Global Perspectives on Astaxanthin: From Industrial Production to Food, Health, and Pharmaceutical Applications [1 ed.] 0128233044, 9780128233047

Citation preview

Global Perspectives on Astaxanthin

This page intentionally left blank

Global Perspectives on Astaxanthin From Industrial Production to Food, Health, and Pharmaceutical Applications Edited by Gokare A. Ravishankar Ambati Ranga Rao

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN : 978-0-12-823304-7

For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Acquisitions Editor: Erin Hill-Parks Editorial Project Manager: Sam W. Young Production Project Manager: Selvaraj Raviraj Cover Designer: Matthew Limbert Typeset by SPi Global, India

Professor Atul R. Mehta (1929–2018)

This volume is dedicated to Prof. Atul R. Mehta, a doyen in the field of research on secondary metabolites from cultured plant cells. He was a remarkable scientist who contributed immensely for the in vitro production of phytochemicals—phenolics, flavonoids, steroids, alkaloids, and many bioactive metabolites of value economic value. Born on August 31, 1929, in Petlad, Khera District in Gujarat, India. He obtained BSc and MSc in Botany from Maharaja Sayajirao University (MSU) of Baroda during 1951 and 1953, respectively. He continued as a lecturer in Botany in the same university and worked on taxonomy of grasses. His love for experimental botany took him to University of Swansea, UK, to work for his PhD under the supervision of Prof. Herbert E Street, a world renowned plant cell culture scientist.

Dedication

For his postdoctoral study, Dr. Mehta later joined Prof. John E. Staba’s laboratory, at the University of Minnesota, Minneapolis, USA, to work on plant secondary metabolites. He rejoined MSU Baroda in 1963 as reader and then rose to the position as Professor and Head of Botany Department in the year 1981. During his distinguished career in Baroda, he guided 25 students for PhD degree, who have now occupied positions in universities, research labs, and industries in several countries. Incidentally, Dr. Gokare A. Ravishankar is one of his doctoral students. Mehta taught Algae and Plant Tissue Culture besides his masterly subjects, plant physiology and biochemistry. He inspired his students to take up career in basic and applied biotechnology, which was his favorite subject. He received a number of projects from the Government of India as competitive grants. He was also keen on the environmental impact of Naramada Dam construction, which turned his attention to aquatic biology as the major topic of his research. He superannuated from M.S.U in 1989. He continued his active research in medicinal plants and bioactive molecules at Gujarat Agricultural University at Anand, as emeritus scientist of CSIR, where he established a state of art laboratory for plant tissue and cell culture. Prof. Mehta was recipient of the Jaikrishna Indrajit Award by the Hari Om Ashram, in 1978, and was awarded the Prof. Vishwambhar Puri Medal by the Indian Botanical Society in 1991. He was selected to deliver the Prof. Philip R. White Memorial Lecture at Jadavpur University in 1965 and the first Prof. H. E. Street Memorial Lecture at Nagpur University in 1982. He also delivered the Prof. A. R. Chavan Memorial Lecture at the M.S. University of Baroda in 1998. He was an elected member of the International Association of Plant Tissue Culture (IAPTC) at the University of Calgary, Canada, and also its National Correspondent for many years. He organized an international workshop sponsored by UNESCO International Crop Research Organization, besides conducting an NBTB sponsored National Training Programme for scholars and faculty between 1990 and 1994.

vi

Dedication

Mehta served in the National Committees like DST, DBT, UGC, ICAR, and CSIR, in India, as an expert member. Besides, he visited several universities and delivered lectures as a National Professor. He was a core team member of the All India Coordinated Project on Medicinal and Aromatic Plants of ICAR. Mehta was a fond lover of classical music and used to sing songs/ghazals. Known for his cricketing talent, he represented the M.S. University cricket team and also the drama group. Mehta always attributed his success to his late wife Yashaswini Mehta. He breathed his last on December 23, 2018 at Vadodara, Gujarat. He is survived by a son and daughter. An inspiring teacher and a dedicated researcher, Mehta was an embodiment of nobility of character, generosity, and benevolence. It is a matter of privilege and honor for the editors to dedicate this volume in memory of this great scientist and a noble soul.

vii

This page intentionally left blank

Contents Contributors.................................................................................................. xxvii About the editors.......................................................................................... xxxiii Preface ....................................................................................................... xxxvii Acknowