.8 Popular Marketed Biofertilizers in Indian Market Range of microorganisms used as biofertilizers (and carrier materials) and available to the farmers in India are, as given in Table 18.3. Table 18.4 shows prices of different biofertilizers. 1) Rhizobium—Nitrogen fixer bacterial strain: Rhizobia are soil bacteria capable of fixing the nitrogen inside the root nodules, especially in legumes, e.g., Rhizobium, Mesorhizobium, Bradyrhizobium, and Azorhizobium sps. They form symbiotic interactions with legumes due to flavonoid chemotactic released by legumes on contact [28, 29]. 2) Azotobacter—Nitrogen fixer bacterial strain: Azotobacter chroococcum spp. is used for increased crop production, better availability of plant nutrition, and increased soil fertility. Azotobacteria genus can synthesize series of hormones such as auxins, cytokinins, and GA-like substances which up-regulates the growth of plant. It also stimulates rhizospheric microbes, provides protection to the plants from phyto-pathogens, improves nutrient uptake capacity and is effective biological nitrogen fixation [30–32]. 3) a) Phosphorus solubilizer, e.g., Bacillus, Pseudomonas, and Aspergillus. Wide range of soil microorganism shows innate capacity to solubilize and mineralize the insoluble soil phosphate and on easy release of soluble phosphate on Table 18.4  List of prices of different biofertilizers. S. no.

Types of biofertilizers

Price

1

Am Fungi

Rs. 250/kg

2

Azospirillum

Rs 60/kg

3

Azospirillum

Rs 420/Packet

4

Azotobacter

Rs 300/Liter

5

PGPR

Rs 350/kg

6

Rhizobium

Rs 420/Packet

7

Liquid biofertilizer

RS 800/20 kg

554  Biofertilizers metabolism, thereby increasing the bioavailability of the applied inoculants for seeds and crops, and is proven tested for global food production with no environmental side effects [33, 34]. b) Phosphate mobilizer, e.g., VA-mycorrhiza (VAM) like Glomus. Some soil bacteria show phosphate solubilizing capacity to transform the accumulated insoluble phosphates from different inorganic sources to soluble form making it available to plants [35]. 4) Plant growth-promoting biofertilizers (PGPR), e.g., Pseudo­ monas sp. Pseudomonas shows plant growth-promoting properties and also reduces insect and pathogen damage [36–38]. 5) Biofertilizers for Micro nutrients, e.g., Bacillus subtilis. Bacillus sps. are widely known to act as PGF, plant growth promoters such as auxins, and aids in synergistic plant growth by solubilizing insoluble phosphates [39].

18.9 Recent Trends in Biofertilizer: Liquid Biofertilizer The above limitations of biofertilizer have been overcome by the use of the liquid biofertilizer to some extent. Due to fewer shortcomings, it is more acceptable to farmers and the marketers. Liquid biofertilizers are primarily the suspensions of the agriculturally efficient MOs, which can fix atmospheric N2 and also solubilize the insoluble phosphates present in soil for high bioavailability. Liquid fertilizers are showing increasing share in consumption and production in the market and acting as suitable alternative for chemical as well as organic fertilizers. Microorganism present in the biofertilizers not only enhances the plant growth but also produces healthy rhizosphere [40, 41]. Liquid biofertilizer does not require any carrier for its application. Potential applications of such biofertilizer is seen in present modern agriculture practices, called as soilless farming systems (hydroponics).

18.9.1 Specialties of Liquid Biofertilizer Such biofertilizer has the potential to reduce the usage of chemical fertilizer from 15% to 40%. Liquid biofertilizer are easily absorbed by the plants and has fast results. Small-scale farmers find it quite lucrative option since it helps

Biofertilizers Industry Profiles in Market  555 in reducing the weather dependency. As compared to the solid matrix–based fertilizer, the shelf life of the liquid biofertilizer is higher ranging from 1 to 2 years. Liquid biofertilizers are expected to meet the requirements of Indian farmers needed in production of organic crops as per the share to be contributed in the competitive global market. As the liquid biofertilizers are prepared by fermentation, its identification becomes better chances of survival of viable counts on seeds and soil. Treatment when applied is recorded. It is quite tolerant to high temperature and UV radiation. Being easy to use makes it highly acceptable among farmers [42, 43]. Such fertilizers are capable of generating high commercial revenues. Some of the best ruling companies of the liquid fertilizers in global market includes Agrium Inc., Yara International ASA, Sociedad Química Y Minera De Chile (SQM), K+S Aktiengesellschaft, Plant Food Company, Inc., Kugler Company Rural Liquid Fertilizers (RLF), Compo Expert GmbH, Agroliquid, Haifa Chemicals Ltd., and Israel Chemical Ltd. (ICL), Liquid formulation of biofertilizer plays vital role in helping to solve the increasing shelf life in microorganisms. In current study, the liquid biofertilizer is best way of sustainable agriculture for crop production [43].

18.10 Conclusion and Future Scope Undoubtedly, due to increasing population, rise in production of food grains and the usage of biofertilizers for sustainable agriculture has been widely adapted by various regions of India. Fertilizers have shown growth in double digits in past 5 years. Despite this growth rate, average consumption in India is much less than most of emerging and developed countries. Although not all states of India have adopted the biofertilizers as an integral part of the crop production, still it gives a lot of scope and innovative possibilities for future growth. We can expect in near future more number of government, non-government campaigns for educating and spreading biofertilizers literacy and importance to the farmers. Social media like television radio and customized workshop are contributing immensely toward the awareness of better and sustainable fertilizers. Contract farming is also helping and supporting farmers, largely affecting the total fertilizer consumption in the country. Still, the chemical fertilizers occupy the major share of fertilizer consumption in country, but the potential and future scope of biofertilizer is very large. Keeping in view of the potential hazards due to chemical fertilizers, biofertilizers can be the remarkable alternative sources for meeting the

556  Biofertilizers nutrient demand of the crops. Especially in the biofertilizers, beneficial bacteria such as Rhizobium, Azotobacter, Azospirillium, and Mycorrhizae are best suited, reliable, and essential in crop production. It has been estimated that major opportunities for biofertilizers growth lies in the emerging economy.

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558  Biofertilizers 26. D.L.N Rao, Biofertilizer situation in India and future thrusts. Indian Council of Agricultural Research (ICAR), pp 1–3, 1999. 27. A. Sharma, B.K. Upadhyay, Marketing Promotion Policies in Agriculture (Special Reference to National Fertilizer Limited). Marketing Promotion Policies in Agriculture in India, 152, 8–15, 2007. 28. K. Majumdar, Bio-Fertilizer use in Indian Agriculture Aripex Indian J. of Research, 4, 277–380, 2015. 29. N. Boonkerd, P. Singleton, Production of rhizobium biofertilizer. Biotechnology of Biofertilizers, Narosa Publishing House, New Delhi, pp. 122–128, 2002. 30. M.S. Singh Effect of Rhizobium, FYM and chemical fertilizers on legume crops and nutrient status of soil-a review. Agric. Rev, 26, 309–312, 2005. 31. S. A. Wani, S. Chand, M. A. Wani, M. Ramzan, K. R. Hakeem, Azotobacter chroococcum – A Potential Biofertilizer in Agriculture: An Overview, In book: Soil Science: Agricultural and Environmental Prospectives, Edition: 1, Chapter: Azotobacter chroococcum—Potential Biofertilizer in Agriculture: An Overview, Publisher: Springer International Publishing pp. 333–345, 2018. 32. N. Milosevic, B. Tintor, B.C. Protic, R. Cvijanovig Effect of inoculation with Azotobacter chroococcum on wheat yield and seed quality. Rom. Biotechnol. Lett. 17, 7352–7357, 2012. 33. E. T. Alori, B. R. Glick, O. O. Babaola, Microbial Phosphorus Solubilization and Its Potential for Use in Sustainable Agriculture, Front. Microbiol. 8, 971– 978, 2017. 34. D. Nassal, M. Spohn, N. Eltlbany, S. Jacquiod, K. Smalla, S. Marhan, E. Kandeler,Effects of phosphorus-mobilizing bacteria on tomato growth and soil microbial activity, Plant and Soil, 42717–37, 2018. 35. S. B.Sharma, R. Z.Sayyed, M. H. Trivedi and T. A Gobi, Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. Springer Plus, 2, 587–600, 2013. 36. Wani, P. A., Khan, M. S., and Zaidi, A. (2007). Co-inoculation of nitrogen fixing and phosphate solubilizing bacteria to promote growth, yield and nutrient uptake in chickpea. Acta Agron. Hung. 55, 315–323, 2007. 37. G. Gupta, S. S. Parihar, N. K. Ahirwar, S. K. Snehi and V. Singh,Plant Growth Promoting Rhizobacteria (PGPR): Current and Future Prospects for Development of Sustainable Agriculture, J. Microb. Biochem. Technol. 7, 96–100, 2015. 38. A. C. Gange, K. R. Gadhave, Plant growth-promoting rhizobacteria promote plant size inequality, Scientific Reports, 8, 13828–13830, 2018. 39. J.U. Itelima, W.J. Bang, M.D. Sila, I.A. Onyimba, O.J. Egbere, A review: Biofertilizer - A key player in enhancing soil fertility and crop productivity, Microbiol. Biotechnol. Rep. 2, 22–28, 2018. 40. Chandra, K., Greep, S., Ravindranath, P., Srivathsa, R.S.H., Liquid Biofertilizers, Ministry of agriculture department of agriculture & co-­ operation, Government of India. 4, 190–194, 2020.

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19 Case Study on Biofertilizer Utilization in African Continents Osikemekha Anthony Anani1 and Charles Oluwaseun Adetunji2* Laboratory for Ecotoxicology and Forensic Biology, Department of Biological Science, Faculty of Science, Edo State University, Uzuairue, Edo State, Nigeria 2 Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Department of Microbiology, Edo State University Uzuairue, Auchi, Edo State, Nigeria 1

Abstract

Beneficial microorganisms have been identified to play a crucial role toward the maintenance of a sustainable agriculture and their role in maintaining adequate balance of the ecosystem structure and soil function. The presence of these beneficial microbial consortia has been recognized to improve the bioavailability soil and crop soil nutrients as well as the efficiencies of the soil such as soil structure. The utilization of chemical fertilizers in the improvement of crops in farms has been documented to enhance soil quality and soil function but, whenever they are applied in excess, it led to increase in health and environmental hazards. Moreover, numerous people in Africa are currently experiencing high level of malnutrition and several nutritional challenges, and high level of soil infertility is couple with the problem of daily increase in population. Therefore, the application of biofer­tilizer will play a crucial role as a typical example of sustainable alternative that could help in mitigating all the aforementioned challenges and the effects of these synthetic fertilizer. a crucial role as an alternative to mitigate all the aforementioned challenges and the effects of these synthetic biofertilizer. Therefore, this chapter intends to discuss the current situation on the application of biofertilizer in Africa and their mechanism of action. Different types of biofertilizer that have been introduced were also highlighted. Moreover, specific examples that were cited where biofertilizer has led to increase in agricultural production were discussed extensively. Keywords:  Africa, biofertilizer, malnutrition, health, environment, hazards, ecosystem, and soil functions *Corresponding author: [email protected]; [email protected]; [email protected], 0000-0003-3524-6441 Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (561–574) © 2021 Scrivener Publishing LLC

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562  Biofertilizers

19.1 Introduction It has been observed that the level of agricultural productivity is gradually decreasing in Africa which could be linked to the factors such as pest and diseases, water stress, potassium, and low soil fertility due to potassium and nitrogen. Several efforts have been intensified through active research and development to discover innovation that could help to mitigate all the highlighted challenges. All the aforementioned challenges have become worsen through several agricultural problems such as bad climatic conditions, climatic changes, high level of depletion of the soil nutrient and high level of soil erosion, and leaching as well as high level of continues cropping that has aggravated the high level of declining soil fertility [1, 2]. Majority of the farmers in Africa utilize or improve fallow system for rejuvenation of the unfertile soil but this is no more sustainable because of the high level in the number of human population experienced in every part of the world due to rise in population pressure [3, 4]. The increase in the cost of synthetic fertilizer and high level of environmental dilapidation associated with the extensive application of synthetic fertilizer has led to high rate of environmental contamination majorly from phosphates and nitrates. This has increased the level of hazards in human health and environment, most especially in the developing countries, majorly in Africa [5]. Therefore, there is need to search for a sustainable biofertilizer that could be a replacement to the synthetic fertilizer. This continued application of synthetic fertilizer has affected the level of soil biotic community which has led to increase in the level of pesticide residue in some of these crops which eventual led to a weaken crop system [6]. There is a great demand for safer food with high quality that will be available in a higher quantity with adequate amount of nutrients. This will go a long way in meeting the demand of the ever increasing population. There is also a greater need to reduce the high level of synthetic fertilizer most especially in the African continent [7]. Hence, there is a great demand for food in most of these African countries because numerous parts of the population are still experiencing high level of malnutrition couple with a high level of population in these regions. This has necessitate a need to sustainable agriculture and high level of intensification through the application of eco-friendly fertilizer as a replacement for inorganic fertilizer [8].

Biofertilizer Utilization in Africa  563 Several researches have intensified effort on the application of some ecofriendly, sustainable, and cost-effective bio-inoculants such as Arbuscular mycorrhizal fungi and rhizobial. These beneficial microorganisms have been identified as sustainable biotechnological solutions that could be utilized as effective soil fertility management techniques that could improve the level of soil fertility due to their eco-friendly and capability to serve as a replacement to inorganic fertilizers [9]. Numerous researches have been carried out that involve the utilization of rhizobial inoculants [10, 11] and Arbuscular mycorrhizal fungi [12, 13]. It has been discovered that the bioinoculant entails living cells of numerous types of microorganisms with the capability to absorb nutrient to crops through various biological processes [14]. Microbial inoculants have been recognized as a formulation that entails beneficial microorganism that could play an active role in the maintenance of the ecosystem for a sustainable agriculture. It has been discovered that microbial inoculants are eco-friendly that could serve as substitute to pesticides and synthetic fertilizer [15]. They entail active microorganisms which could indirectly or directly enhance microbial action by stimulating the movement of nutrient in the soil [16]. Therefore, this chapter intends to provide a comprehensive review on the level of production of biofertilizers in Africa, and the latest information regarding production, commercialization, and their eventual application were discussed.

19.2 Specific Examples of Biofertilizer for Crop Improvement, Environmental Bioremediation, and Their Advantages and Challenges in Africa This section looked at the utilization of biofertilizers for the improvement of crop productivity, environmental stress (abiotic and abiotic factors), their success story, as well as their benefits and limitations in Sub-Saharan African with little documentations in other continents. Poor fertility of agricultural soils in African continent in conjunction with incompetent management has led to low crop productivity. To boost these problems, a sustainable form of biofertilizer with low economic effect and little or non-toxicity to the agricultural soils is highly recommended. In a review, Raimi et al. [17] looked at the influence of biofertilizers on soil fertility as an alternative for increasing subsistence agricultural productivity.

564  Biofertilizers The authors reported that recent strains of microbes (fungi, blue-green algae and bacteria) such as Bacillus, Pseudomonas, Azospirillum, Azotobacter, and Rhizobium play vital role in sustainable agriculture because of the benefits they portend (enhance crop yield and nutrition) via growth stimulating substances, bio-control actions, nutrient solubilization, and biotic N2 fixation. In conclusion, the authors suggested the utilization of biofertilizer derived from microbes as the best candidate for and effective soil boosters for nutrient deficient soil, ecosystem permanency, and improved food security, as well as poverty. They encourage subsistence farmers and stakeholders to adopt this technology for a prospective food sustainability. Masso et al. [18] did a review of the impasse of food management and security in Sub-Saharan Africa. The authors recounted that to meet the basic dietary wants, nutritious food, safe food, and food security must be at the top chain. That there is a clarion call to improve the soil nitrogen value which the African continent has recorded about 80% deficiencies, leading to malnutrition and food insecurity. In other vein, excess N2 load in H2O can result to eutrophication, leaching, and soil erosion. However, there is paucity of research conducted to enhance N2 in soil for good food yield and production, as well as the improvement of adaptation vigor to environmental stressors. The gap of their study with other research clearly looked at the exploit of N2 in reducing pollution. In conclusion, they recommend N2-based biofertilizers as a conjugate for crop improvement and ecosystem stress mitigation to farmers and relevant stakeholders. Carrapiço et al. [19] did a review of the future utilization of Azolla as a potential biofertilizers for boosting agricultural produced. The authors reported that Azolla plant contains consortia of endosymbiotic organisms inhabiting the small leaf cavities. In addition, in the plant’s cavities, nitrogen-­fixing bacteria, cyanobacteria (Anabaena azollae), play an important association of agricultural concern as well when it has been used for field purpose. The authors also looked at the impending challenges and applications of Anabaena azollae as biofertilizer in Sub-Saharan Africa, in which about 75% of workforce relies on agriculture as one of the major economic hubs second to crude oil in that region. The ecology of the plants was also highlighted and documented. The potential utilization of Anabaena azollae in improving agriculture sustainably as well as the risk involved with the combination of it with chemical fertilizers for soil efficiency and fertility were also discussed. Globally, nutrients from plants have installed important healthy derivatives to the teeming population of African continents. Of recent, there has been a shift from chemical fertilizers in boosting soil fertility and productivity. However, the impact in the food chain cannot be overemphasized.

Biofertilizer Utilization in Africa  565 In this context, in a review, Itelima et al. [20] looked at the potential of biofertilizer as a major player in improving crop efficiency and soil fruitfulness. The authors recounted that biofertilizers have been known to be a substitute for crop efficiency and soil fruitfulness for possible farming and sustainable production. They said that the utilization of useful microorganisms as potential biofertilizers has become a supreme value in the agrochemical sectors for the management of plant-soil deficiencies and abiotic stress. Some of the microbes used for this purpose are bacteria, ecto- and endomycorrhizal fungi, cyanobacteria, phosphate-solubilizing microorganisms/rhziobacteria, etc. In conclusion, the authors stated that to enhance biotic/abiotic factors, plant development and growth, and nutrient and water uptake, biofertilizer is the best means. More so, biofertilizers is cheap, eco-friendly, and eco-sustainable and should be recommended for soil and crop fertility and productivity. In a review, Jain [21] looked at organic farming using biofertilizer as a way to sustainable agriculture. That inorganic fertilizers, NPK (nitrogen, potassium, and phosphorus), have been known to increase plant productivity. However, the health and ecological risk it portends cannot be overemphasized. Recent advances in agriculture technology have proven that biofertilizers have potentials that can be harness, such as the ability to make nutrients accessible to crops, ability to improve plant and soil tolerance to abiotic stress, and its non-toxic and eco-friendly nature over the widely used inorganic ones. More so, biofertilizer is a promising tool for long-term commercialization for both farmers and producers as well as the stakeholders in the agrosector. Of recent, farmers in developing countries use inorganic fertilizers for the boosting of soil quality for a better crop produce. However, excessive usage of it can result to spur acidification of the soil and can project high risk to food security, the atmosphere, and groundwater via sustainable farming. In a review, Shina [22] looked at the prospects and potential of biofertilizer as a substitute for inorganic fertilizers. The authors stated that biofertilizers are renewable, eco-friendly, and cheap. That biofertilizers can improve nutrient disposal to soil and crops as well improving crop yields and the health of plants. Biofertilizers do not have issues with alkalinity and salinity that results to soil erosion or leaching, because of their organic nature. The authors, in conclusion, stated that biofertilizer will assist in the management and conservation of plants, animals, and soil quality because they tend to bring stability in a long by providing nutrients and preventing ground water pollution and erosion. The growth of human population has risen geometrically in recent times, and this will toll hard on the food security and safety. Thence, to

566  Biofertilizers further strengthen these gaps, Mahanty et al. [23] looked at the prospective sustainable potential of utilizing biofertilizers in plants growth and development. The authors recounted the widely use commercial chemical fertilizers in modern farm activities to boost crop production. The ecosystem damages as well as the health risk it portend along the food chain were highlighted. However, they stated that biofertilizer exploitation has been recognized as one of the best alternatives to conventional fertilizers based on their safety in usage and probable enhancement of plant growth. Microorganisms like cyanobacteria, fungi, and bacteria have been known to promote growth and development of plants. Hence, their capacity in providing or stimulating nutrient in the soil for plant survival is extensive agricultural outcome. The evaluation of the ecology and bioremediation techniques for a sustainable agriculture was discussed. Girigiri et al. [24] tested and evaluated the potential remediation of crude oil pollutants suing nitrogen-fixing bacteria and phosphatesolubilizing bacteria. Five different polluted soils with crude oil of various designed [A, B, C, D, and E (500 g PS + 50 g NFB, 500 g PS + 50 g PSB, 500 g PS + 50 g NFB + PSB, 500 g PS + 50 g NPK, and 500 g PS only: control)] were used in this study. Total petroleum hydrocarbons (TPHs) were investigated for 4 weeks as well as the toxicity of the organic fertilizers. The results of the ecotoxicology evaluation showed that the biofertilizers were in the proportion of 100:0, 75:25, 50:50, 25:75, and 0:100, correspondingly. The nitrogen-fixing bacteria were identified to be Rhizobium sp. and Azotobacter sp. However, the phosphate-solubilizing bacteria were identified as Bacillus and Pseudomonas, respectively, utilizing the 16S protein gene sequence. The evaluation of the TPHs showed various variations as 97.8, 97.5, 94.3, 92.1, and 34.6, correspondingly, in nitrogen-fixing bacteria, phosphate-solubilizing bacteria together with nitrogen-fixing bacteria, phosphate-solubilizing bacteria, NPK treatments, and the control. The findings from the study showed that there was significant difference at P < 0.05 between the designed outfits as when compared to the control. The result of the testing evaluation showed that the concentration (25.75) had the best support for the growth and development of plant. In conclusion, the authors recommended biofertilizer as an efficient remediating tool for crude oil decontamination as well for the enhancement of soil fertility. In agricultural ecology, microorganisms have been known to play immense role in the ecosystem structure and function. The entire microbial consortia improve the bioavailability soil and crop soil nutrients as well as the efficiencies. The use of chemical fertilizers in the improvement of crops in farms have too an extent aid soil quality and soil function. But persistence usage has, in recent time, been discovered to upset negatively the ecosystem structure and

Biofertilizer Utilization in Africa  567 functions. In the course of this, Otaiku et al. [25] did a review of the effects of biofertilizers on agricultural soil and Manihot esculenta Crantz. The authors recounted that benefits of the utilization of biofertilizers like elicit nutrients competence in soil and crops improve plant suitability and aid in the traceability of issues of inorganics impacts of agricultural inputs. They stated that strains of fungi and bacteria, Clostridium spp., Bacillus spp., Aspergillus spp., and Mycorrhizal sp., aid in the enhancement of PSB in the soil and the mutualistic association of soil-plant nutrients rations, which has been a major challenge in terms of mineralization and biomass for Manihot esculenta plant. The authors concluded that this will aid the management of climatic susceptibilities and carbon impounding, Suliasih and Widawati [26] tested and evaluated the influence of conjugates of inorganic and organic fertilizers with biofertilizers in the boost of the growth of soil beneficial microorganisms and Caesalpinia pulcherrima plant. The experiment was performed in a greenhouse setting with different media; biofertilizers vs. Rhizobium sp. (combination of five segregates), Azospirillum sp. (combination of five segregates), and Rhizobium sp. (combination of five segregates) were used with effluent/POME sourced from palm oil or Muller solution (inorganic fertilizers). These media were arranged in triplicates using a randomized complete block design. The outcome of the experiment showed that Caesalpinia pulcherrima, which were put in the media with biofertilizer (Rhizobium, Azospirillum, and Azotobacter), revealed better growth when compared to the control treatment. Caesalpinia pulcherrima treated with an amalgamation of biofertilizer with Muller solution and POME had well-developed growth than the plants inoculated with inorganic and organic fertilizers, correspondingly. Findings from their study indicated that the use of biofertilizer had more impact than organic fertilizers. There were significant development and growth between Muller solution and the POME conjugates with the biofertilizer. The combination of organic fertilizers with the biofertilizers produced better bacterial consortia than the chemical (inorganic) fertilizer. In conclusion, the author recommended the utilization of biofertilizers in the management and improvement of crops and soil nutrient deficiencies. In a review, Okur [27] looked at the beneficial effect of microbes when used as biofertilizers. The authors recounted that biofertilizers are prepared latent strains of microbial cells that aid plant nutrient uptake and growth, as well as other ecological and biological interaction therein the plant and soil. In addition, biofertilizers stimulate some bacterial processes in the topmost and inside the soils, so that nutrients’ availability and assimilation are maintained. The author in conclusion stated that the use of biofertilizers is very effective in the management of crop disease, ecological stress, and soil

568  Biofertilizers nutrient deficiencies because it has important combined renewable nutrient that can supplement the inorganic ones and cheap to purchase. Different approaches have been used in the management of soil deficiencies and ecological stress in farming activities. Previously, inorganic-based fertilizers have been employed in soil management but the aftermath impact was discovered to be deleterious to the plants as ecosystem. In the light of this, in a review, Sahoo and Narendra [28] looked at the major role of biofertilizers in sustainable farming in the improvement of crop yield, ecological stress, and soil fruitfulness. The authors stated that biofertilizers are recommended as a first class fertilizer because of the eco-sustainability and food safety role they play in the farming sector. Cyanobacteria, ecto- and endomyycorrhizal, and PGPRs (plant growth promoting rhizobacteria), as well as many beneficial microbes aid in the improvement of plant toward tolerance toward ecological stress like biotic and abiotic factors, nutrient availability, bioavailability, and uptake. This study also looked at the influence of biofertilizers in the mediation between protection and defense of diseases and pests, nutrient profile, crop productivity and growth, and cellular response and pathways in the enhancement of crop. The authors in conclusion opined that through the understanding of the physiology of biofertilizers, it will boost the sustainable farming in Africa as well as decrease the impacts of inorganic fertilizers. The utilization of biofertilizers in African continents is very low as compared to other continents. The general possibility of this is based on non-awareness of the possible usefulness and application in the agricultural sector, absence of regulatory bodies to supervise impact in the environment when used, and expertise in the field of agricultural microbiology. Based on this, Masso et al. [29] did a review of the sustainable application of biofertilizers for agriculture purposes in Sub-Saharan Africa. The authors stated that acceptance of high value biofertilizers in the agricultural section in Africa will aid crop yield, growth and development and nutrient uptake. More so, they will also elicit nutrient availability, bioavailability, and uptake and combat ecological stress factors. Some benefits in the application of the biofertilizers were discussed such cost effective, eco-friendly, and non-toxic nature. In conclusion, the authors stressed that rapid awareness should be emphasized for the need to utilize this technology for a sustainable food economy in Sub-Saharan African. Abdullahi et al. [30] tested and evaluated the conglomerate influence of poultry manure and biofertilizer on the consortia of microorganism, nutrient uptake, and growth and development on Sesamumindicum L. (sesame plant). The authors utilized the following microbes: Glomusmossea, Arbuscular mycorrhiza fungi, and Azospirillum with PM (poultry manure) for the experiment on sesame under a field setting. Four treatments with corresponding

Biofertilizer Utilization in Africa  569 control: Azospirillum + Arbuscular mycorrhiza as the first treatment, Azospirillum + Arbuscular mycorrhiza + 5 ton ha−1 poultry manure as the second treatment, 10 ton ha−1 poultry manure as the third as the first treatment, and the last treatment serves as control, were used for the designed experiment. Several growth parameters of plants such as biomass of the roots, shoots, branches, leaves, as well as their height and numbers were ascertain in this study. The results from the biological controlled experiment showed that the conjugates, PM and the biofertilizer (5 ton/ha), significantly improve the uptake of potassium, phosphorus, and nitrogen of the experimental plant as well as the parameters of growth by the Arbuscular mycorrhiza fungi in both the non-inoculated and inoculated plants. The percentage of colonization ranges from 6% to 62.8%. The result obtained indicated that there was a substantial difference at p < 0.05 in the percentage colonization of the inoculated plant roots and the spores of the fungi increased in population in all the experiment treatments groups. The highest inhabitants (28.56 × 106 CFU/g soil) of the Azospirillum and the soil population (69.3 AM spores/g) worth were identified to be more than the treatment group. Findings from their study showed that PM and biofertilizers (bioorganic) produced maximum nutrient uptake and growth in the soil containing Arbuscular mycorrhizal and Azospirillum inhabitants when compared to the selective application of PM and biofertilizer at 10 ton/ha. In conclusion, the authors recommended the amalgamation of PM and biofertilizer for a sustainable farming of sesame. In a review, Berruti et al. [31] looked at the success and benefits of utilizing AMF (Arbuscular mycorrhizal fungi) for biofertilizer. The authors stated that AMF are biotrophs which are made up of consortia of obligate microorganisms in the root of higher plants, about 80% colonization in plants. That they are potentially used as biofertilizers because of the role they play in photosynthesis, protection of plants against parasite and diseases, nutrient bioavialability, and high water retention in plants. AMF serve as a perfect alternative to convention fertilizers. The application transcriptomics and genomics have led to a giant breakthrough in recent advances in biotechnology where the genome of the AMF has been altered to adapt to green house, laboratory, and field application. The authors in conclusion proposed that experiments on AMF biofertilizer should focus more on vital factors that may improve or militate against the process of inoculation. In a review, Herrmann and Lesueur [32] looked at the challenges of the quality and formulation of biofertilizers. The authors stated that the rising demand of biofertilizers in the world shows that they are efficiency and counterproductive. However, they lamented so that many adulterated forms have been found in large-scale market of recent which made farmers to lose confidence in their value. In view of this, the way forward is via

570  Biofertilizers origination of the inoculant with various engineered strains of microbes that can withstand adverse environmental conditions and boost the nutrient uptake of nutrients. The authors also looked at the various components in the formulation of good worth of biofertilizers. Various techniques were also highlighted. The quality of novel biofertilizers was also reviewed. In conclusion, the authors proposed that major problems militating against the novel formulation should be looked on to. The consistent tilling of the soil for agricultural purpose has reduced the quality of farm produces. The ecological and health impacts of the use of conventional fertilizers are not devoid from it also. All these factors or impacts might impede the general productivity and wellness of plant. However, in a review, Shina [22] looked at the limitations and benefits of biofertilizers in farm practice. The author stated that there is the need to boost the quality of farm produce via the biofertilizers because of the economic and ecological benefits they portend to farmers and producer. Moreover, they can boost the safety of food as well as the supply in the food value chain to the end consumers. In all, they stated that biofertilizers are cheap, eco-friendly, able to tolerate adverse biotic and abiotic conditions, and non-toxic. In conclusion, the authors recommend this novel biotechnology for the boosting of sustainable agriculture.

19.3 Conclusion and Future Recommendations This chapter has provided detailed information on the application of biofertilizer for the improvement in the level of soil fertility toward enhancement or increase in crop production. The current situation on the application of biofertilizer in Africa and their mechanism of actions were discussed. Different types of biofertilizer that have been introduced were also highlighted. Moreover, specific examples that were cited where biofertilizer has led to increase in agricultural production were discussed extensively. Therefore, there is a need to isolate and characterize numerous beneficial microorganisms that could serve as a biofertilizer which could lead to increase in food production. There is a need to utilize agricultural wastes for the mass production of these essential strains for effective production of biofertilizer.

References 1. Sleper, D.A. and J.M. Poehlman, Breeding Field Crops. 5th Edn., WileyBlackwell, New York, USA., pp. 424, 2006.

Biofertilizer Utilization in Africa  571 2. Bekunda, M.A., A. Bationo and H. Ssali, Soil Fertility Management in Africa: A Review of Selected Research Trials. In: Replenishing Soil Fertility in Africa, Buresh, R.J., P.A. Sanchez and F.G. Calhoun (Eds.). Soil Science Society of America, Madison, USA., pp: 63–79, 1997. 3. Sserunkuuma, D., J. Pender and E. Nkonya, Land management in Uganda: Characterization of problems and hypotheses about causes and strategies for improvement. Project on Policies for Improved Land Management in Uganda, International Food Policy Research Institute, Uganda, March, 2001. 4. Sanchez, P.A., Soil fertility and hunger in Africa. Science, 295: 2019–2020, 2002. 5. Tilman, D., J. Fargione, B. Wolff, C. D’Antonio and A. Dobson et al., Forecasting agriculturally driven global environmental change. Science, 292: 281–284, 2001. 6. Gyaneshwar, P., G.N. Kumar, L.J. Parekh and P.S. Poole, Role of soil microorganisms in improving P nutrition of plants. Plant Soil, 245: 83–93, 2002. 7. Boiffin, J., E. Malezieux and D. Picard, Cropping Systems for the Future. In: Crop Science: Progress and Prospects, Struik, P.C. J. Nosberger and H.H. Geiger (Eds.). CAB International, Oxford, UK., pp. 261–279, 2001. 8. Pretty, J., Agricultural sustainability: Concepts, principles and evidence. Philos. Trans. R. Soc. B, 363: 447–465, 2008. 9. Sharma, S.B., R.Z. Sayyed, M.H. Trivedi and T.A. Gobi, Phosphate solubilizing microbes: Sustainable approach for managing phosphorus deficiency in agricultural soils. SpringerPlus, Vol. 2. 10.1186/2193-1801-2-587, 2013. 10. Tran, Y.T., Response to and benefits of rhizobial inoculation of soybean in the south of Vietnam. Proceedings of the 4th International Crop Science Congress, September 26-October 1, 2004, Brisbane, Australia, 2004. 11. Ndakidemi, P.A., F.D. Dakora, E.M. Nkonya, D. Ringo and H. Mansoor, Yield and economic benefits of common bean (Phaseolus vulgaris) and soybean (Glycine max) inoculation in Northern Tanzania. Aust. J. Exp. Agric., 46: 571–578, 2006. 12. Sumathi, C.S., V. Balasubramanian, N. Ramesh and V.R. Kannan, Influence of biotic and abiotic features on Curcuma longa L. plantation under tropical condition. Middle-East J. Sci. Res., 3: 171–178, 2008. 13. Ceballos, I., M. Ruiz, C. Fernandez, R. Pena, A. Rodriguez and I.R. Sanders, The in vitro mass-produced model mycorrhizal fungus, Rhizophagus irregularis, significantly increases yields of the globally important food security crop cassava. PLoS One, Vol. 8. 10.1371/journal.pone.0070633, 2013. 14. Ahmad, F., S. Uddin, N. Ahmad and R. Islam, Phosphorus-microbes interaction on growth, yield and phosphorus-use efficiency of irrigated cotton. Arch. Agron. Soil Sci., 59: 341–351, 2013. 15. Babalola, O. O., and Glick, B. R. The use of microbial inoculants in African agriculture: current practice and future prospects. J. Food Agric. Environ. 10, 540–549, 2012.

572  Biofertilizers 16. Suyal, D. C., Soni, R., Sai, S., and. Goel, R. “Microbial inoculants as biofertilizer,” in Microbial Inoculants in Sustainable Agricultural Productivity, ed. D. P. Singh (New Delhi: Springer India), 311–318, 2016. 17. Adekunle Raimi, Rasheed Adeleke, Ashira Roopnarain, Soil fertility challenges and Biofertiliser as a viable alternative for increasing smallholder farmer crop productivity in sub-Saharan Africa. Cogent Food & Agriculture, 2017. 18. Masso C, Fredrick Baijukya, Peter Ebanyat, Sifi Bouaziz, John Wendt, Mateete Bekunda, and Bernard Vanlauwe. Dilemma of nitrogen management for future food security in sub-Saharan Africa – a review. Soil Research, 55, 425–434, Review, 2017. 19. Carrapiço F, Generosa Teixeira & m. Adélia Diniz. Azolla as a biofertiliser in Africa. a challenge for the future Revista de Ciências Agrárias, 23 (3–4): 120–138, 2000. 20. Itelima JU, Bang WJ, Onyimba IA, et al. A review: biofertilizer; a key player in enhancing soil fertility and crop productivity. J Microbiol Biotechnol Rep. 2(1):22–28, 2018. 21. Jain G. Biofertilizers - A way to organic agriculture. Journal of Pharmacognosy and Phytochemistry SP4: 49–52, 2019. 22. Shina S, Biofertilizer: Usage, Potential and Prospects as Alternative To Chemical Fertilizer in Nigeria, National Cereals Research Institute, Moor Plantation, Ibadan, Nigeria p. 1-6, 2018. 23. Mahanty T, Surajit Bhattacharjee, Madhurankhi Goswami, Purnita Bhattacharyya, Bannhi Das, Abhrajyoti Ghosh, Prosun Tribedi. Biofertilizers: a potential approach for sustainable agriculture development. Environ. Sci. Pollut. Res, 2016. 24. Girigiri B, Caroline Nchedu Ariole, Herbert Okechukwu Stanley. Bioremediation of Crude Oil Polluted Soil Using Biofertilizer from Nitrogen-fixing and Phosphate-solubilizing Bacteria. American Journal of Nanosciences. Vol. 5, No. 4, pp. 27–38, 2019. 25. Otaiku AA, Mmom PC2 and Ano AO3. Biofertilizer Impacts on Cassava (Manihot Esculenta Crantz) Cultivation: Improved Soil Health and Quality, Igbariam, Nigeria. World Journal of Agriculture and Soil Science, 2019. 26. Suliasih and Widawati, S. The Effect of Biofertilizer Combined with Organic or Inorganic Fertilizer on Growth of Caesalpinia pulcherrima and Bacterial Population in Soil IOP Conf. Series: Earth and Environmental Science 166 (2018) 012024, 2018. 27. Okur, N. A Review: Bio-Fertilizers- Power of Beneficial Microorganisms in Soils. Biomed J Sci &Tech Res 4(4)- 2018. BJSTR. MS.ID.001076. 28. Sahoo RK and Narendra T. Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity. Microbial Cell Factories, 2014, 13:66 http://www.microbialcellfactories.com/ content/13/1/66. 29. Masso, C, Juliet R. Awuor Ochieng1, 2 Bernard Vanlauwe1 Worldwide Contrast in Application of Bio-Fertilizers for Sustainable Agriculture: Lessons

Biofertilizer Utilization in Africa  573 for Sub-Saharan Africa. Journal of Biology, Agriculture and Healthcare www. iiste.org. Vol. 5, No.12, 34, 2015. 30. Abdullahi, R1., Sheriff, H. H2 and Lihan, S3. Combine effect of bio-­ fertilizer  and poultry manure on growth, nutrients uptake and microbial population associated with sesame (Sesamumindicum L) in North-eastern Nigeria. IOSR Journal Of Environmental Science, Toxicology And Food Technology (IOSR-JESTFT) Volume 5, Issue 5, PP 60–65, 2013. 31. Berruti A, Lumini E, Balestrini R and Bianciotto V. Arbuscular Mycorrhizal Fungi as Natural Biofertilizers: Let’s Benefit from Past Successes. Front. Microbiol. 6:1559, 2016. 32. Herrmann L and Lesueur D, Challenges of formulation and quality of biofertilizers for successful inoculation. Applied Microbiology and Biotechnology 97(20), 2013.

20 Biofertilizers: Prospects and Challenges for Future Tanushree Chakraborty and Nasim Akhtar* Department of Biotechnology, GITAM Institute of Technology, GITAM Deemed to be University, Gandhi Nagar Campus, Rushikonda, Visakhapatnam (A.P.), India

Abstract

Biofertilizer, the microbial inoculant is an eco-friendly alternative to chemical fertilizer, protects lithosphere, improves biosphere by protecting air, water, soil pollution, and eutrophication and enhances yields of agriculture produce. It helps to enrich the soil with macro- and micro-nutrient and also by releasing plant growth regulators. The main nutrients required by plants are nitrogen, phosphorus, and potassium. The microbial inoculants of biofertilizers enhance plant growth and yields by secreting siderophores, antibiotics, enzymes, antifungal, and antibacterial substances and/or by releasing the hormones and overcoming disease and stress. Biofertilizers are classified as N-fixing, phosphate solubilizing, phosphate mobilizing, potassium solubilizing, potassium mobilizing, and sulfur oxidizing. The first commercial outbreak of biofertilizer is launch of “Nitragin” in 1895. For the preparation of biofertilizer, microbes need to be mass-cultivated, maintaining culture pH, temperature, and the cell count. To deliver and proper efficacy of biofertilizer, it requires a suitable carrier material. In future, focus should be made to create mutant and genetically engineered microbes as biofertilizer that has better response compared to wild type as a sustainable solution to agriculture. Keywords:  Biofertilizer, bateria, carrier material, cyanobacteria, mycorhiza, N-fixers, P-solubilizers

*Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (575–590) © 2021 Scrivener Publishing LLC

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576  Biofertilizers

20.1 Introduction Present-day scenario is escalation of population, which comes with demand of healthy foods. Food production becomes a challenge because of problems like degradation of productive land, expensive throughput, shortage of agricultural necessities, variability in climate, and loss of soil exuberance [1]. Healthy food demand creates stress on agriculture and its sustainability. For sustainable growth of agriculture, “green revolution” plays a very important role. It focuses on use of eco-friendly techniques to reduce the loads of harmful artifacts on environment. This “green technology” concept made researchers to find a new way for continuous use of environment without degrading its stability. One of the solutions of this problem to maintain sustainability is the use of eco-friendly and biodegradable technology in agriculture in tandem with nature as an alternative for chemical fertilizers [2]. Soil an important component of lithosphere which supports life directly and indirectly. It is differentiated into three layers, viz., top layer, subsoil layer, and parent layer. Top layer supports the growth of plants because it is the layer that contains micro- and macro-inorganic nutrients, organic matters, air, and water [3]. Each component of soil is present in a proportion so as to make it fertile. Soil also contains microorganisms that help to make it fertile and supply the available nutrients which are unusable by plants directly. It is top layer of the soil which is involved in the nutrient cycling by the presence of microbes [4]. Nitrogen is an important macronutrient of plants. It helps in DNA and RNA formation, formation of heme of chlorophyll, cytokines, as osmoregulation, etc. Plants obtain nitrogen in the form of nitrates, ammonium, and urea [5]. It enters soil through biological nitrogen fixation, thundering, organic matter decomposition, and artificially by fertilizers [6]. Phosphorus is also a macronutrient required in large amount by plants. It provides many functions like it helps in legume growth, nodule number and mass, it is part of membrane in the form of phospholipids, and helps in the crop yield and quality. Phosphorus is also found to be an essential element of nuts as well as fruits [7]. The form of phosphorus which is usable by the plants is phosphates. Plants get phosphorus through fertilizers. Potassium is a macronutrient required by plants in large amount after nitrogen. It has many functions like it helps in water uptake, growth of root, turgor maintenance, and stomatal and transpiration regulation and thereby helps in yield and growth of plants [8]. It also helps in enzyme catalysis, production, and aggregation of some vitamins like Riboflavin

Biofertilizers: Prospects and Challenges  577 and Thiamine. Potassium is very useful for proper working of guard cells, to conduct the process of photosynthesis and synthesis of protein, and to improve fruit quality. Potassium also helps to provide resistivity against bacterial and fungal diseases [5, 6]. Plants consume potassium as ions which enter soil naturally by decaying organic matter and manually by fertilizers [9]. For the sustained development and output of producers, nutrients are very essential required in macro- and micro-quantities by the plants. The high yielding varieties developed as part of “Green Revolution” demands continued nutrient supply, irrigation water, insecticide, and pesticides. The monoculture practice for the continued cultivation of these cultivars year after year resulted in nutrient deficiency in the soil and accumulated salts due to over irrigation, leaching, and contamination of underground water and atmosphere as well as acidification of soil. Due to excessive and extended use of synthetic fertilizers, plants’ root becomes weak and prone to various diseases [10]. To solve these problems, an environment-friendly alternative is an immediate need to sustain the agricultural productivity. This atmosphere-friendly substitute is a biofertilizer which is developed by using favorable microorganisms [11]. Agriculture is one of the main sources of earnings in the whole world. It has been estimated that ⅓ of workers are employed in the field of agriculture [9] for the production of more and more food grains to feed growing population of the world. The “Green Revolution” of the last century is highly dependent over the use of synthetic fertilizers, irrigation, insecticide, and pesticides [12]. This has demonstrated significant improvement in the food grain production but finally resulted recession. The main drawback of green revolution is the reduction of crop production in developing countries due to soil infertility [8]. It is mainly caused by fertilizers which are used extensively to increase crop yield for the necessity of ever growing populations. Chemical fertilizers have many harmful effects on environment like soil infertility, loss of genetic diversity, emission of gases like nitrous oxide and leaching of chemicals [10]. Additionally, industrial effluents such as heavy metal contamination and petrochemical discharge are also a major threat to the soil fertility. Hence, there is a need for an alternative solution for these problems [11]. These chemical fertilizers are very harmful for biotic and abiotic components of biosphere. It contaminates the abiotic supports system as well as directly or indirectly threats to human and other life forms. Its continuous use disturbs the whole ecosystem, making it unfavorable for survival of life forms. Some of its effects discussed by [13] are as follows:

578  Biofertilizers • Methamoglobinaemia is a disease which leads to blue coloration of skin and damage to respiratory and vascular system, and cancer is caused by taking vegetables that are grown in NO3 rich soil. • Excessive use of single chemical fertilizer can lead to deficiency of other important compounds, for example, excessive K treatment leads to deficiency of ascorbic acid and carotene of plants. • Proteins of the crops get diminished when crops are grown with NO3 fertilizer. • Lack of protein in human diet leads to malnutrition. The demand of growing population forced farmers to cultivate more and more crops from same section of land every year. To meet the needs of ever increasing population, farmers depend on chemical fertilizers which resulted in eutrophication, soil acidification, and soil barrenness and also weaken the plant roots system [10]. This has augmented the shifting of stereotyped conventional farming toward the eco-friendly organic agriculture with the use of soil microbe and organic fractions as biofertilizers [12, 14]. Being nutrient-rich, these biofertilizers offer many advantages over chemical fertilizers; these are eco-friendly, cost effective, secure food safety, which helps to increase biodiversity of soil [8]. Various microbes are in practice nowadays that are used as an alternative for chemical fertilizers [2]. Use of microbes as an alternative to chemical fertilizer enhances the growth of plants by various ways as by solubilizing and mobilizing phosphates, zinc, and potassium, by fixing nitrogen, or by releasing plant growth regulators. Biofertilizers also help plants to survive under stressful environment [15, 16]. These microbes are popularly known as plant growth-promoting bacteria (PGPB) due to their growth-promoting potentials and are in practical use since 1950s. However, some of its limitations including vitality, efficiency, adaptability, and inconsistency have hampered its large-scale commercial application. Since then, many approaches were applied to improve and overcome these constraints [17]. Apart from chemical fertilizers, industrial effluents are also a major threat to the soil, because it includes heavy metal contamination and petrochemical discharge, which can also be solved by the use of biofertilizers. The organisms used in biofertilizer have the potential to overcome metal stress by reverting there ill-effect [11]. These microbes enrich the soil with macro- and micro-nutrients through fixing of nitrogen, solubilization of phosphate, release of bound potassium, secretion of plant growth-promoting substances, antibiotic production,

Biofertilizers: Prospects and Challenges  579 and mineralization of inorganic and organic matter of soil [4, 8]. The use of biofertilizer is a long-lasting, environmental-friendly alternative which does not reduce the fertility by reducing soil acidification, imparts more flavor, taste, and aroma in the product, and protects from eutrophication [18]. It helps to control plant diseases, for example, pythium-induced root rot, rhizoctonia-induced root rot, parasitic nematode, and chilli fusarium wilt [19]. It helps in binding the particles of soil together to prevent loss of soil due to erosion and desertification and increases the water holding capacity [20].

20.2 Definition Biofertilizers are defined as substances that are microbial inoculants that help in the growth of plants and maintain the sustainability of environment [21, 22]. Biofertilizers consist of biologically active microorganisms that are bacterial and fungal strains, which help to increase, add, conserve, and convert the nutrients from unusable form to usable form [23]. It helps to increase biodiversity by adding useful bacteria and fungi to the soil [24]. Biofertilizers include microorganisms such as symbiotically associated arbuscular mycorrhiza fungi (AMF), nitrogen-fixing organism, phosphate-­ solubilizing organisms, and decomposers of soil organic stock [23]. They enhance yields by secreting substances to promote plant growth and development and release antibiotics for protection from harmful microbes [4]. It participates in the nutrient cycle and maintained sustainable and healthy environment; hence, biofertilizer forms the backbone of integrated nutrient management [25]. As crop survival and grain production depend on the environmental conditions like biotic and abiotic factor, biofertilizers are proved to improve the response of plants in stress conditions [26].

20.2.1 Helper Bacteria There are some bacteria that do not fit into any category of biofertilizer but can be considered as a new group of biofertilizers. These bacteria improve plant growth by enhancing the plant-microbe interaction by themselves behaving as third party [27]. For example, rhizospheric actinomycetes which have been isolated from legumes as well as actinorhizal nitrogenfixing nodules stimulate nodulation and thereby help in nitrogen fixation finally resulting to plant growth. Such type of tripartite association is still not well understood but clearly demonstrated that such type of association can improve biofertilizer efficiency [28].

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20.2.2 The Point of Difference • Organic fertilizer is the product of plant or animal origin like animal manure and green manure. • Biofertilizer is the microbial inoculants that help to increase soil fertility and crop productivity [29]. • Biopesticides are the microbes that protect plant by eliminating harmful effects of pest insects [13]. • Bioenhancers are microbial supplements that accelerate growth of plant by releasing hormones [30].

20.3 Advances in Biofertilizer The fertility and productivity of soil is restored by biofertilizers due to the presence of nitrogen-fixing microbial inoculants. Rhizobium found in root nodule of leguminous plant is in use as biofertilizer since long time. Apart from it, Azotobacter is also used for growth promotion. Azospirillum is another potential microbe to be used as biofertilizer. Other organisms are blue-green algae which are in use on regular basis as biofertilizer. Compost production is an example that shows that biofertilizers are efficient and environment-friendly because it increases the yield of crops by enhancing the soil minerals through the decomposition of organic residues and agricultural byproducts [8, 31]. The first commercial outbreak of biofertilizer started in 1895 with the launch of “Nitragin” by Nobbe and Hilther. The blue-green algae and Azotobacter were further applied as biofertilizer [32]. The use of Azospirillum as biofertilizer is the recent phenomenon. While the Vesicular-Arbuscular Micorrhizal (VAM) roots are the new addition with respect to biofertilizer. Commercial production of Rhizobium was first started in 1956 [13]. Bradyrhizobium as inoculants in leguminous plants was first used in Malaysia. Halim [31] at University Putra Malaysia (UPM) studied the potential of Mycorrhiza and contribution of nitrogen from Azospirillum in oil palm seedling [31]. Since then, various researches were done, and different microorganisms are proposed to be use as the potent biofertilizer. Many Rhizobium species are used as nitrogen fixers in biofertilizers along with that Azotobacter chroococcum. A few nitrogen-­ fixing Cyanobacteria are also potent biofertilizers applied in agriculture field. Many microorganisms function to enrich phosphorus; one such example is Bacillus megaterium. Fungus such as Aspergillus fumigates can also fix phosphorus. The plant growth-promoting Rhizobacteria (PGPR) is also shown to solubilize phosphorus when applied along with vesicular

Biofertilizers: Prospects and Challenges  581 arbuscular Mycorrhiza (VAM) fungi in biofertilizers. Similarly, potassium is being made available to plants by Bacillus mucilaginous used in the biofertilizer [31].

20.4 Preparation of Biofertilizer The enhancement of growth and productivity of crop plants by the application biofertilizers is attributed to the microorganism present in it through the increased supply of nutrient through decomposition of organic matter, fixation of atmospheric dinitrogen, mineralization of salts, and solubilization of bound phosphorus [23]. These specific and selected strains of microorganisms are cultured in the laboratory at large scale and added with the suitable carrier for the preparation of biofertilizers [33]. Biofertilizer is also used in liquid form in combination of microorganisms and its nutrients which acts as protectant for cell or for formation of cysts and resting spores to resist or tolerate adverse environmental conditions [13]. The modern biofertilizer contains microorganisms that are beneficial to the plants and also keep the environment sustainable along with the organic manures [20]. Preparation and use of biofertilizers is eco-friendly and also costeffective with natural and innate microbes of plant and soil [8]. The microbes of biofertilizers induce plants for the production of growth hormones like auxin, cytokinin, gibberellic acid, and ethylene in involved in the general and reproductive growth with enhanced productivity. The rhizobacteria with profound plant growth-promoting (PGPR) activity has special attention in this regard [21, 22]. Many of them are directly involved fixing of nitrogen, mobilization, and solubilization phosphate, potassium, sulfur, and iron. They help plant growth indirectly by removing the pathogens by secreting siderophores, antibiotics, enzymes, or fungicides [34–38]. The growth of plants and its productivity by the microbes of biofertilizers occurs in any one of the three ways: i. indirectly by suppressing the plant disease serves as bioprotectant, ii. promotes plant growth by improving the nutrient absorption function as biofertilizers, and iii. increases plant growth by secreting phytohormones called as biostimulants [39].

20.5 The Carrier Materials Carrier materials are the substances that are used to carry biofertilizer for field application. These are added to escalate the efficacy of biofertilizers.

582  Biofertilizers A good carrier material should have the properties such as cheap, easily available, easy sterilizable, easy to process, free of sticky substances, nontoxic to microbial growth, possesses good moisture absorbing capacity [40], have high water retention capacity, contains adhesion capacity to seeds, with efficient soil pH buffering activity, and very high in organic matter content [41]. Carrier materials offer some advantages in providing the smooth handling, long-term repository and improved efficacy to the biofertilizers. Many substances are in use as carrier materials such as saw dust, talcum dust, manure, and earthworm cast [8]. Carrier materials should be sterilized to make the inoculants free of contamination. Most suitable method used for sterilization of carrier materials is gamma radiation without any change in its properties (physical and chemical) as biofertilizer. Autoclaving is another effective sterilization method but it will have deleterious effects on the properties of carrier material with reduced potential as biofertilizer [33].

20.6 Production System of Biofertilizer To formulate biofertilizer, many things need to be considered like microbes characteristics, methods of application, and storage. There are six steps in production of biofertilizer. These include i. selection of active microbe, ii.  isolation and choosing of target microorganism, iii. choice of propagation technique, iv. selection of carrier substances, v. phenotypic testing, and vi. wide-ranging tests [8]. In biofertilizer preparation, the first thing is the choice and types of microorganism to be used as single organism or combinations of inoculants. The next step is to culture the chosen microorganism in the laboratory. Then, the carrier material has to be chosen. The carrier material is selected according to the need of the biofertilizer like if the need is powder form of biofertilizer, then carrier material to be used as apioca flour or peat. The next step is the selection of propagation method. Propagation method depends on the growth parameters of microorganism. Last but not the least, biofertilizer is tested under various environmental conditions to check its potentiality and constrains [8]. For applying to the soil, 500–800 g of biofertilizer is amalgamated with 10–15 kg of farmyard manure. The content is mixed thoroughly stored properly before adding to the soil as effective biofertilizer for plants. This step is important because biofertilizer requires organic manure for their growth and activity [13].

Biofertilizers: Prospects and Challenges  583

20.7 Mechanism of Growth-Promoting Activity of Biofertilizers Different microorganisms have different mechanisms to promote plant growth. For example, Azospirillum increase plant growth by secreting auxins, gibberellins, and ethylene. It also produces some antibiotics that help in control of plant diseases. Along with these, it also produces vitamin B complex [13]. Some bacteria induce phytohormone synthesis like Paenibacillus polymyxa  that induces IAA production in roots of lodge pole pine [42]. Rhizobium and Bacillus also synthesize IAA complex [13]. Phototropic prokaryotic bacteria produce IAA and gibberllic acid. It also adds vitamin B12 to the soil, thereby making soil more efficient with respect to aeration and water binding capacity complex [13]. Azospirillum brasilence Sp245 enhances root growth of tomato by IAA through nitric oxide synthesis. Nitric oxide function is an intermediate in the production of IAA that helps in root growth [27]. Whatever mechanism biofertilizer uses, it should reach and enter rhizosphere demonstrate its action on plants through the interaction of roots [43]. The growth-promoting characteristics of biofertilizer are viewed on two modes of action based on biochemical and genetic makeup of the microorganism used [44], and their action of [23]. The two modes of action of biofertilizer are described as follows: i. direct: when biofertilizer supplies nutrients or growth promoters to enhance the plant growth; and ii. indirect: when biofertilizer inhibits or eliminates the harmful situation of plant growth [45]. Biofertilizers along with increasing growth parameters also help plants to tolerate various stress situations. To fight with salt stress Rhizobium trifoli is a good candidate if it is coninoculated with Trifolium alexandrium. This combination of microbe also showed satisfactory biomass nodule number [46]. To cope with various biotic as well as abiotic stress, Pseudomonas aeruginosa also showed satisfactory results [46]. Cotton plant showed increased rate of germination with the help of Pseudomonas putida RS-198. It also helped to increase the growth parameters of cotton like height and weight. It enhances these properties under high-salt situation by enhancing the intake of ions like Ca2+, Mg2+, and K+ and also by decelerating consumption of Na+ [9]. There are some strains of Pseudomonas that help to increase the tolerant capacity of plants via 2,4-diacetylphloroglucinol. To cope with extreme temperature and salinity, Mycobacterium phlei also showed convenient results [46]. AMF can also provide presentable plant growth in case of high-salt environment [42]. There are other microbes

584  Biofertilizers like Piriformospora indica that are useful biofertilizer for salinity environment [42]. Combination of fungi like arbuscular fungi and bacteria that are nitrogen fixers can help legume crops to withstand water scarcity condition. For the growth of seedling and for the germination of seeds in drought condition, Pseudomonas  Spp. was showed to be found satisfactory [47]. Arbuscular mycorrhiza can give positive result with respect to increase of potentiality of photosynthesis and anti-oxidative property [48]. Infection of Fusarium can be controlled by treatment of banana plant with Bacillus subtillis N11 when added with compost [9]. Azospirillum spp. produces a compound called cadaverine that help rice seedlings to survive osmotic stress [49].

20.8 Advantages and Limitations Biofertilizers have great potential in maintaining the sustainability of the environment by reducing contamination of air, water, and soil caused by chemical fertilizer. It increases microbial biodiversity and enhances the fertility of soil, helps plant to survive in various stress conditions, and manages disease in plants [40]. However, certain limitations of biofertilizers hindered its wide acceptance and application at the commercial scale. The supply of lower-nutrient content compared to chemical fertilizer, variation, or deviation of its efficacy on storage and external condition of light, temperature, and humidity [50], unsuitable carrier materials, inappropriate microbial strains, lack of quality assurance, unskilled and inefficient staffs [13], low volume production, and faulty inoculation techniques [51], mutations of the strains and market availability are the major limitations in the industrial scale production and commercialization of biofertilizers.

20.9 Future Aspects Being eco-friendly in nature, biofertilizers are the best alternative and solution to the many current problems of modern production system and can proved to be a boon for the sustainability of agriculture. Despite some of the limitations related to its production, usage, applications, and commercialization, it has created a new era in the field grain production with a hope of a second green revolution. AMF, which is present in 90% of symbiotic association with plants, are still not culturable in laboratory conditions. As this fungus is very important with respect to soil conservation agricultural management, so, there is a need for extensive research and development

Biofertilizers: Prospects and Challenges  585 devoted to discover a suitable culturable technique for its growth in laboratory. A little success in this direction with some possibility of its culturing in carrot root organ can prove to be very good beginning [52]. The potential of many other microorganisms are yet not explored to be used as biofertilizers. So, in coming future, the search of such other useful microorganisms will always be in demand when the aim is sustainability of agriculture. Some limitations are there with the use of biofertilizer that it gets affected by environmental conditions and native microbes of the soil. So, the focus should be diverted to overcome these limitations in future to get maximum response of biofertilizer in all environmental conditions and soil type. Azospirillum that helps to enhance plant growth by N fixation was later found that it enhances plant growth by demonstrating its effect on root development and architecture. Later, it was showed that glutamine synthetase (GS) mutants of Azospirillum give more growth when compared to parental type [53]. In the similar way, many more mutants can be generated, tested, and compared with parental types in future to get better results than wild-type microbes. Exploring and devoting focus to find more tripartite associations to enhance the competency of biofertilizers. Some enzymes involved directly in the plant growth and developments are not found in all native microorganisms. For example, 1-aminocyclopropane-­ 1-carboxylate (ACC) deaminase involved in the elimination of ethylene in turn promotes plant growth and productivity. The gene for this enzyme was identified and isolated from Pseudomonas putida and inserted into other bacteria to make them PGPB [54]. Therefore, we should hunt for such genes and apply biotechnology tools to form genetically engineered plant growth-promoting microbes in near future. This will definitely provide a new path toward imperishable agricultural growth.

20.10 Conclusion As the population is increasing, therefore to meet the population demand, fertilizers also come into play. The demand of fertilizers after the “Green Revolution” has escalated the food grain production due to the application of chemical of fertilizer. To increase crop production and yield, chemical fertilizers play an important role, resulting in tremendously increases in its demand. But as time passes, it was found that chemical fertilizers have many disadvantages including the pollution of air, water, soil, plant disease infestations, and emergence of new pests, finally reducing the productivity of the same piece of cultivable lands. These factors have become a challenge for the sustainability of agriculture for future. These problems have

586  Biofertilizers created the necessity of developing biofertilizer as a possible alternative solution for the sustained agriculture production. Since, biofertilizers are microbial inoculants that offer many advantages without such drawbacks. These microorganisms help roots to utilize compounds of soil effectively which are normally unusable by the plants. These microbes in biofertilizers releases hormone in the soil that helps in the growth of plants. Hence, biofertilizer is a potential tool that utilizes renewable inputs to increase fertility of soil with microorganisms that add organic nutrient to the soil and support agriculture production. Being bio-inoculants, the response of biofertilizer is also dependent on environmental conditions. The type of soil, nature of soil, native microbial content, composition of soil, etc., affect the working of biofertilizers. Apart from external and internal factors, the in-depth study of soil microbial ecology and its dynamics are essential to improve biofertilizer technology. A single microbial strain applies a different mechanism to enhance plant growth and yield whose dynamic changes when used in combinations with different strain. Therefore, it becomes very difficult to analyze the exact mechanism of biofertilizer that stimulates plant growth and over all yields. The knowledge of genetic, molecular biology, and application of biotechnological tools can help to design much better biofertilizer for future. Developing new technologies for efficient production system with beneficial and modified microbes, better handing system, and enhanced storage shelf-life would certainly improve the wide acceptability and application of biofertilizers as a sustainable alternative to the current agriculture production system. Hence, there are enormous future prospects for new startup in the area of biofertilizer.

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Biofertilizers: Prospects and Challenges  587 5. Ifokwe, N.J., Studies on the production of biological fertilizer from domestic wastes and Azolla Pinata. Unpublished M.Sc. Thesis. Department of Plant Science and Technology. University of Jos, pp. 10–45, 1988. 6. Barak., Essential elements for plant’s growth, pp. 1–5, Nature publishers, 1999. 7. Scalenghe, R., Edwards, A.C., Barberis, E., Marsan, F.A., Agricultural soil under a continental temperature climate susceptible to episodic reducing conditions and increased leaching of phosphorus. Journal of Environmental Management; 97: 141–47, 2012. 8. Khosro, M. and Yousef, S., Bacterial bio-fertilizers for sustainable crop production: A review APRN. Journal of Agricultural and Biological Science; 7(5): 237–308, 2012. 9. Itelima, J.U.,  Bang, W.J.,  Sila, M.D.,  Onyimba, I.A.,  Egbere, O.J.,  A review: Biofertilizer - A key player in enhancing soil fertility and crop productivity; 2(1): 22–28, 2018. 10. Chun-Li, W., Shiuan-Yuh, C., Chiu-Chung, Y., Present situation and future perspective of bio-fertilizer for environmentally friendly agriculture. Annual Reports, pp. 1–5, 2014. 11. Pal, A.K., Mandal, S., Sengupta, C., Exploitation of IAA Producing PGPR on mustard (Brassica nigra L.) seedling growth under cadmium stress condition in comparison with exogenous IAA application. Plant Science Today; 6(1): 22–30. 2019. 12. Santos, V.B., Araujo, A.S.F., Leite, L.F.C., Nunes, L.A.P.L., Melo, W.J., Soil microbial biomass and organic matter fractions during transition from conventional to organic farming systems. Geodderma; 170: 227–31, 2012. 13. Mazid, A. and Khan, T. A., Future of biofertilizers in Indian agriculture: an overview. International Journal of Agriculture and Food Research: 3(3): 10–23, 2014. 14. Youssef, M.M.A. and Eissa, M.F.M., Biofertilizers and their role in management of plant parasitic nematodes: A review. Biotechnology Pharmaceutical Resources; 5(1): 1–6, 2014. 15. Li, Y., Shi, H., Zhang, H., Chen, S., Amelioration of drought effects in wheat and cucumber by the combined application of super absorbent polymer and potential biofertilizer. Peer J., 7: e6073, 2019. 16. Ojuederie, O.B., Olanrewaju, O.S., Babalola, O.O., Plant Growth Promoting Rhizobacterial Mitigation of Drought Stress in Crop Plants: Implications for Sustainable Agriculture. Agronomy; 9: 712, 2019. 17. Ji, S., Kim, J., Lee, C., Seo, H., Chun, S., Oh, J., Choi, E., Park, G., Enhancement of vitality and activity of a plant growthpromoting bacteria (pGpB) by atmospheric pressure non-thermal plasma. Scientific Reports; 9:1044, 2019. 18. Knobeloch, L., Salna, B., Hogan, A., et al. Blue babies and Nitrate contaminating well water. Journal of Science; 2(1): 6–24, 2009. 19. Mahimaraja, S., Dooraisamy, P., Lakshmanan, A, et al. Composting technology and organic waste utilization. Journal of Science; 1(3): 332–560, 2008.

588  Biofertilizers 20. Swathi, V., The use and benefits of bio-fertilizer and biochar on agricultural­ soils, unpublished B.Sc. thesis, Department of Chemical and Biological Engineering. Chalmers University of Technology Goteborg Sweden, pp. 20–24, 2010. 21. Sogut, S. and Cig, F., Determination Of The Effect Of Plant Growth Promoting Bacteria On Wheat (Triticum Aestivum L.) Development Under Salinity Stress Conditions. Applied Ecology and Environmental Research; 17(1): 1129–1141, 2019. 22. Gamez, R., Cardinale, M., Montes, M., Ramirez, S., Schnell, S., Rodriguez, F., Screening, plant growth promotion and root colonization pattern of two rhizobacteria (Pseudomonas fluorescens Ps006 and Bacillus amyloliquefaciens Bs006) on banana cv. Williams (Musa acuminata Colla). Microbiology Research; 220: 12–20, 2019. 23. Vessey, J.K., Plant growth promoting Rhizobacteria as bio-fertilizers. Journal of Plant and Soil; 225(43): 571–86, 2003. 24. Raja N. Bipesticides and biofertilizers: ecofriendly sources for sustainable agriculture. Journal of Biofertilizer Biopesticide; (3): 112–15, 2013. 25. Adesemoye, A.O and Kloepper, J.W., Plant-microbes interactions in enhanced fertilizer use efficiency. Applied Microbiology Biotechnology; 85: 1–12, 2009. 26. Yang, J.W., Kloeppe, J.W., Ryu, C.M., Rhizosphere bacteria help plants tolerate abiotic stress. Trends Plant Science: 14(1): 1–4, 2009. 27. Wall, L.W., Biofertilizers: Present and future use of transgenic microorganisms, Biosafety and the environmental uses of micro-organisms. Conference Proceedings OECD, pp. 23–34, 2015. 28. Solans, M., G, Vobis., Wall, L.G., Saprophytic actinomycetes promote nodulation in Medicago sativa – Sinorhizobiummeliloti symbiosis in the presence of high N. Journal of Plant Growth Regulations; 28: 106–114, 2009. 29. Vishal, K.D. and Abhishek, C., Isolation and characterization of Rhizobium leguminosarum from root nodules of Pisums sativum L. Journal of Academic and Industrial Research; 2(8): 464–67, 2014. 30. Khan, T.A., Mazid, M., Mohammad, F., Sulphur management: An agronomic and transgenic approach. Journal of Industrial Research & Technology; 1(2): 147–161, 2011. 31. Halim, A.N.B. (2009). Effects of using enhanced bio-fertilizer containing N-fixer bacteria on patchouli growth, Thesis faculty of Chemical and Natural Resources, Engineering University Malaysia Pahang, 145 pp., 2009. 32. Kribacho., Fertilizer ratios, krishak and bharati cooperative Ltd, Journal of Science, 5(8), pp. 7–12, 2010. 33. Hari, M. and Perumal, K., Booklet on Bio-fertilizer (phosphabacteria). Shri Annm Murugapa Chettiar Research Centre Taramani Chennai, pp. 1–6, 2010.

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21 Biofertilizers: Past, Present, and Future Mukta Sharma1* and Manoj Sharma2† 1

Department of Chemistry, Faculty of Engineering and Technology, Manav Rachna International Institute of Research & Studies, Faridabad, Haryana, India 2 Global Drug Safety Department, Win Medicare Pvt. Ltd., New Delhi, India

Abstract

The continuous upsurge in population across the world has complemented an increase in the demand for the food and other agriculture based produces. This has also resulted in the advancement of new agritechnologies which, in turn, has exponentially increased the usage of chemical fertilizers to produce high yield. Chemical fertilizers which mainly comprise of N (nitrogen), P (phosphorus), and K (potassium) are produced at a big scale in industrial setting. The chronic usage of chemical fertilizers is not only toxic to the soil condition but also, as they get assimilated into the food chain, these chemically derived fertilizers elicit deleterious effects on human health in addition to reducing the crop yield and making soil infertile. These issues have vigorously demanded the approach to sustainable farming. Sustainable farming can be achieved with the biofertilizer use as these preserve the soil environment through various mechanisms including nitrogen fixation, solubilization of potassium and phosphate, and degrading organic matter in the soil. Although biofertilizers seems to be a good option but these too possess detrimants. Prospectively, the main thrust is upon improvement in the biofertilizer performance and reduction in cost of production so that environmental problems can be solved in most efficient and economic way. For this, newer technologies, for example, coating, co-encapsulation, fermentation, lyophilization, and inoculation, are now into progression. In future, the development of fertilizers using chemical, organic, and microbial sources (biofertilizers) has a wide acceptance for eco-friendly and economically viable agriculture. The chapter highlights the development and recent advances in biological fertilizer and its performance.

*Corresponding author: [email protected] † Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (591–606) © 2021 Scrivener Publishing LLC

591

592  Biofertilizers Keywords:  Biofertilizer, chemical fertilizer, sustainable farming, nitrogen fixation, inoculation

21.1 Introduction Globally population is increasing at an alarming rate and so is the demand of food and agriculture products. The increase in demand has necessitated the need and dependency on the use of fertilizers that are chemical in nature as these are known to increase the productivity. Not only chemical fertilizers but also the pesticides exhibit a significant role in the production of food and its significant yield [1]. Chemical fertilizers are produced and manufactured in industrial setting. These primarily comprise of N (nitrogen), P (phosphorus), and K (potassium) in the defined cocentrations. Although, on one hand, the constant usage leads to high productivity and the increased yield; however, on the other hand, their use also pose severe threat to environmental problems including but not limiting to degradation in quality of soil, ground water and water at the surface, air pollution, diminished biodiversity, and a crushed ecosystem. It also causes mistreatment and misuse of restricted resources of phosphorus, groundwater pollution with nitrates, and damage to the aquatic ecosystems [2–4]. This has necessitated the efforts to be channelized and vigorously focused to produce high quality nutrient rich food items for achieving sustainable means to safeguard the eco system through adopting measures for bio-safety. This demand draws attention for fertilizers that are bio-organic (bio fertilizers) in nature as these provide an alternative to chemical based fertilizers (agro chemicals) [5]. These biofertilizers act because of bacteria that are beneficial to soil and promote the growth of plant. For sustainable farming, there is a high demand for such microorganisms as these facilitate safe production of crop and avert pollution to environment. These beneficial microorganisms are either commonly known as biofertilizer agents or biological control. Biofertilizers keep the soil environment enriched with all types of nutrients (micro and macro) by maintaining symbiotic relationship with plants. This is achieved via decomposition of organic matter, process of nitrogen fixation, mineralization or phosphate and potassium solubilization, release of substances that regulate plant growth, and antibiotic production [6]. The non-usable nutritional elements can be mobilized to usable form with the use of biofertilizers. During past two decades, the biofertilizers have gain more importance and have become one of the important aspect for integrated approach on nutrient management. When

Biofertilizers: Past, Present, and Future  593 fertilizers are applied, generally, 10% to 40% of the fertilizer is taken up by plant, whereas 60% to 90% is lost. In such situations, inoculants of microbes are more significant in their integrated approach to manage nutrients for sustainable production and safe environment. Biofertilizers significantly benefit crop productivity as they contribute in nutritional maintenance cycle of the crop or plant and reproduce when used as inoculant of soil or seed [7]. Currently, the use of microorganisms that are beneficial to the plants are used widely as their use enhance and maintain the fertility for a longer period and further facilitate the crop nutritional requirement [8]. The production of biofertilizers (microbial fertilizers) does not merely get influenced by factors like information on microorganisms and plant physiology but it also heavily depends upon technical aspects such as the process of fermentation, formulation type, type of microorganism population, and their release system. Owing to biofertilizer acceptability in the ecosystem and their significant contribution in the modern agriculture technology, it becomes evident that while developing a bioformulation that may be stable, a unified knowledge on technical and microbial aspects shall be applied [9]. Recently, extensive studies have been conducted on plant interaction with soil and other different type of microorganisms. Although such studies provide knowledge on the relationship between plant, soil, and type of microorganisms and further explore method for their use in agriculture [10–12], the factors such as type of technology, inoculate formulation, and the carrier for production of inoculate limit their wider use [13]. Hence, the utility of microorganisms in agriculture in the form of biological fertilizers, the features and characteristics of formulations, for example, encapsulated formulations, polymeric formulations, inorganic, organic, culture production, techniques of inoculation, bulk sterilization, and techniques of seed coating, need to be explored extensively [13, 14]. This chapter provides an overview on previous, current technological status, and future potential of biofertilizers in terms of new approaches in increasing nutrient profile, decreasing environmental stress and bacterial inoculations.

21.2 Biofertilizer: A Brief History Historically, in 1888, a Dutch scientist initially identified the biofertilizers. Thereafter, in 1895, the use of biofertilizer was initiated by Nobe and Hiltner with the introduction of Nitragin and with a laboratory culture of Rhizobia [15] followed by discovery of blue-green algae,

594  Biofertilizers Azotabacter, etc. Although the biofertilizers such as BGA (blue-green algae), Azospirillum, Azotobacter, and Rhizobium are in use since past many years, however, recently, biofertilizers such as Azorhizobium, Azotobactor, Vesicular Arbuscular Microrrizae, and Azospirillum have become more popular and have drawn much attention. There is also a history of microbial inoculums that has been applied in the crops and their knowledge has passed over generations. It all started with production of compost culture at small scale which provided an insight in the capabilities of biofertilizers [16]. During late 1940s, the production of microbial inoculants was started at industrial level in Malaysia which gradually got picked up in 1970s. A research was progressed by the MRB (Malaysian Rubber Board), a Government Research Institute, on inoculums of Rhizobium for leguminous crops. In 1980s, research activity was advanced by University Putra Malaysia (UPM) on Mycorrhiza. The study was conducted for evaluation of nitrogen contribution from Azospirillum to seedlings of palm oil. The advancements in the research conducted on biofertilizers lead to the preparation of biofertilizers as inoculants as carriers of microorganisms.

21.3 Biofertilizer Classification Typically, biofertilizers are not like the commonly used chemical fertilizers as these comprise of cultures of the soil microorganism (dormant or live cells) that are maintained artificially. Biofertilizers can be used as inoculants of soil or microbes that are added for improving soil fertility and plant productivity as these contain microorganisms that facilitate the nutrient accessibility to plants. The extract from plant produce has numerous applications including prohibiting corrosion for further application in agriculture industry and agriresearch as these supplant unsafe and toxic inhibitors of chemical nature [17]. The examples of microorganisms that are used as biofertilizers include blue-green algae, fungi, and bacteria. These microorganisms are added to the plant rhizosphere to enhance soil activity [8, 18]. On the basis of function and nature of microorganisms, biofertilizers can be grouped as follows: a) Nitrogen fixers: These are the organisms that fix the atmospheric nitrogen by converting nitrogen molecules in air into the ammonia or other related nitrogen containing compounds in the soil. In this way, atmospheric nitrogen (N2) is converted into the nitrogenous compounds that can be

Biofertilizers: Past, Present, and Future  595 utilized by plants through a process called biological nitrogen fixation (BNF). These are further categorized as Symbiotic and non-symbiotic nitrogen fixing bacteria. The examples of symbiotic nitrogen fixing bacteria include rhizobiaceae family bacteria for example Rhizobium, Azorhizobium, Bradyrhyzobium, Mesorhizobium, and Sinorhizobium which are collectively recognized as rhizobia. The rhizobia develop a symbiotic relationship with plants of leguminosae family and non-leguminous trees. The examples of nitrogen fixing bacteria that are non-symbiotic include cyanobacteria (for example, Anabaena and Nostoc), Azotobacter, Azospirillum, Diazotrophicus, and Gluconaacetobacter. The non-symbiotic types exist as free living, endophytes, and in associative form [8]. b) Phosphorus solubilizing biofertilizers: There is a significant number of microbial species that exhibit capacity for solubilization of phosphorus. These include fungi, bacteria, actinomycetes, and algae to some extent. The examples of bacteria include Arthrobacter, Bacillus, Chryseobacterium, Gordonia, Pseudomonas, Rhodococcus, Serratia, and others. The examples of fungi that have been reported as solubilizers of phosphorus include Aspergillus, Penicillium, Trichoderma strains, and strains of Rhizoctonia solani [18]. c) Phosphorus mobilizing biofertilizers: These are the microorganisms (for example, mycorrhizal fungi) that facilitate and enhance the uptake of phosphorus. These act through a mutual symbiotic associationship which is based on transfer of nutrient between roots and soil fungi. The plants produce sugar through process of photosynthesis and supply the fungi with these sugars. The fungi hyphae network enhances the capacity of plant for absorption of nutrients and water. The example includes arbuscular mycorrhizae which is also known as endomycorrhizae. This is also considered as the most prevalent and common type of mycorrhizae. Other examples include Rhizoctonia solan (Orchid mycorrhiza); species of Amanita, Boletus, Laccaria, Pisolithus Ectomycorrhiza, and Pezizella ericae (Ericoid mycorrhizae) [19]. d) Biofertilizers as micronutrients: These include microorganisms that have the capacity to dissolve silicate, for example, silicate-solubilizing rhizobacteria (SSR). The

596  Biofertilizers silicate-solubilizing rhizobacteria facilitate silicate mineral solubilization so that silicon and potassium is readily available for crops [20]. e) Plant Growth-Promoting Rhizobacteria (PGPR): The bacteria that are present near and around the rhizosphere (plant root) are categorized as rhizobacteria that promote growth of the plant. Since these have capability for facilitating growth of the plant, these are also known as plant growth-promoting bacteria or PGPB. These either inhibit plant disease, facilitate nutrient absorption, or act as biostimulants for production of phytochromes, for example, species of Bacillus and Pseudomonas [20].

21.4 Different Paradigms of Biofertilizers 21.4.1 Impregnation of Fertilizers and Fertilizer Use Efficiency Fertilizer use efficiency (FUE) is a critical parameter to measure impact of fertilizer management and effect of soil-water-plant relationship on crop production. Since the agrimanagers are mainly focused to fulfill the constant growing food demand of society, FUE signifies the probability of loss of nutrient in the environment from the existing systems of cropping and further paves the way to fill the gap. It should be understood that measures of FUE are not the measures of loss of nutrient as soil retains the nutrient, and thus, low FUE may not essentially be detrimental to the environment while high values of FUE does not signify the safety to environment [21]. Due to their applications in agriculture, the routine usage of mineral fertilizers cannot be avoided and their response on crop yield can be envisaged from the FUE of crops. The factors such as soil erosion, leaching, and dentrification contribute to the loss of mineral fertilizers, thereby not only causing loss to economy but also poses problems to ecosystem. As the use efficiency of fertilizers containing nitrogen and phosphorus is poor accordingly, there is an extensive requirement for the use of bacteria that promote growth of plants (plant growth-promoting bacteria) as these have progressive impact on environment and growth of the plant [22]. In recent years, the development of fertilizers impregnated with bacteria is in progress as a novel approach to enhance the plant nutrient use efficiency. In comparison to other approaches, plant growth-promoting bacteria (PGPB) impregnation with mineral fertilizers is a novel concept and advanced strategic approach to improve growth of plant and crop yield.

Biofertilizers: Past, Present, and Future  597 The impregnation of fertilizers with bacteria is a coating of chemical fertilizers with different mineral types and plant with bacteria that promote the growth of plant (PGPB). This is done to enhance the quantity of rhizosphere useful microflora and also the efficiency of fertilizers applied [22]. It is envisaged that impregnation of fertilizers, i.e., coating of chemical fertilizers with microbes will commence acceptance of nutritional sources (natural/synthetic) that may lead to better knowledge of FUE enhanced by microbes [23].

21.4.2 Inoculants of Mixtures of Microorganisms An important aspect is to make usage of mixtures of microorganisms rather than using microorganism singly as this approach in the form of mixed inoculants enhances the crop potential due to altered mode of action of different microorganisms present in the inoculum [24]. In this way, the usage of inoculant of microbial mixtures along with organisms causing nitrogen fixation, phosphorus dissolution, and mobilization of potassium seems to provide a significant alternative [25]. The research activities on inoculants of mixture of microorganisms have also shown that when different microorganisms with different benefits are mixed together, these prove to be beneficial at a larger extent in comparison to using inoculants of single microorganisms [26, 27]. The incorporation of different microbes with different capabilities into common biofertilizers gives rise to potential and promising yield in a desirable and innovative manner. Thus, continuous investigation of effective strains of microorganisms that are beneficial for crops is important for growth of plant and soil strength as microbial mixture produces wide spectrum of effects and increased efficacy.

21.4.3 Different Formulations of Inoculants The inoculation development of an efficacious strain comprises of microorganism, a carrier, and an additive. The formulation of a bioinoculant is usually in the liquid and solid form which poses problems related to low microbial viability at the time of storage and application. Since there are many parameters that altogether affect the microbial cell viability; hence, the bacteria require formulations that are more protective and also that enhance the bacterial efficacy at the target site. For commercialization, the inoculant performance is the parameter of paramount importance as the poor quality of inoculant and inadequate formulation poses major hurdle for the wide acceptability and use of inoculants. The interactions

598  Biofertilizers of flora and fauna with inoculants also poses a big challenge for commercialization. Hence, for wide use, the formulations of inoculation must show stability at the time of production, supply, transport, and storage [28]. The surroundings of farming conditions such as droughts, high temperature, acidic soils, saline soils, and soil erosion shall be considered for making formulations. In such circumstances, the usage of local strains of plant growth-promoting bacteria that pose resistance to any physical or chemical stress will be a useful approach. The process of developing an inoculant formulation shall be on the basis of basic doctrine of plant biochemistry, material science, and microbiology. The application of the knowledge based on these areas facilitate production of sustainable mechanism to agriculture and ecological solutions. The development of an adequate formulation demands continuous research in the formulation sciences as adequate formulation always poses challenge with a limited success in bio-inoculant commercialization. The main factors on which a strategy for development of formulation mainly depends include availability of equipment, convenience of farmer, inherent characteristics of plant, application technology, site of action, cost, technique of application, and inoculant colony [4, 14].

21.4.4 Inoculant Carrier For long-term exposure of microbial fertilizers with plants, a sustainable carrier is required for development of formulation of microbial fertilizers. The optimal requirement of the technology for production of carrier formulation and inoculum is significant for the effective application [13]. Though there may be significant difference in the carrier raw material and the type of formulation, the final inoculant product will always be in the form of powder, granules, slurry, and liquid and can be applied to seedlings, leaves of the plant, compost, seed, and soil [14]. The carrier for inoculant shall be present abundantly in nature, sterile, physically and chemically uniform to the best possible extent, possess capacity for holding water, and shall be appropriate for different strains of bacteria. Assimilation of microorganisms used in biofertilizers with the coating technologies can be used as a tool to reduce the overall cost and volume of the fertilizers, leading to more profit to farmers. Sustainable agriculture can be achieved by combining microorganisms with the advanced technologies for coating as the most appropriate economical coating material and inoculant carrier are the need of hour for present and prospective research on bio-organic chemical fertilizers. It has been observed that the biological

Biofertilizers: Past, Present, and Future  599 activity of biofertilizers can be enhanced by providing a suitable medium and making them deliver to the target areas [29]. The formulations containing water in oil emulsion type are also used for storage and for further application on crops using irrigation systems [30]. The suitable carriers widely used in bacterial formulations include mica, lignite, mud filter, talc, perlite, zeolite, vermiculite, bentonite, saponites, coal, inorganic soil, sawdust, wheat bran, rice bran, husk of rice, manure from poultry, waste from banana, mineral and vegetable oils, and polymers like polyacrylamide, alginate, carrageenan, kaolin, alginate-kaolin, silicates, cellulose gels and powder formulations, clays, liquids, and peats [14, 29, 31]. The powder and granular formulations require media such as saponites, mica, wood pulp, lays, polysaccharides derived from seaweed extracts, gums, alginates, starches, plant extracts, microbial gums, clays, pulses flour, lignin, and bran, whereas solid formulations require charcoal, bentonite, lignite, and peat [32]. The formulations in the form of powdered solid and granular forms are required for seed coatings [14].

21.4.5 Biofertilizer Carriers and Liquid Formulations In present time, biofertilizers in the liquid form are considered to the best option in comparison to biofertilizers that require some carrier as a medium. The liquid formulations of biofertilizers mainly use carriers that are liquid, for example, oil, water, solvents, suspensions, and emulsions. Liquid formulations include concentrates, suspensions, and emulsions (oil-based products) [32]. Liquid inoculants are mainly developed using suspensions (oil-in-water), emulsions, and oils derived from mineral or organic oil. For the delivery and storage of microorganisms through liquid formulation, the emulsions (water-in-oil) are used primarily. The emulsion is widely used to deliver and store microorganisms through liquid formulations. In liquid formulations, sugars such as sucrose, mannitol, water, and other chemical nutrients are required as medium for plant growth. For this, biofertilizers in the liquid form will be most appropriate as these formulations ensure the organism survival in the long way and thus create a sufficient environment for the crop. Accordingly, strategies and advancements for suitable carrier development and formulation technology refinement are the need of the hour [33]. Liquid biofertilizers pose easiness in their application and act as an alternative to other biofertilizers that are mainly based on carrier-based medium. Liquid biofertilizers facilitate an increased plant yield, recovering health soil and production of sustainable food.

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21.4.6 Controlled Release Techniques: Encapsulation, Lyophilization, and Drying The conventional systems used to apply active compounds to the plant lack specificity in terms of delivery to the target site. Controlled release systems are thus required for the control release of active substances to promote growth of the plant and to further minimize loss of active ingredients which may arise due to soil pH and local climate conditions [34]. The effective technologies for controlled release systems mainly include lyophilization and encapsulation. The encapsulation method encompass providing a layer, thereby shielding the microorganism. Microbial immobilization enhances nutrient shelf life and increases field efficiency. Encapsulation is required for controlled release into soil [14] as this can release inoculants of microbes varying in different composition and morphology [4, 32]. The advanced technology of microencapsulation creates a protective shell or forms a capsular covering around the cells, tissues, or active plant ingredients, thereby ameliorating nutrient shelf life and efficiency of application [32]. For example, bioencapsulation of rhizobium bacteria keeps the soil bacteria protected and facilitates a slow and prolonged release [35]. In comparison to other formulations, the application of seed and soil in the form of microencapsulation (controlled release pattern) exhibits superiority as it improves the bacterial survival with prolonged effect [32]. Microencapsulation exerts a protection to bacteria against non-conducive environmental conditions. It has been reported that complications associated with liquid and dry form of inoculants can be overcome by encapsulating freeze-dried bacteria in beads of alginate in dried form [14]. This is due to the fact that the bacteria in encapsulated form is dry, easily biodegradable, and simple to use and facilitates a gradual bacterial release for long duration. Generally, kaolin and bacteria are mixed with alginate to form beads which are then lyophilized. The material that is widely used for bioencapsulation of microbes includes polymers of natural and synthetic origin, carbohydrates, proteins, starches, polystyrene, polyurethane, polyacrylamides, and clay. Although there are advantages, the main disadvantages associated with technique of microencapsulation include the requirement of special equipment for spray drying, chronological steps for proper encapsulation, time of encapsulation, and the cost of encapsulation material [32]. The long-term protection and preservation can be achieved by microbial drying process as it involves low cost of storage and distribution and is subjected to less contamination. The most commonly used methods for drying of microorganism for their use in biofertilizers include

Biofertilizers: Past, Present, and Future  601 spray-drying, vacuum-drying, air-drying, fluid bed-drying, lyophilization, and freeze-drying [4].

21.5 Biofertilizers: Current Status Current research activities have suggested fertilizer development using different type of metabolites, microorganisms, and inert materials as these can upsurge stability of biofertilizer, further prolong effectiveness and life in the natural soil conditions [36, 37]. The fertilizers impregnated with bacteria are vital as these increase the quantity of beneficial microorganisms and efficiency of fertilizer. In recent time, the utility of compost rich with germs is also gaining importance in mobilization of nutrients along with the biofilms which are also in use for producing plant inoculum [38, 39]. The residue from agroindustries contain rock phosphates and other microorganisms. The use of this residue can minimize the cost of production and enhance the efficiency of use of nutrition [13]. The carriers like biofilm-based carriers, polymer-based carriers, natural carriers, macrocapsule, and silica gel encapsulation are being developed to further facilitate the biofertilizer development [39]. The studies have reported the attachment of bacteria with micro-grains of alginate and coated with silica membranes [40]. During recent time, highly technical and economically feasible models have been proposed for concept based design of production plants for liquid biofertilizer. This is envisaged to further provide the way and incite for agriresearchers and engineers in analysis, optimization, development, and evaluation of new methodologies for production of biofertilizers and technologies [41].

21.6 Biofertilizers: Future Paradigm In a prospective manner, there is a need to collect information on interaction of plant. This facilitates the capability in different type of organisms to enhance the growth of the plant and their possible benefits as biofertilizers. It is well evident that a good accomplishment can be achieved from microbial fertilizers if local strains are selected and used for target crops. The inoculum comprising of different combinations of bacteria possesses great value when different microorganisms with different benefits can be integrated to the crop plant. This aspect of integrating different microbes into one product, thereby promoting a large number of other effects is

602  Biofertilizers necessitated. In agricultural practices, the microbial use can be improved. For this, local strains, coating materials, and good protectant can be used for reducing cost of production and providing a suitable carrier. The advancement in new technologies that enhance the shelf life of biofertilizer, development of carriers, and techniques of encapsulation remains critical for usage and their applications in the production of biofertilizers. Thus, the technique of coating of microbes can be used in addition to techniques of bioencapsulation or carrier-based techniques which are available currently, thereby making formulations of microbial mixture and chemical fertilizers more efficient. For sustainable agriculture and seeking long-term environmental solutions, the inoculant formulations based on the principles of plant biochemistry, plant pathology, and plant microbiology will be important. As it is a big challenge to possess strains that are efficacious, the local strains can be used as these elicit the performance in every agricultural conditions. Thus, integration of organisms among each other and with plants can lead to high beneficial results. An approach has been made to distinguish and match inoculants as the inoculation efficiency differs on various parameters including but not limiting to different species of plants, soil, and climate [14]. Not only the advancement in formulations that are specific to strains but also the utility of modern tools for monitoring viability of cell is a significant step in exploiting potentially beneficial strains of plant [4]. The biomass of microbes can be used for development of species that produce siderophore and as an inoculum to colonize the plant root systems that are deficient in iron [42]. In future the enrichment, amelioration of chemicals with microbial and organic contents and the integration of microbes, chemicals, and organo compounds in a single formulation will have a significant impact with a hope to agriscientists to adopt measures for agricultural practices that are environmentally and economically friendly. Accordingly, the development of an appropriate system with the integration of microbial, chemical, and bio-organo compounds in a single formulation for management of nutrition in the plants for sustainable agriculture is the need of hour. The major thrust shall be on continuous research and adoption of these innovative methods for more economic, environmental friendly, and sustainable agriculture practices.

21.7 Conclusion The utility of biofertilizers for sustainable agriculture is the need of hour as it provides the ways for sustainable practices and application of sources

Biofertilizers: Past, Present, and Future  603 of fertilizers that have less deleterious impact on the plants and environment. Current research activities make use of different type of metabolites, microorganisms, and inert materials to increase biofertilizer stability and further extend effectiveness and life in the field. In future, the technique of coating of microbes can be used in addition to techniques of bioencapsulation or carrier-based techniques for making formulations of microbial mixture and chemical fertilizers more efficient.

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22 Algal Biofertilizer Muhammad Mudassir Iqbal1*, Gulzar Muhammad2†, Muhammad Shahbaz Aslam1, Muhammad Ajaz Hussain3, Zahid Shafiq4 and Haseeba Razzaq2 Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Lahore, Pakistan 2 Department of Chemistry, GC University Lahore, Lahore, Pakistan 3 Institute of Chemistry, University of Sargodha, Sargodha, Pakistan 4 Institute of Chemical Sciences, Bahauddin Zakariya University, Multan, Pakistan 1

Abstract

Increasing demand for agricultural products due to escalating population and concern about the quality of food, environment, and human health necessitates exploration and development of throughput products and strategies for significant increment in yield, decrement in cost, utilization of wasteland for cultivation, and environment protection and improvement. Algal biofertilizers are natural recyclers and reservoirs of nutrients, increase plant growth, and offer all said advantages. Recently, various algae have been explored for their impact on cultivation, soil, and environment, and novel industrial processes have been developed for extensive scale cultivation of algae and production of algal biofertilizers. The chapter introduces the diverse nature of algae as a biostimulant. The biochemical compounds of algae, which impact the growth of plants, are also essential parts of the discussion along with the functions of algae as biofertilizers. Moreover, recent advancements to exploit, develop, and manufacture novel algal biofertilizers with innovative strategies for sustainable agriculture are also discussed in the chapter. Particular emphasis is given on algal strains to produce biofertilizers, the methods to obtain algal biomass, and strategies for subsequent use for plant growth. Finally, processes and technologies to cultivate algae and manufacture algal biofertilizers on a mass scale are discussed. Keywords:  Algae, algal fertilizers, biofertilizers, recycling, biostimulant, sustainable agriculture *Corresponding author: [email protected] † Corresponding author: [email protected] Inamuddin, Mohd Imran Ahamed, Rajender Boddula, and Mashallah Rezakazemi (eds.) Biofertilizers: Study and Impact, (607–636) © 2021 Scrivener Publishing LLC

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608  Biofertilizers

22.1 Introduction Algae are photosynthetic microorganisms that are used as biofertilizers in the field of crop cultivation [1, 2]. The biomass obtained from algae holds nutrients and organic substances in larger quantities and promotes the slow release of nutrients from fertilizers and soil and increases the absorption of nutrients from fertilizers [3]. Products obtained from organic material are biostimulants and enhance the development and growth of many crops grown under normal conditions or stress. Biofertilizers contain living microorganisms or natural substances capable of increasing the nutrient status of soil and hence increase plant growth. Both characteristics of microalgae improve agricultural sustainability and are increasing the intrigue of agrochemical industries and farmers in microalgae [4, 5]. An increase in the crop yield for sustainable agriculture depends on inorganic fertilizers. However, the excessive use of inorganic fertilizers is a menace not only for the environment but also for human beings. Thus, there is a need to explore microorganisms such as microalgae cyanobacteria and mycorrhizal fungi capable of symbiosis with plants to produce biofertilizers [6, 7]. Ample use of chemical fertilizers results in soil and groundwater contamination, leading to intrusion in the confined ecosystem, which decreases crop yield over time. The disadvantages of chemical fertilizers prompt demand for the discovery of surrogate nutrition sources to augment the yield of food crops and causing less contamination of the environment [8, 9]. Biofertilizers show the capacity to provide the nutrient elements converting from unusable form to useable form through the biological process and help in germination and initial growth of plants [10]. The use of organic biofertilizers is not only cost-effective but also the best approach to save the natural environment. The microalgae help plants fix nitrogen, thus proving its worth as biofertilizer [11]. The use of blue-green algae is cost-effective, decreases the requirement of synthetic fertilizers, increases grain yield, and dwindles pollution [12]. Biofertilizers are an essential part of biological nutrient supply systems and nitrogen fixation. Biofertilizers are a renewable source of nutrients and useable as a supplement with synthetic fertilizers and recently have become popular among small and borderline cultivators [13, 14]. Improved effectiveness of the product is a test for the fertilizer industry, achieved by more effective use of existing fertilizers and inventing and introducing new fertilizers. Among recently developed technologies, the crucial strategies are the capability to control and slow the release of nutrients with advantages of their ability to improve efficiency for a more

Algal Biofertilizer  609 extended period and evading the loss due to leaching and volatilization [15]. Although seaweeds are extensively used in the cultivation of crops to improve the fertility of the soil, seaweeds demand a further investigation to explore the bio-stimulatory effect of seaweeds and underlying mechanisms [16]. Several countries use marine algae as manure, and the potential of seaweeds in current crop cultivation is extensively investigated recently for the development of new applicable products from seaweeds in the form of liquid fertilizer and biomass [17]. The central importance of fertilizers to augment the revenue from profitable crops demands industrial production of cost-effective and broad range fertilizers [18]. At present, algae are the focus of scientific research worldwide to exploit as successful biofertilizers for sustainable agriculture. The chapter reviews the biodiversity of algae and biochemical constituents of algal biofertilizers with the effects on plant growth, yield, soil, and environment. The new novel schemes for mass cultivation of algae and methods of mass production of novel biofertilizers from algal biomass are also discussed in detail. Moreover, strategies to apply as-developed algal biofertilizers to crops for the impact of algal biofertilizers on cultivation are focused.

22.2 Algae and Algal Biofertilizers 22.2.1 Algae is a Polyphyletic Functional Group Algae are microorganisms capable of carrying out photosynthesis and include organisms from very distinct domains of life. Microalgae and diatoms belong to eukaryotes, while cyanobacteria belong to prokaryotes. Almost 30,000 species of both unicellular and complex multicellular algae are expected to present naturally [19, 20]. The term “microalgae” is not based on phylogeny; it is based on function. Microalgae take in microorganisms from two phylogenetically distinct groups, eukaryotes, and prokaryotes. Microalgae is the polyphyletic functional group of autotrophic organisms capable of photosynthesis. They are colorful due to the presence of photosynthetic and other pigments. Microalgae inhabit the aqueous environment, both fresh and marine. Microalgae are commonly unicellular and are capable of living in colonies [21]. Microalgae are classified as Chlorophyta, Rhodophyta, Phaeophyta, Euglenophyta, Pyrrophyta, and Chrysophyta. Chlorophyta, Rhodophyta, and Phaeophyta are commonly known as a green alga, red alga, and brown alga, respectively. Cyanobacteria are solitary oxygen-producing prokaryotes through

610  Biofertilizers photosynthesis [22]. Blue-green algae, known as cyanobacteria, are microorganisms coming to the fore for improvement of enduring cultivation [23, 24]. Cyanobacteria are the original form of life in the time course of evolution and are considered as triumphant and lasting prokaryotic microorganisms on earth [25]. Nostoc, Anabaena, and Phormidium populate the aerobic section in the paddy rice field [26]. Other common genera include Aulosira, Cylindrospermum, Fischerella, Lyngbya, and Plectonema [27].

22.2.2 Multifaceted Role of Algal Biofertilizer in Sustainable Cultivation Algae as biofertilizers perform various functions and increase the quality and fertility of the soil resulting in increased plant growth and yield. Algal fertilizers increase the productivity of soil through carbon sequestering and nitrogen fixation. They also augment the soil quality by improving the structure and reclamation of land. Biofertilizers escalate plant growth through the production of plant growth hormones and help plants in defense processes, colonize plant tissue, and control the pests [2]. Different roles of algal biofertilizers in promoting sustainable cultivation are depicted in Figure 22.1. Biomass from blue-green algae is industrially important for the production of biofertilizers, food, energy, secondary metabolites, cosmetics, and therapeutics, as illustrated in Figure 22.2 [25]. Blue-green algae inhabit the earth, fresh and saltwater worldwide, and improve the bioavailability of phosphorous to plants through organic acids Biofertilizer inoculation

Effect on soil physicochemical and biochemical properties

Effect on microbial community

Increase in nitrifiers Increased soil aggregation Increased in fungal community Increased soil porosity

Figure 22.1  Effects of biofertilizers on the physicochemical properties of soil. (Reprinted with permission of Elsevier from [28]).

Algal Biofertilizer  611 CYANOBACTERIA (Blue Green Algae)

Agriculture • • • • •

Biofertilizers - Nitrogen Fixation Soil Fertility Improvement Wastelands Reclamation Bio-Control Crop Productivity Improvement

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Stress Agriculture Management Healthy Agro-ecosystem Quality Foods Food Security

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Bioremediation CO2 Sequestration CH4 Oxidation Augmentation Bio-Fuels (Bio-Diesel) Food Supplements (Wonder Food)

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Alternative Energy Resource Wastewater Treatment Climate Change Mitigation Clean & Safe Environment

Sustainable Agriculture Development & Environmental Development

Figure 22.2  Potential functions of cyanobacteria in sustainable agriculture and the environment. (Reprinted with permission of Elsevier from [25]).

and compounds that regulate the growth of the seeds [29]. Cyanobacteriabased biofertilizers are used in the cultivation of many food crops such as barley, oats, tomato, radish, cotton, sugarcane, maize, chili, and lettuce [30]. Heterocystous cyanobacteria fix nitrogen and decompose matter to release nutrients, while non-heterocystous cyanobacteria and green algae release nutrients from the soil, hence making nutrients in the land available for plants [2]. Blue-green algae are cost-effective to grow and need very simple inorganic nutrients and convert dinitrogen into ammonia known as nitrogen fixation. Blue-green algae increase grain yield through nitrogen fixation by 10%–30%. There are 40 blue-green algae biofertilizer production centers in India [31]. Cyanobacteria produce hydrogen gas, excrete ammonium ions, use carbon dioxide, and nitrate to produce algal biomass [32]. Cost-effective, readily available, and safe for ecosystem fertilizers are obtained from Diazotrophes, which are blue-green algae. Biofertilizers from Diazotrophes possess the ability to manage nitrogen deficiency in plants, increase gaseous content of the soil, hold water, and increase vitamin B12 [33].

612  Biofertilizers

22.2.3 Biostimulants From Algae Both macronutrients and micronutrients are essential for plant growth. The macronutrients include nitrogen, potassium, phosphorous, calcium, and magnesium, while micronutrients include iron, zinc, copper, molybdenum, boron, and chlorine. Essential nutrients are imperative and prerequisites in specific amounts to obtain the maximum yield of cultivated crops [34]. Marine algae are nonfossil biogenic resources with applications in fields of food, therapeutic, and botany and serve as raw material for industry. A critical feature of marine algae is the presence of bio-­stimulant substances in seaweeds and derived products [16]. Marine alga is a reservoir of biomolecules and minerals. Besides, marine algae contain phytohormones, osmoprotectants, and antimicrobial compounds in larger quantities. The recent increase in the use of seaweed extract is the prerequisite of organic farming. Marine macroalgae stimulate germination of the seed, increase the length of shoot and root and overall health and growth of the plant, improve the water and nutrient uptake, help plants to resist salinity and plant pathogens, increase fertility, and recover soil from contaminations [17]. Green algae Coelastrella oocystiformis and Chlorolobion braunii uptake potassium and are prospective biofertilizers. The two selected algae are different in shape and are well suited to study the effect of shape on potassium uptake. The potassium absorption capacity of tow algal strains is assessed by growing algal strains on different concentrations of potassium from 200 to 2,000 parts per million for 30 days under 5,000 lux. After harvesting when the algal biomass becomes dry, ash is produced from algal biomass and tested for potassium content. Analysis of ash content shows potassium uptake of Chlorolobion braunii (40.66%) is higher than Coelastrella oocystiformis (30.5%) grown on 400 parts per million potassium ion concentration. Coelastrella oocystiformis takes up 74.8% potassium at this concentration [35]. Algal extracts contain macromolecules like carbohydrates, proteins, oils, fats, and polyunsaturated fatty acids. Additionally, the algal extracts are rich in various diverse types of biologically active compounds. Polyphenols, tocopherols, vitamin C, and mycosporine-like amino acids show anti-­ oxidative properties. Carotenoids and chlorophylls act as light-absorbing pigments. Phycocyanin and phycoerythrin are phycobilins. Phycobilins show essential capacities to neutralize bacteria, fungi, viruses, oxidation, inflammation, and tumor. The aim of worldwide research recently is the development of innovative methods of extracting active bio-stimulants. The extraction methods use enzymes, microwaves, high pressure,

Algal Biofertilizer  613 supercritical fluid, and ultrasound to increase the effectiveness and stability of biostimulants extracted from seaweeds [36].

22.3 Techniques of Application of Algal Biofertilizer 22.3.1 Algal Extracts as Biofertilizer Algal extracts increase the efficiency of essential nutrient sources by stimulating plant growth [37]. Low cultivation yield, scantiness of water, escalating contamination of the environment, and demand to reduce the use of inorganic fertilizers prerequisite practice of novel algal extracts and irrigation strategies [1]. Algal extracts of Chlorella sp. increase the uptake of nitrogen, phosphorous, and potassium from the soil by Broccoli plants and stimulate the overall growth of plant grown on sandy loam soil. To evaluate the efficiency of algal extract, different combinations of organic, compost, bio, and mineral nitrogen sources were studied. The algal extract in combination with nitrogen sources impacts plant growth. The impacts on vegetative parts include increase in height of plant, number of leaves per plant, leaf area, and weight of fresh shoot. The impact on biochemical compounds includes increase in both chlorophyll a and b and vitamin C content. The efficiency of extract becomes even more prominent when the algal extract is sprayed on leaves of plants [37]. The algal extract contains both macro (nitrogen, phosphorous, and potassium) and micronutrients (manganese, iron, copper, and zinc), organic acids, hormones, and amino acids compulsory for plant growth. The algal extract is either applied to soil or in the form of spray on plants. The spray of extract from the leaf of algae shows a noteworthy influence on the growth of vegetative parts and the yield of flowers in freesia plants. The spray of extract from algae leaves is useful when sprayed after 15, 30, and 45 days after germination. Freesia plants show a 20% increase in height when treated with algal leaf extract, with the increasing average number of leaves on the plant by 33%. The algal extract increases the length of the flowering stem (25%) and doubles the number of inflorescences. The extracts described above also enhance the number of florets per inflorescence (50%) and total chlorophyll (31%) [38]. To meet the worldwide demand for nutrition, accessibility of water is getting attention and requires an increase in water availability for cultivation. Possible negative effect on plant physiology and yield is a disadvantage of an excellent approach to augment the water proficiency by the use of deficit irrigation. Much is not known about the impact of marine algal

614  Biofertilizers extracts on the resistance of onion plants grown in areas with less availability of water to draught. The effect of foliar spray of extracts from marine algae Amphora ovalis on onion cultivated in commonly available soil water (80%) and stress with 50% available soil water is investigated in field experiments with split-plot design in four replicas. The NPK uptake by onion plants and the size of bub increase considerably with statistical significance by the application of algal extract as a foliar spray. During drought, plants shift energy utilization from chlorophyll synthesis to start production of proline and phenols, which are known to resist water to derive Algal extract also decrease the impact of drought by increasing chlorophyll synthesis [39]. Part of extracts from algae in metabolic practices of Cynara cardunculus L. known as cardoon plant is imperative. Various phenolic compounds are present in cardoon plants and are known for different medical uses. Effect on growth, seed production, and chemical composition of cardoon plant is investigated analysis of growth indicator parameters like chemical constituents, height, leaf count, fresh and dry mass of plant, and seed number and weight after foliar application of different concentrations four algal extracts from Spirulina platensis, Chlorella vulgaris, Amphora coffeaeformis, and Scenedesmus obliquus. The use of Spirulina platensis extract increases the morphological properties of cardoon significantly as compared to control plants. Scenedesmus obliquus extract increases fixed oil and total carbohydrate content of cardoon plant. Chlorella vulgaris extract promotes flavonoids production and protects cells against the free radical activity. Amphora coffeaeformis extract produces phenolic compounds considerably higher in plants as compared to control plants, as indicated by increased chlorogenic, caffeic, and vanillic phenolic compounds [40]. The extracts from marine algae are used as biofertilizer in horticulture. The application increases overall plant growth and yield. The effect of Ascophyllum nodosum extracts on the growth of Vitis vinifera cv. Feteasca Alba plants are investigated. The extract from marine algae is applied in three different concentrations. In the experimental investigations, an algal excerpt shows the effect on fertility coefficients, length and diameter of the shoot, and area of the leaf. Absolute fertility coefficient and relative fertility coefficient are higher in the plants grown on algal extract concentrates with high concentration as compared to plants grown on lower concentrations of algal extracts. The spray of the algal extract on leaf also shows the increased growth of vegetative parts of plants [41]. Extracts from Codium tomentosum and Sargassum vulgare, respectively, are used as a liquid fertilizer to increase the germination of seeds and growth in the seedling of wheat. Effect of marine green and brown algae

Algal Biofertilizer  615 extracts on germination, growth of the seedling, and chlorophyll content is studied by applying the liquid fertilizer in various concentrations from 10% to 50% using distilled water. First seeds are soaked in liquid fertilizer for 12 h and then transferred to Petri dishes. For control experiments, seeds are soaked in water only. It was noted that 98% and 97% seeds germinate in 20% algal extract of Codium tomentosum and Sargassum vulgare, respectively. The application of liquid fertilizer stimulates the growth of vegetative parts as evident from increased length of shoots and roots and increases the dry weight of seedlings. Algal fertilizer increases photosynthetic pigment and carotenoid constituents of leaf [42]. Extracts from marine algae provide nutrients and stimulate plant growth and are a potential alternative to chemical fertilizers in crop cultivation. A liquid extract from brown algae Stoechospermum marginatum prepared in different concentrations is sprayed on leaves of brinjal, Solanum melongena seedlings grown in experimental pots and natural conditions. The analysis of growth and chemical parameters after 30 and 180 days shows the increased length of shoots and roots, area of the leaf, and mass of the fresh and dry plant. Alga also improves the chemical constitution and increases the proportion of moisture, chlorophyll, proteins, and reducing sugar. The activity of ascorbic acid and nitrate reductase also increases by application of algal biofertilizer. Lower concentration of algal biofertilizer increases fruit number and mass of fruit. In comparison, a higher level of algal biofertilizer shows an inhibitory effect on plant growth indicators, as revealed by Figures 22.3 to 22.7 [43]. Green algae Chlorella sp. increase rooting and development of willow plants Salix viminalis by stimulating metabolism significantly. The plant cuttings are grown in the presence of either sonicated or un-sonicated biofertilizers. There are two methods to apply biofertilizer to cuttings of plants. In one approach, plant cuttings are rooted in universal horticulture substrate. Then, rooted cuttings are soaked in biofertilizer for 4 days in a vegetation chamber. In the second method, untreated plant cuttings are rooted and grown in substrate moistened with biofertilizer. The control plant cuttings are grown in water GA3, IBA, Bio-Algeen S90, and environmental sample. The application of biofertilizer to the willow plant cutting stimulates root formation because of the stability of cytomembranes. Another reason is the advancement in physiologic processes (photosynthesis, transpiration, and conductance) and biochemical factors (dehydrogenases, RNase, acid, and alkaline phosphatase). The biofertilizer decreases intracellular carbon dioxide in rooted cuttings and plants [44]. Cellular extract and biomass from green algae Acutodesmus dimorphis both show properties of biostimulants and are used as biofertilizers to

616  Biofertilizers

Light, Nutrient, CO2 and H2O

nobacteria Cya Nutraceutical

Bioremediation

Biofertilizer

Bioenergy

Soil fertility

Increased soil aggregation

Increased soil porosity

Figure 22.3  Development of sustainable agriculture practices by utilization of beneficial outcomes of cyanobacteria. (Reprinted with permission of Elsevier from [25]).

Con

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Figure 22.4  Influence of liquid extract of Stoechospermum marginatum on the total plant height of brinjal. (Reprinted with permission of Springer Nature from [43]).

Algal Biofertilizer  617

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Figure 22.5  Influence of liquid extract of Stoechospermum marginatum on the leaf area of brinjal. (Reprinted with permission of Springer Nature from [43]). Fruit weight

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Figure 22.6  Influence of liquid extracts of Stoechospermum marginatum on fruit weight and a number of fruits/plant of brinjal. Numbers present above each bar are percent over control. (Reprinted with permission of Springer Nature from [43]).

increase seed germination, plant growth, and fruit production in Roma tomato plants. The effect of biofertilizer on seed germination is investigated by the application of culture or aqueous extract of microalgae in different concentrations from 1% to 100%. The impact on the growth of

618  Biofertilizers Influence of Stoechospermum marginatum on fruit weight of brinjal

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Figure 22.7  Influence of liquid extract of Stoechospermum marginatum on the fruit weight of brinjal. (Reprinted with permission of Springer Nature from [43]).

plant and fruit is investigated by foliar spray of algal extract and the use of biomass of green algae. Treatment with algal culture and concentration of algal extract more significant than 50% shows faster germination in seeds with a time of two days before the control group increases the height of the plant, the number of flowers, and branches on one plant. The seedlings are treated two times 22 days before seedling transplantation. The treatment before 22 days increases the growth of the plant, the number of branches, as well as flowers [5]. Microwave-assisted extracts from Polysiphonia, Ulva, and Cladophora are obtained by keeping powdered algal biomass with water in Teflon chambers inside a microwave oven at 25°C–60°C and characterized for the presence of biostimulant compounds. Microwave-assisted extracts from seaweeds contain high concentrations of polyphenols and a lack of fatty acids. The concentration of polyphenols in extract decreases with temperature while that of micro and macroelements increases with temperature. Microwave-assisted extract from seaweeds impacts seeds germination and growth of Lepidium sativum plant without affecting the morphology of plants. Microwave-assisted extracts from seaweeds are potential biofertilizers for agriculture [45]. Extract from marine green algae Ulva intestinalis impacts germination and root elongation in Arabidopsis thaliana plants by stimulation and decreases the formation of lateral roots. Investigations show that a higher concentration of extract is inhibitory and reduces seed germination and root growth. The impact of the algal extract on seed germination and root growth follows different stimulation mediators (hormones). The presence

Algal Biofertilizer  619 of a higher amount of Al3+ in Ulva suggests the role of ethylene and cytokinin in the decreased growth of roots. The results of investigations concluded a careful use of biofertilizers for maximum yield [46]. Foliar application of algal extract and two different strategies of irrigation seeping and aerosol are investigated for impact on wheat plant growth. The use of algal extract from blue-green algae Spirulina platensis on the leaf is very efficient on plant growth parameters. Seeping and aerosol irrigation systems are efficient significantly, however not for the area of the leaf, height of the plant, and length of spike [1]. The impact of the application of algal extract on leaves of freesia plant is investigated. The algal extract is applied in three replicates. In the first experiment, the algal extract is applied three times with an interval of 15 days. Secondly, three different concentrations of the extract are used on 10 days old seedlings. The algal extract considerably increases the growth and flower production in plants due to higher leaf and photosynthetic area. Increment in the length of shoot containing the flowers and clusters of flowers and inflorescence is also observed [47]. Algae are mixed in groups for use as biofertilizers on tomato plants. Algal biofertilizers are applied as an extract on seed primers and leaves through the spray. Biofertilizers from mixed algae increase macromolecules and biochemicals that stimulate plant growth [48]. The use of multicellular blue-green microalgae Spirulina platensis extract as a foliar spray or on soil increases the growth of Spinacia olerasea L. (spinach). The application of algal extract increases the values of growth indicator parameters by increasing the photosynthetic pigment content A Algal biofertilizer reduces 20% nitrogen need for plants [49].

22.3.2 Addition of Algal Strains and Algal Biofertilizer to Soil Marine microalgae strains increase the seed germination and early growth of maize plants and hence augment the yield. For this purpose, C. vulgaris and Spirulina platensis are added to soil in combination with the cattle manure for 2.5 months. The maize plants supplemented with biofertilizers are grown in greenhouses. The increase in growth and yield shows the ability of marine microalgae as prospective fertilizers for the cultivation of maize crops [18]. Use of S. platensis along with cattle manure followed by C. vulgaris increases growth and yield of the onion by expanding the availability of nutrients required for plant growth of onion. The strategy increases plant height, number, and weight of leaves per plant, fresh and dry weight of the plant. The use of marine strains also increases leaf area, neck thickness, size, and weight of the onion bulb, thereby increasing

620  Biofertilizers the yield. Biofertilizer also increases pigment content and improves the biochemical composition of onion for soluble sugars, total phenols, free amino acids, and indoles [10]. Use of marine algae Ulva lactuca, cystoseira spinose, and Gelidium crinale as biofertilizer in the soil increases growth and yield of Brassica napus (canola) grown in the greenhouse. Seaweeds are harvested in low tide by hand. Washing with seawater and brushing of weeds removes epiphytes and sand particles. Rinsing with tap water removes the resident salts. The plants are air-dried for 6 days, and a mechanic grinder grinds seaweed in fine powder, which is used as biofertilizer. Three grams of algal powder mixed with 1.0 kg of soil is irrigated for 7 days at a frequency of two times a day. The combination of three seaweeds decreases the process of inhibition of chlorophyll a and b, total carbohydrate accumulation, and hormones that increase growth. The combination increases antioxidants and osmoprotectants. The antioxidants include phenols, flavonoids, and anthocyanin, while total carbohydrates and proline are osmoprotectants raised by the application of a blend of seaweeds. The increase in the growth of canola is due to the rise in growth-promoting hormones such as indole acetic acid, indole butyric acid, gibberellic acid, and cytokinins [50]. Biomass obtained from microalgae applied with phosphate fertilizers increases the uptake of phosphorous by plants. For this purpose, the microalgae are cultivated on wastewater. Investigation of total phosphorous content of Pennisetum glaucum L. (millet) grown on different proportions of algal biomass mixed with triple phosphate shows that 12% algal biomass releases maximum phosphorous both with and without the greenhouse. A further increase in the proportion of algal biomass decreases the values of parameters investigated in the plant. However, the use of 12% microalgae biomass with triple phosphate does not show an effect on phosphorous diffusion in the soil [3]. Marine microalgae Chlorella vulgaris and Spirulina platensis stimulate the growth of rice plants and upsurge yield of rice by 20% when applied under surface irrigation in clay loamy soil as an alternate of costly chemical fertilizer [11]. Algaefert is a commercial biofertilizer based on an extract of Ascophyllum nodosum. Algaefert is used with microbien and phosphorein Jatropha curcus L. seedlings, and the effect of biofertilizer on growth and chemical contents of seedlings is investigated. Three fertilizers are added to pot after 30 to 60 days of planting 200-g DAP or compost per container. Application of algae, microbien, and compost considerably increases the length and diameter of the stem, leaf number per plant, and area of leaf for Jatropha curcus L., which are growth indicators. Three fertilizers also increase chlorophyll a and b, a carotenoid pigment, carbohydrates, phenols, and indoles.

Algal Biofertilizer  621 The use of compost decreases indole and phenol content in Jatropha curcus seedlings [51]. Microalgae grow and derive nutrients from wastewater, and the biomass is used to cultivate crops. Unicellular microalgal or filamentous macroalgal biomass is grown on sewage. In both cases, the algal biomass is mixed with vermiculite/compost that acts as a carter. Triticum aestivum (wheat) crop is cultivated on either of the algal biomass. Filamentous macroalgal biomass increases the values of available nitrogen, phosphorous, and potassium in soil and nitrogen-fixing potential significantly. Unicellular microalgae biomass increase microbial biomass carbon considerably. Both types of algae grow NPK constituents in roots, shoots, and fruit with an approximate value of 4% nitrogen in grains. The biofertilizer also increases dry plant mass upon treatment at the harvesting step [52]. Algal biomass obtained from wastewater management in a nutrient recycling approach is added to soil to increase fertility. Less is known about how the significant addition of alive algae to the soil in higher quantity impacts the biochemical composition of the land, mainly the algae able to form soil biofilms. Soil biofilms affect the interaction between plants and microorganisms. Chlorella is a genus of unicellular algae employed in wastewater management systems, especially Chlorella sorokiniana. The algae in the form of a suspension of cells increase bacterial biomass in soil. Increased CO2 evolved from land suggests the presence of bacteria in the soil. Algal biofertilizer, in the form of the film, also increases the total carbon constituent of soil [53]. Chlorella vulgaris, a microalga, shows growth stimulation in tomato and inhibits nematode parasite development [54]. C. vulgaris is added to the culture medium used to grow to germinate tomato and cucumber seeds, and the root and shoot growth is observed for a couple of weeks. The seeds germinated in 0.17 and 0.25 g/L algal suspension show increased seed growth. Tomato root growth is almost two times in 9 days, and that of cucumber is more than two times in 12 days as compared to control, as revealed by Figures 22.8 and 22.9 [55]. Biomass products obtained from Chlorella sp. Neochloris conjucta and Botryococcus braunii are used as biofertilizers for the cultivation of corn and impact plant growth and nutrient uptake from soil. The material remains after the digestion of microalgae is applied to corn plants in four different concentrations. Increasing concentrations of Botryococcus braunii and Neochloris conjucta decreases the growth of plants and uptake of nutrients. Chlorella sp. increases plant growth and nutrient uptake considerably [56]. Biofertilizer obtained from Scenedesmus obliquus biomass increases the germination ability and growth of wheat and barley seeds [57].

622  Biofertilizers 50 45 40 Length (mm)

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Figure 22.8  Effect of culture medium containing Chlorella grown for 3, 6, 9, and 12 days on growth parameters of seed germination of tomato. (Reprinted with permission of Creative Commons from [55]).

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Figure 22.9  Effect of culture medium containing Chlorella grown for 3, 6, 9, and 12 days on growth parameters of seed germination of cucumber. (Reprinted with permission of Creative Commons from [55]).

Algal Biofertilizer  623 The blue-green algae are applied to the soil, and the effect on soil fertility and germination of mustard seeds is investigated. Blue-green algae blended with clay is placed over the surface of pots containing mustard seeds. Analysis of soil before and after the application of blue-green algae shows that blue-green algae increase soil quality that impacts seed germination [58]. Nostoc linkia, Anabaena variabilis, Aulosira fertilisma, Calothris sp. Tolypothrix sp., and Scytonema sp. are significantly effective nitrogen fixers for rice agriculture [59]. The recommended quantity of flacks of blue-green algae is 10 kg per hectare. The blue-green algae are used to prepare fields of rice in acidic soil, and seedlings are transferred in the area with standing water. After 10 days, the fields are given a shot of 0.05 kg/m2 of biofertilizer comprising a blend of various strains of blue-green algae. The results show that rice plants are bright and healthy with thick green leaves, which are higher in number than control. Blue-green algae increase the yield of rice grains, both in numbers and mass by 10% [31]. Anabaena sp. is used with NPK on rice grown in greenhouses and fields with sandy loam soil and compared with control plants. Blue-green algae increase phosphorous and potassium content of seeds and plants considerably in greenhouse plants. The seed mass of rice is increased considerably by the application of bluegreen algae. The use of blue-green algae in the rice field decrease the cost of synthetic fertilizer and show potential as biofertilizer. The rice plants are now planted directly instead of planting seedlings. However, the underlying mechanism for the stimulation of growth of the plant by blue-green algae is still uncovered [60]. Blue-green algae are part of a natural ecosystem found in standing water of rice fields and thus are compatible and cost-effective biofertilizers for rice agriculture [61]. Fly ash, soil, and montmorillonite, alone or in different combinations, are used as a carrier of biofertilizer. Four blue-green algal strains, Anabaena variabilis, Nostoc muscorum, Tolypothrix tenuis, and Aulosira fertilissima, are grouped to produce cost-effective algal biofertilizer. The biofertilizer is inoculated, and plants are observed for 90 days. The group of algal strains increases nitrogen and carbon significantly with the use of a mixture of fly ash and soil in 1:1 as carrier [62]. Blue-green algae are used as biofertilizer in rice agriculture. Blue-green algae are now used for the cultivation of other crops too. The impact of blue-green algae on the performance of nitrogen reductase in Capsicum annum is investigated. Blue-green algae are used either alone or with vermicompost. The use of blue-green algae with vermicompost increases nitrate reductase activity [63]. Blue-green algae are obtained from beds of plants with medical applications cultivated on medium devoid of nitrates. Blue-green algae can convert atmospheric nitrogen into a form useable

624  Biofertilizers by plants. Some cyanobacteria produce hormones used by plants. Impact of blue-green algae Nostoc carneum, Wollea vaginicola, and Nostoc punctiforme on plant growth and production of essential oils by Matricaria chamomilla L. plants are investigated in the greenhouse by application of blue-green algae in a suspension to the soil. Blue-green algae in suspension are given to plant after 15 days of the plantation. Blue-green algae impact the yield of essential oil and root elongation significantly. Highperformance liquid chromatography analysis shows that blue-green algae produce indole acids that act as hormones and increase plant growth [64]. Use of technologies allows controlled and slow release of nutrients and increases the efficiency of algal fertilizer product by extension in action for a longer duration and decrease loss due to leaching and volatilization. A slow-release biofertilizer is obtained by the incorporation of microalgae in the polymeric urea-formaldehyde matrix. Techniques of titration, ultraviolet, and atomic absorption spectroscopy allow the quantitative analysis of macro and micronutrients in the microalgae. The methods of structural characterization infrared spectroscopy and scanning electron microscopy will enable the analysis of polymeric urea-formaldehyde matrix and incorporated chlorella spinosis. The formulation shows a slow release of nitrogen, phosphorous, and potassium in vitro with maximum release of 28%, 26%, and 46%, respectively, for 30 days. The novel formulation is based on nutrients of natural origin and shows better results in vivo as compared to conventional commercial fertilizer [15]. De-oiled waste from microalgal biomass increases growth and yield in long-lasting farming of rice crops as compared to chemical fertilizer and vermicompost. Wastewater and flue gas are used to grow Scenedesmus sp. microalgae in open raceway ponds. Microalga, chemical fertilizer, and vermicompost are applied directly to the soil either alone or in a combination of two according to the recommendation to meet 100% nitrogen supply. The combination of microalga and chemical fertilizer shows the highest increase of overall plant growth, including height and tiller number and grain yield and weight [8]. Date palm (Phoenix dactylifera L.), a plant capable of surviving at high temperatures and less water availability, grows in arid and semiarid areas worldwide. It helps to stop the conversion of land into desert. Mixed planting and the use of chemical fertilizer to meet demand results in decreased quality of fruit and less valuable product. The yield of fruit is also affected by the long generation time. For large-scale production plantlets coming out of proliferating callus, grown in vitro cultures are planted in soil producing true-to-type clonal plants to meet the demand. The survival of date palm plantlets transferred from the culture medium to soil increases by

Algal Biofertilizer  625 the use of microalgae Tetraselmis sp. The biofertilizer helps plants in acclimatization from culture to soil. Microalga is added to the soil without synthetic fertilizer or to soil amended with manure, and the growth of plants is observed for 3 months. Microalga increases plant survival to 100% by enhancing overall plant growth through improvement in root formation, an increase in the length of root, number of leaves, and stem girth, and an increase in chlorophyll content. Microalgae also increase the NPK in the soil, which are essential nutrients for plant growth [65].

22.4 Cultivation of Algae and Production of Algal Biofertilizer Agriculture of algae on a large scale for the manufacture of byproducts and bioenergy has increased recently [66]. Large-scale cultivation of microalgae requires nutrients in large quantities, so the production of cost-­ effective biofertilizer by the cultivation of algae is not possible economically for the industry. Cheap source of nutrients is required for mass scale cultivation of algae. The exploitation of wastewater in algae cultivation not only provides cheap nutrients abut also manage water and recycle the non-­ renewable sources like phosphorous. Wastewater possesses both organic and inorganic nutrients, including carbon, nitrogen, and phosphorous, in abundance. Wastewater from domestic use, soybean and potato processing industry, carpet industry, and aquaculture is used for the cultivation of algae. Solid waste from agriculture accomplishments and industries based on agriculture, food, dairy, and poultry manure is rich in nutrients and is used in the cultivation of algae for the production of biofertilizers [2]. Taking advantage of the uptake of nutrients from waste in the biotechnology industry by microorganisms to produce useful products containing bioactive substances through biorefinery constitutes a cyclic economy based on biotechnology. Scenedesmus obliquus efficiently uptakes nitrogen, phosphorous, and chemical oxygen demand from brewery waste. Brewery waste is used to grow Scenedesmus obliquus. The resulting biomass is subjected to a biorefinery for the manufacture of biofertilizer, and the process also produces other useful products and compounds. The byproducts include phenols, flavonoids, hydrogen, oil, and biogas [57]. For the production of biofertilizers, the purified strains of microorganisms are grown in large fermenters or photobioreactors. The process exploits the capability of algae to carry photosynthesis. Either sunlight or artificial light is used for the large-scale manufacturing of microorganisms in photobioreactors. The use of sunlight for photosynthesis is

626  Biofertilizers advantageous for being free. The bioreactors are an open system or closed system. The disadvantage of contamination in open bioreactors is solved by using closed photobioreactors. Moreover, the closed system increases the surface-to-volume ratio and cell densities of cultivated microorganisms [25]. The strategy for the production of biofertilizer from blue-green algae is shown in Figure 22.10. For the commercial manufacture of algal biofertilizer, few locally adapted  strains of blue-green algae are inoculated in an open photobioreactor. An open photobioreactor is of different types such as tank made of cement, less deep steel trays, pits with plastic lining, or an open field. A mixture of fertilizers, soil, lime, and insect killer chemicals is spread on photobioreactor before inoculation. Fertilizer acts as a nutrient source. Lime adjusts the pH of soil. Suitable temperature for the growth of bluegreen algae is 35°C to 35°C temperature. After separation of dry algal biomass from the soil, it is a grind and packed [67]. High-rate algal ponds (HRAPs) originally meant for water treatment are combined with the manufacture of biofertilizer. In another scheme, the HRAP is combined with the production of biogas and biofertilizer. Both projects, along with a third scheme, the activated sludge system is illustrated in Figure 22.11 [68]. There are various methods for the manufacture of extract biofertilizers from seaweeds. Conventionally, water, alkalis, or acids are used to Mother culture

Fermenters for large scale production

Broth is mixed with sterilized carrier

Culture Quality check

Low temperature storage

Slant

Broth in small flask

Broth in big flask

Packaging in polythene bags

25°C Curing in controlled temperature room

Dispatch to Farmers

Figure 22.10  Mass culture of biofertilizer and the steps. (Reprinted with permission of Elsevier from [25]).

Algal Biofertilizer  627 (a) Background system

Construction materials production

Energy production (electricity and heat)

Electricity and Heat

Foreground system

CHP unit

Emissions to air

EFFLUENT BIOGAS

INFLUENT

Primary Settler

Emissions to water

Secondary settler

HRAPs

Microalgal Biomass

Thickener

Anaerobic digestion

Thermal pre-treat.

Sludge

Emissions to air/soil Input Output

DIGESTATE (agriculture)

Transportation

Avoided product

(b) Background system

Construction materials production

Energy production (electricity and heat)

Chemicals Production

Foreground system Emissions to air

INFLUENT

HRAPs

EFFLUENT Secondary settler

Centrifuge

Emissions to water

Microalgal Biomass Biofertiliser production

Emissions to air/soil Input Output

BIOFERTILISER (agriculture)

Transportation

Avoided product

(c) Background system

Construction materials production

Chemicals Production

Energy production (electricity and heat)

Foreground system Emissions to air

INFLUENT

Primary Settler

Activated sludge reactor

EFFLUENT Emissions to water

Secondary settler Sludge

Thickener

Centrifuge

Sludge

Incineration

Input Output

Transportation

Avoided product

Figure 22.11  Flow diagrams and system boundaries of the wastewater treatment alternatives: (a) HRAP system for wastewater treatment where microalgal biomass is valorized for energy recovery (biogas production) (Scenario 1); (b) HRAP system for wastewater treatment where microalgal biomass is used for the nutrients recovery (biofertilizer production) (Scenario 2); (c) activated sludge system (Scenario 3). HRAPs; high-rate algal ponds. (Reprinted with permission of Elsevier from [68]).

628  Biofertilizers prepare extracts, or cells are lysed by passing through mills at lower temperatures by physical means to develop a suspension [69]. Bio-stimulant extraction methods include enzyme-assisted extraction, microwave-­ assisted extraction, supercritical fluid extraction, and pressurized liquid extraction. The use of innovative extraction methods gives increased yield, reduces process time, and is economical as compared to custom methods based on solvent extraction [70]. Marine algal bioactives and methods of removal are shown in Figure 22.12. The scheme of extraction from algae for three innovative processes is shown in Figures 22.13 to 22.15. HO

Enzymes Microwaves Ultrasound waves Supercritical fluid Pressurized liquid

O

E X T R A C T I O N

C

O

Fucoxanthin

HO

OH

MARINE ALGAL BIOACTIVES HO H2C O3SO

OOOCH2

OSO3

Fucoidan

OH

Phloroglucinol

O OH H3C O

O OSO3

O

Figure 22.12  Marine algal bioactives and methods of extraction. (Reprinted with permission of American Chemical Society from [70]).

Transducer

Ultrasonic probe Ultrasound Generator h–depth of probe in liquid

Jacketed glass beaker

Figure 22.13  Schematic diagram of ultrasound-assisted extraction for the preparation of extract from algae. (Reprinted with permission of American Chemical Society from [70]).

Modifier

Extraction cell

Condenser

Algal Biofertilizer  629

CO2 Reservoir

Separators I & II

Pump

CO2 Pump

Heat Exchanger

Figure 22.14  Schematic diagram of supercritical fluid extraction for the preparation of extract from algae. (Reprinted with permission of American Chemical Society from [70]).

Purge Valve

Pressure Relief Valve Oven

Pump

A

B

Extraction cell

C

Solvents Nitrogen Waste vial

Collection vessel

Figure 22.15  Schematic diagram of pressurized liquid extraction for the preparation of extract from algae. (Reprinted with permission of American Chemical Society from [70]).

630  Biofertilizers

22.5 Conclusion Increasing demand for food and agriculture-based products requires sustainable cultivation and higher yields. The extensive use of chemical fertilizers is expensive and harms soil, ecosystem, and environment directly and human health indirectly. Recent studies show comprehensive advancement to explore novel algal strains and the development of algal fertilizers. Production of algal biofertilizers is cost-effective and secure. Algal biofertilizers increase yield, are safe to the ecosystem and environment, and are the potential alternative of chemical fertilizers for sustainable crop cultivation. Moreover, algal biorefineries add valuable byproducts besides the production of biofertilizers.

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Index

α-proteobacteria, 430, 441, 448, 449 β-proteobacteria, 452 1-aminocyclopropane1-carboxylate deaminase, 452 Abiotic, 186, 194 Abiotic stress, 39–41, 48, 65–67, 75, 77 cold stress, 65–66, 71–73 drought stress, 65–66, 70–71 heat stress, 65–66, 73 heavy metal, 65–66, 70–71 salinity, 41, 65–66, 68–70 ACC deaminase, 11, 13, 352, 355 Acidification, 577–579 Actinomycetes, 437, 579 antibiotic production, 346–347 biocontrol agent, 345–346 drought resistance, 354, 355 heavy metal toxicity, 352–354 hydrogen cyanide, 349 lytic enzyme, 349–351 nitrogen fixation, 338–340 phosphate solubilization, 340–342 plant stress busters, 351–352 potassium solubilization, 342 properties, 337 salinity resistance, 355 siderophores, 348–349 soil ecology, 342–345 Actinorhizal, 4–5 Advantage, 394, 404 Adverse, 393, 394

Aerotaxis, 313 Africa, 561 African cassava mosaic virus (ACMV), 202 Agricultural health, 294 Agriculture, 393, 400, 402–405, 429, 431, 435, 453, 576–578, 580, 584–586 Agrobacterium, 379 Aleksandrove media, 429, 430, 447, 460 Algae, 608, 609 Algaefert, 620 Algal extracts, 612 Alginate, 240–241 Alternative, 394, 401, 403, 405, 406, 408 Amalgamation, 109 Ammonia (NH3), 128, 193, 447 Ammonium, 576 Amphora, ovalis, 614 coffeaeformis, 614 Anabaena, 130, 439, 448, 452 Anaerobe, 5 Antheridia, 195 Antibacterial, 432, 433 Antibiotic, 429, 435, 441, 575, 578, 579, 581, 583 Anticancerous, 429, 431, 435, 458 Antifungal, 435 Antimalarial, 429, 435, 439, 441 Antioxidant, 435, 446

637

638  Index Antioxidants, 620 Antiviral, 435, 446 Apoptosis, 195 Application, 393–397, 399–404, 408 Appressoria, 198 Arabidopsis thaliana, 618 Arbuscular mycorrhiza fungi, 579 Arbuscular mycorrhizal fungi (AMF), 151, 162, 163, 166, 186–187, 278, 262, 435 Arbuscular, 187 Arbuscules, 8 Arhar, 129 Arthrospira, 436, 445 Ascomycota, 8, 198 Ascophyllum nodosum, 614 Ascorbic acid, 578 Aspergillus, 434 Aspergillus fumigates, 580 Aulosira fertillissima, India, 130 Aulosira, 439, 445, 446 Autoclave, 432 Autoclaving, 109, 582 Autoregulatory mechanisms, 191 Auxin, 434, 442, 457, 581, 583 Auxins, 133, 194 Auxotrophic valine, 385 Avermectins, 347 Azolla as biofertilizer, 134–135 Azolla carolinii, 134 Azolla filiculoides, 134 Azolla maxicana, 134 Azolla microphylla, 134 Azolla nilorica, 134 Azolla pinetta, 134 Azolla rubra, 134 Azospirillum, 122, 126, 260, 267, 270–275, 378, 395, 406, 431, 432, 435, 436, 440, 449, 451, 494, 495, 498, 504, 580, 583–585 Azospirillum biomass, 321 Azotobacter, 108, 111, 114, 122, 126, 128, 378, 429, 440, 442, 455, 580

Azotobacter chroococum, 130 Azurol sulphonate agar, 429, 439, 440, 442–444, 455 Bacillus, 190–191, 379, 580, 581, 583, 584 Bacillus licheniformis, 448 Bacillus megatherium, 130 Bacillus radicicola, 129 Bacillus subtilis, 191, 438 Bacillus thuringiensis, 439, 444, 452, 455 Bacillus velezensis, 437 Bacteria, 199 nitrogen fixing, 595 non-symbiotic, 595 plant growth promoting, 596, 598 rhizobacteria, 595, 596 symbiotic, 592, 595 Bacteria-fungi consortium, 165 Bacterial colonization, 317, 322 Bacterial magnetic particles, 386 Basidiomycota, 8, 198 Beneficial endophyte interactions, 194 Beneficial interactions, 185 Beneficial microbes, 39–41, 43, 45, 49–50, 52–56, 58, 61, 66, 184 Beneficial microorganisms, 186 Benefits of different biofertilizers, 542 Bioavailability, 152, 154 Biochar, 453 Biocompatible, 239–294 Biocontrol, 1, 3, 6, 10, 12–13, 16 antibiosis nutrient competition, 12 hydrogen cyanide (HCN), 12 induced systemic resistance (ISR), 12–13 lytic enzyme production, 12 Biocontrol agents, 345 Biodegradable, 116 Biodiversity, 578, 579, 584 Bioenhancer, 580

Index  639 Biofertilizer, 157, 160, 238–308, 267, 269–270, 336, 337, 393–408, 561 formulations, 215 Biofertilizer carriers, biochar, 234 peat, 232–233 talc, 233 vermiculite, 235 wheat bran, 233 Biofertilizer formulation, alginate formulation, 504 bionanofromulation, 505 fluidized bed dryer formulation, 504 liquid inoculants formulation, 503 peat formulation, 502 polymer-based formulation, 504 Biofertilizer formulations, fluid bed dried, 243–244 granular, 236 liquid, 236 mycorrhizal, 244–245 polymer entrapped, 239 powder, 230 Biofertilizer market segments, based on crop, 544 based on microorganism, 544 based on region, 544 based on types, 544 Biofertilizer market, 414 Biofertilizers, 39–79, 186, 191, 203, 375, 377, 491–493, 608, 610 carrier, 599 current status, 601 future paradigm, 602 history, 593 liquid formulation, 599 Biofertilizers and biofertilizer technology, 541 Biofertilizers market drivers in India, 546 Biofertilizers, organic farming by, benefits of biofertilizers, 126 benefits of organic farming, 140–143 biofertilizer application, 126–127

classification of, 128–136 disadvantages of organic farming, 143–144 introduction, 122–123 organic farming, 136–139 traditional agriculture vs. organic and inorganic farming, 139 Biofilm, 454, 460 Bioinoculants, 204, 309, 326, 329 Biological nitrogen fixation (BNF), 3, 5 Biomass, 522–527, 530, 533 Biomass productivity, 382 Biopesticides, 186, 203, 580 Bioprospecting, 167 Bioprotectant, 456, 581 Bioreactors, 625 Bioremediation, 186, 203, 417, 424, 431, 443 Biostimulant, 18, 432, 581, 599 amino acids, 18 fulvic acids, 18 humic acids, 18 microbial inoculants, 18 seaweed extracts, 18 Biosynthesis, 194 Biotechnology, 257, 268–269 Biotic, 186 Biotic stress, 40–41, 52 fungal and bacterial pathogens, 52–56 insect pests, 58–61 nematodes, 61–63 viral pathogens, 56–58 weeds, 64–65 Biotin, 133 Biotrophic, 195–198 BIS standards, 420 Black pepper, 431 Blue-green algae, 126, 130–131, 493, 498, 580, 610, 619, 623 Bradyrhizobium, 580 Bradyrhizobium japonicum, 436 Brassica napus, 620

640  Index Brewery waste, 437, 441 Brown algae, 615 Burk’s media, 438 Burkholderia, 191 Burkholderia phytofirmans, 448 Cadaverine, 323, 584 Calcium, 133 Calcium carbonate, 451 Calcium phosphate, 434, 458 Calothrix, 444, 447 Carbohydrates, 186–187 Carboxysomes/polyhedral bodies, 432, 433 Cargo plasmids, 386 Carotene, 578 Carotenes, 431 Carrier, 431, 436, 575, 581, 582, 584 Carriers, inoculant, 491 polymer-based, 504 seed-coated, 500 solid-based, 501, 502 sterilized, 500 Cassava brown streak virus (CBSV), 201 Cation exchange capacity, 315 Cedrus deodara, 132 Cell growth, 522, 525 Cell immobilization, 239 Cellular, 393 Cellular extract, 615 Cellulase, 429, 433, 434, 439, 440, 459 Cellulomonas turbata, 435 Cereal, 451 Charcoal, 439, 443 Chelation, 157 Chemical fertilizers, 393, 394, 397, 398, 399, 401, 403, 404, 405, 408, 413, 414, 491–493, 499, 508, 510, 518 Chemoautotrophs, 7

Chemotaxis, 312, 313, 316, 317 Chitinase, 429, 433, 434 Chitins, 191 Chlorella, 435, 441, 449 sp., 613 vulgaris, 614, 619, 621 Chlorophyll, 396, 404, 434, 438, 450, 576 Chromosomal transformation, 384 Citricoccus zhacaiensis, 355 Cladophora, 618 Claroideoglomus etunicatum, 431, 436, 438, 444, 454 Classification biofertilizer, micronutirents, 595 nitrogen fixers, 595–596 phosphorus mobilisers, 595 phosphorus solubilisers, 595 plant growth promoting rhizobacteria, 596 Climate change, 39–40, 65, 73 Clostridium, 128 Coal, 436 Codium tomentosum, 614 Coinoculation, 325, 429, 433 Coir waste, 429, 444–446, 457, 460 Colonization, 190–191, 195, 199, 202 Commercial biofertilizers in Indian market, 547 Compositions, 393, 397 Compost, 128, 429, 433, 580, 584 Compound, 394, 395, 397, 403 Concomitant, 436, 437, 439, 453, 457 Congo red, 458 Conservation, 584 Cortex penetration, 187 Cortical cells, 187 Cow dung manure, 449 Crab shell, 438 Crop, 458 Crop diversity, 138 Crop growth, 393, 405, 408 Crop production, 152, 163, 167 Crops productivity, 393

Index  641 Crops, 396–398, 400, 401, 403, 405, 408 Crotalaria juncea, 430–434, 436, 438–441, 443, 446, 450–456, 459 Current, 393, 401 Cyanobacteria, 126, 130–131, 372, 393, 394, 395, 401, 455, 580, see Bluegreen algae Cyanobacterial, 393 Cyanophycin granules, 429, 431–435 Cyanotoxins, 373 Cylindrospermum nostoc, 130 Cynara cardunculus L., 614 Cytokines, 194, 576 Cytokinin, 581 Cytoplasmic cleavage, 195 Date palm, 624 Decomposer, 579 Decomposition, 153, 155, 576, 580 Deficiency, 431 Denitrification, 128, 310 Desertification, 579 Deterioration, 152 Diazotroph, 5, 310, 327, 330 Diazotrophes, 437, 450, 611 Dinitrogen (N2), 193 Dinitrogenase reductase, 113 Dinoflagellates, 371 Diterpenoid, 11 Dolomite, 432 Dunaliella, 454 Dworkin and Foster minimal agar, 434

Endomycorrhiza, 186–187 Endophyte, 450, 460 Endophytic, 4–5, 184, 191, 194, 436, 437, 450, 452 Endophytic microbes, 194 Endosphere, 185 Endosymbiotic theory, 372 Endotrophic mycorrhiza, 132–133 Energy, 184 Environment, 561 Environmental, 294–295 pollutants, 165 pollution, 160 stress, 162 sustainability, 159–160 Environmental impact, 518 Environmental safety, 257, 270 Enzymes, 151, 156, 164, 167, 185, 187, 191, 436, 437, 450, 452 Ethylene, 11–13, 352, 581, 583, 585 1-aminocyclopropane-1-carboxylic acid (ACC), 11 Eucalyptus, 132 Eucalyptus grandis, 429, 431, 435, 440, 441, 443, 451 Eutrophication, 152–153, 575, 578, 579 Excessive fertilization, 153 Exophytic, 184 Exopolysaccharides (EPS), 200 Extracellular polymeric substances, 438, 442 Extraction methods, 628 Exudates, 316

Earthworms, 135–136 Ecofriendly, 293, 294, 296 Ecological diversity, 184 Ecology, 185 Ecosystem, 186, 449, 561, 563, 564, 566, 568 Ectomycorrhiza, 186–187 Ectotrophic mycorrhiza, 132 Encapsulation, 430, 431, 438, 451, 454, 458

Facultative anaerobes, 128t Farmyard manure, 438 Fatty acids, 186 Female oogonia, 195 Fermenter, 440 Ferrous sulfate, 433 Fertility, 394, 397, 399, 400, 405, 407 Fertilizer use efficiency, 596 Filamentous fungi, 186 Filter mud, 448

642  Index Fixation, 153 Flocculation, 317, 321 Flowering, 396 Fluorescent antibody technique, 320 Fluoroquinolone, 429, 433 Foliar application, 323, 332 Food chain, 184 Food supplements, 458 Frankia, 339 Freesia plant, 619 Fungal pathogens, 198 Fungal symbiont, 186 Fungi, 198 Fungicide, 432 Funneliformis geosporum, 430, 431, 442 Funneliformis mosseae, 436 Fusarium wilt, 436, 445 Gamma radiation, 453, 582 Geminiviridaes, 202 Gene replacement mutagenesis, 386 Genetically altered, 257, 270, 273, 275, 280 Genetically modified organisms, 257, 268 Genome plasticity, 324 Genomic technology, 185 Gibberellic acid, 434, 581 Gibberellin (GA), 3, 11 biosynthesis, 11 catabolism, 11 reversible conjugation, 11 Gibberellins, 194 Glomus, 122 Glomus claroideum, 431, 435, 442, 449 Glomus microagregatum, 436 Glomus mosseae, 436 Glutamate synthase (GOGAT), 4 Glutamicibacter halophytocola, 437 Glutamine synthetase (GS), 4 Glycine, 446

Gram, 129 Gram-negative, 4 Green algae, 612, 615 Green manure, 448 Green payment, 329 Green revolution, 455, 460, 518, 576, 577, 584, 585 Greenhouse gas emissions, 518 Growth, 393–397, 400–404, 406, 408 Growth regulator, 430 Haematococcus, 430, 440, 452 Halotolerant, 434 Hartig network, 8 Haustoria, 198 Hazards, 293–294, 561–562 HCN, 446 Health, 561 Healthy, 393, 397, 398, 399 Helper bacteria, 579 Hemibiotrophic, 195–196, 198 Hemorrhoids, 133 Hereditary trait transmission, 154 Heterocyst, 435, 448 Heterocysts, 374 Hexoses, 186 High-rate algal ponds, 626 Homologous recombination, 386 Hormones, 431 Horticulture, 39–40, 75, 79 Hydathodes, 199 Hydrochloric acid, 429, 431, 432, 435, 437, 439–442, 446, 447, 456 Hydrogen cyanide (HCN), 191, 349 Hydrogen sulfide, 157 Hydroxyapatite, 115 Hypersensitive response (HR), 194 Hyphae, 187, 195 IAA biosynthesis pathway, 11 anthranilate, 11 indole-3-acetamide pathway, 11 indole-3-pyruvic acid pathway, 11

Index  643 L-tryptophan–dependent pathway, 11 L-tryptophan–independent pathway, 11 Immobilization, 153 Impregnation, 596–597 Improvement, 393, 394, 395, 396, 399, 404, 407 Indigo green algae, 122 Indole acetic acid, 344 Indole-3-acetic acid, 449 Induced systemic resistance (ISR), 190–191, 195 Infertility, 577 Inoculant, 109, 437, 575, 579, 580, 582, 586, 593–594, 597–602 Inoculant formulation, 499 Inoculum, 163–165, 199 Inorganic, 394, 396, 403, 406 Inorganic phosphorus, 153, 155 Insecticidal drugs, 127 Insoluble minerals, 9 illite, 9 micas, 9 orthoclases, 9 Insoluble phosphate, 154, 161 Integrated pest management system, 345 Invertebrate herbivores, 184 Iron (Fe), 3, 9–10, 13, 581 Fe-limiting, 9 Jatropha curcus L., 620 Key players of biofertilizers in Indian market, 549 Kluyvera cryocrescens, 429, 430, 433, 434, 436, 439, 440, 442, 453 Lactuca sativa, 451 L-diaminopimelic acid, 437 Leaching, 310, 577 Legume, 576, 579

Legume-rhizobia, 191 Legume-rhizobia interaction, 4 Leguminous plants, 129 Lentils, 199 Light harvesting antennae, 435 Lignin, 431 Lignite, 433, 437 Limitations, 429, 433 Limonium sinense, 430, 439, 449, 450, 455, 460 Lipases, 191 Lipid bodies, 446 Lithosphere, 575, 576 L-malic acid, 191 Luria Bertani media, 431 Lyngbya, 130 Macronutrients, 115, 612 Magnesium, 133 Maiz, 619 Malaysian rubber board, 594 Malnutrition, 561 Mannitol, 129, 187 Manure, 128, 130, 133–134, 137, 580–582 Marine alga, 612, 620 Mastigocladus, 130 Metabolic pathway management, 154 Metabolism of microorganisms, 524 Metabolite, 449 Methamoglobinaemia, 578 Methanol, 438, 460 Methylobacterium, 441 Microaerophile, 5 Microalgae, 105, 441, 609, 619–621 Microalgae cultivation, bioreactor design, 523–525 light energy, 523–525 nutrients, 523–525 pH, 525 temperature, 525 Microbes, 184, 395, 405, 406, 429, 438 Microbial activity, 184, 499, 508

644  Index Microbial fertilizers, 186 Microbial inoculant, 492, 499, 500, 505 Microbial strain, 393–395, 401–403, 406–408 Microbial strains, algal, 222, 223, 225 bacterial, 216 fungal, 225–226 Microbial symbiont, 185–186, 191 Microbiological techniques, 422 Microbiota, 203 Micro-dosing, 429–431, 436, 439, 443, 444, 446–448, 451–453, 456, 460 Micronutrients, 612 Microorganisms, 184–186, 189–190, 193, 593, 594, 597, 600 Mimosidi, 122 Mineral soil, 452, 454 Mineralization, 579, 581 Molybdenum, 131 Monotropa, 131, 132 Motility, 312, 314, 315, 316 Mucilage, 185, 187 Municipal solid waste, 429, 433 Mycorrhiza, 7, 122, 126, 186, 580, 581, 584 arbuscular, 7, 13 arbuscular mycorrhizal fungi (AMF), 7, 8, 14–17 arbutoid, 7 as biofertilizers, 131–134 ectomycorrhiza, 7 ectomycorrhizal fungi (ECMF), 8 endomycorrhiza, 7 ericoid, 7 monotropoid, 7 mycorrhizal, 6–8, 14 orchid, 7 Mycorrhizae, 496 Mycorrhizal, 186 Mycorrhizal fungi, 187–189 Mycorrhizal helper bacteria (MHB), 8 Mycorrhizal symbiont, 186

Mycrorrhizal, 393, 394, 395, 401 Myo-inositol, 156 N, P, K, 396, 397, 398, 400, 404 N2−fixation, 406 Nannochloropsissp, 438 Nanobiofertilizer, 450 Nanobiotechnology, 458–460 Nanomaterial, 458, 460 Nanoparticle, 458, 459 Naphthoquinone, 347 National, 398, 399 National Botanical Research Institute’s phosphate medium, 459 Necrotrophic, 195–198 Nematode, 15, 17, 19 Neotia, 131 Nessler’s reagent, 447 N-fixation, 193–194 Nitragin, 448, 575, 580 Nitrates, 128, 576 Nitrogen, 193, 394, 395, 397, 403, 405, 406, 408, 575, 576, 578–581, 584, 592, 594, 596–597 Nitrogen fixation, 122, 127, 271, 276, 310, 313, 315, 318, 320, 323, 324, 327, 329, 378, 434, 492–495, 498, 508 Nitrogen fixer bacteria, 128–130 Nitrogenase, 3–5, 431, 448 Nitrogen-fixing microorganisms, 193 Nitrogen-fixing organism, 579 Nitrous oxide, 577 Nod factors, 4 Nodulation, 105, 431, 445 Nodule, 431, 436, 576, 579, 580, 583 Nostoc, 130, 431, 437, 441, 443–445, 452 Nutrient, 184, 187, 191, 193, 575–579, 581, 583, 584, 586 Nutrient management, 336 Nutrients recovery by microalgae, 523–525

Index  645 Nutritional content, 49 mineral-biofortified, 49–50 secondary metabolites, 50–51 vitamin content, 51–52 Obligate aerobes/anerobes, 128t Oloceira, 130 Oomycetes, 195–198 Oospores, 195 Optimization, 432, 433 Option, 402, 403 Organic, 394–399, 401–404, 406, 407 acid, 157 phosphorus, 152, 154, 156 Organic acids (OA), 6, 9 acetic acid, 3, 6 citric acid, 6 formic acid, 9 gluconic acid, 6 malic acid, 9 oxalic acid, 9 tartaric acid, 9 Organic carbon, 379 Organic farming, benefit for environment, 137 benefits of, 136–137 methods of, 137 objectives of, 136 techniques for, 137–139 Organic matter, 576, 577, 579, 581, 582 Orthophosphate, 456, 459, 460 Oscillatoria, 373 Osmoprotectant, 438, 449 Osmoprotectants, 620 Osmoregulation, 576 Oxidative phosphorylation, 157 Paenibacillus, 447 Paenibacillus polymyxa, 583 Pantoea agglomerans, 429, 440, 441, 452, 453, 455 Papilionidi, 122 Parasitic nematode, 110

Pathogen, 456 Pathogenic bacteria, 199–200 Pathogenic interactions, 185, 194 Pathogenic microbe associations, 185–187 Pathogenic microbe interactions, 184–186, 188–189, 193, 203 Pathogenic microbes, 194 Pathogenic microorganisms, 186, 189–190 Pea, 129 Peat, 431, 435, 437, 441, 453 Penicillium, 433, 434, 443, 459 Pennisetum glaucum L., 620 People, 393, 398 Peptone, 439, 445 Peroxisomes, 198 Pesticides, 160, 165 Phalaenopsis, 130 Phloem transport, 8 Phosphate, 575, 576, 578, 581 Phosphate compounds, 152, 154, 156 Phosphate solubilization, 310, 320, 323, 329 Phosphate solubilizing bacteria, 448, 456 Phosphate solubilizing enzyme, 438–440, 445, 451, 457 Phosphate solubilizing fungus, 437 Phosphate solubilizing microorganisms (PSMs), 6–7, 16 Phosphates, 439, 445 Phosphate-solubilizing, bacteria, 157–158, 160, 165–166 fungi, 160, 162, 164–165 Phospholipid, 576 Phosphoric acid, 134 Phosphorus, 394, 406, 575, 576, 580, 581 Phosphorus pollution, 151–152, 167 Phosphorus solubilization, 413 Phosphorus solubilizing bacteria (PSB), 13–14, 17 Phosphorus/phosphate, 131, 133

646  Index Photosyntetic microorganisms, bacteria, 522 cyanobacteria, 522–523 fungi, 522 microalgae, 522–523 Photosynthesis, 6, 8, 11, 184, 430–432, 435–441, 444–448, 451, 455–457, 523–524, 529 Phycobilins, 612 Phycobilisomes, 432 Phycocyanin, 431 Phycoerythrin, 431 Phyllosphere, 194, 431 Phylosphere, 184 Phytases, 341, 342 Phytate, 156 Phytochrome, 596 Phytohormones, 1–3, 10–11, 109, 114, 115, 186, 191, 194, 319, 323 abscisic acid, 3, 11 cytokinin, 3, 11 ethylene, 3, 11–13 gibberellin (GA), 3 indole acetic acid (IAA), 3, 11, 18 Phytopathogens, 1–2, 7, 10, 12 Phytophthora, 196 Picea, 132 Picric acid, 441, 448, 460 Pikovskaya agar medium, 448 Piriformospora indica, 584 Planktonic, 447 Plant bacterial pathogens, 199 Plant growth, 393, 394, 395, 396, 397, 401, 402, 403, 404, 406 Plant growth promotion, 318, 324 Plant growth promoting microorganisms (PGPMs), 2–3, 7, 10–13, 191 plant growth promoting rhizobacteria (PGPRs), 3, 14–16, 345 Plant growth-promoting bacteria, 191, 456, 457, 578

Plant growth-promoting rhizobacteria (PGPR), 190–191, 345, 492, 498 Plant pathogen, 402, 408 Plant phenotype, 185 Plant-fungi interactions, 187 Plant-genotype, 185 Plasma, 430, 459 Plasmid transformation, 384 Plectonema, 130 Pollution, 394, 396, 404, 405, 408 Polyacrylamide, 450, 455, 456 Polyhydroxyalkanoate granules, 433 Polymeric urea-formaldehyde matrix, 431 Polyphosphate bodies, 450 Polyphosphate reserves, 383 Polysaccharides, 200 Polysiphonia, 618 Popix, 132 Popular marketed biofertilizers in Indian market, 553 Potash, 134 Potassium (K), 8–9, 133, 394, 395, 397, 431, 575–578, 581 limitation, 9 solubilization, 9 solubilizing microorganisms (KSMs), 9 Potassium ferrocyanide, 429–431, 439, 441, 447, 455 Practices, 393, 403 Preparation, 575, 581, 582 Present scenario of biofertilizer market, 547 Problems in promotion of biofertilizer, 550 Procedure, 398, 400, 401 Process parameters, upstream and downstream processing, 526–527 Production, 393–398, 401, 403–405, 407, 576–586 Productivity, 449, 577, 580, 581, 585

Index  647 Programmed cell death (PCD), 195 Proliferation, 313 Proline, 429, 430, 440, 450, 451, 459 Propagation, 582 Protease, 191, 446, 447, 454 Protoctista, 195 Pseudomonas, 190, 262–264, 266–267, 273, 379, 435, 583–585 Quality, 40–41, 44–49, 55, 57, 60–61, 73, 76, 78, 394, 396, 404, 405, 407 Quality of yield, 407 Quantity, 397, 400, 404, 405, 406, 407 Quorum sensing, 324 Recent trends in biofertilizer: liquid biofertilizer, 554 Rhizobacteria, 191, 309, 310, 320–323, 330, 332, 393, 394, 402, 429, 430, 440, 450, 451, 459, 580, 581 Rhizobia, 4–5, 193 Rhizobium, 264, 275–277, 279–280, 378, 379, 394, 395, 401, 429, 430, 436, 444, 452, 460, 494, 495, 507, 580, 583 Rhizobium bacteria, 129 Rhizobium biofertilizer, 127 Rhizophagus intraradices, 429, 436, 437, 443–445, 454 Rhizoremediation, 186 Rhizosphere, 2, 5, 7, 10, 13–14, 17, 19, 151, 155–156, 158–159, 161–162, 184–185, 189, 194, 579, 583 Rhizospheric residence, 191 Riboflavin, 576 Rice bran, 436 RuBisCo, 439 Salix, 132 Salkowski reagent, 431 Sam, 129

Saprophyte mycorrhiza, 132 Sarcodes, 132 Sargassum vulgare, 614 Scenedesmus, 449 Sclerotinias clerotiorum, 438 Screening, 451 Scytonema, 447–449 Seed treatment, 126, 506 Seedling root dipping, 506 Seedling treatment, 127 Selts (Pade), 127 Serratia marcescens, 432 Setts and Tuta treatment, 127 Shaker, 438 Shelf-life, 433, 456 Shuttle vectors, 386 Siderophore, 320, 323, 326, 434, 450, 458, 575, 581 Siderophores, 9–10, 115, 191, 348 carboxylates, 10 catecholates, 10 hydroxamates, 10 phytosiderophores, 10 Sijalpinidi, 122 Sodium carbonate, 429, 431, 435, 437, 439, 440–442, 444, 448 Soil, 303, 394, 395, 397, 399, 400, 401, 402, 405, 406, 407, 408 Soil application, 507 Soil functions, 561 Soil management, 138 Soil nutrients, 393 Soil treatment, 127 Soil-based biofertilizers, 134 Soil-borne pathogenic, 189 Solanum melongena, 615 Solubilization, 191, 194 Solubilization index, 448 Solubilizing, 395 Sorghum bicolor, 447 Sorghum husk, 135 Spirulina, 130, 437 Spirulina platensis, 619

648  Index Staphylococcus warneri, 432, 434, 438 Starter culture, 439 Stenotrophpmonas maltophilia, 433 Sterilization, 451 Stomach, 199 Streptomyces, 434 avermitis, 347 coelicolor, 348, 350 fimicarius, 350 fumanus, 343, 346 globisporus, 350 griseoflavus, 339 griseus, 341 lydicus, 338, 339 rochei, 348 tendae, 348 Subphylum glomeromycota, 187 Sugary vitamins, 133 Suicide vectors, 386 Sulfur, 575, 581 Super absorbant polymer, 435, 443, 444 Superoxide dismutase, 453 Superphosphate, 131 Sustainability, 576, 579, 584, 585 Sustainable agriculture, 257, 271 fertility and soil quality, 528–530 plant growth, disease and pest control, 531–532 soil organic matter, improvement, and recovery, 530–531 Symbiosis, 446 associative-symbiosis, 320 legume symbiosis, 320 Symbiotic, 584 Symbiotic bacteria, 129 Symbiotic interactions, 187 Symbiotic relationship, 380 Symplasmata, 452 Synergistic effect, 165 Synthetic mutualism, 325 Systemic acquired resistance (SAR), 195

Talaromyces, 457 Teosinte, 198 Thiamine, 133, 577 Thylakoid centres, 439 Titration, 431 Tobacco mosaic virus (TMV), 202 Tolypothrix, 130, 448 Tolypothrix, Japan, 130 Tomato yellow leaf curl virus (TYLCV), 202 Total coding sequence, 325 Toxins, 200, 203 Traditional farming, problems in, 139 Transgenic, 257, 268, 281, 270 Transposon mutagenesis, 386 Trehalose, 187, 194 Trichoderma, 191, 395, 396, 403, 432 Trichome fragmentation, 374 Trifolium alexandrium, 583 Trifolium repens, 439, 455 Tripartite, 579, 585 Tris-minimal salt agar, 444 Triticum aestivum, 448 Trough method, 135 Tryptone, 437 Tryptophan, 449 Tuber (Tuta), 127 Ulva intestinalis, 618 Urea, 576 VAM, 580, 581 Vanadomolybdate reagent, 449 Vegetable oil, 447 Vegetative propagation, 73 cuttings, 73–74 grafting, 74–75 Vermicompost, 135–136, 406, 407 Vermiculite, 433

Index  649 Vesicles, 7 Vesicular arbuscular mycorrhiza (VAM), 122, 126, 135, 186, 187 Vesicular arbuscular mycorrhizae (AM), 13 Viability, 429, 433, 450, 453 Viable, 393 Viruses, 200 Vitality, 450, 453 Waste source, agricultural, 519 industrial, 520 municipal, 520

Waste treatment, conventional, 521–522 emerging, 522–523 Weed management, 138–139 Xanthophyll, 430, 450, 455, 456, 458 Yeast extract, 129 YEMA medium, 431 Yield, 41–50, 52–61, 64–66, 70–73, 79 Zinc, 449, 578 Zoosphere, 195

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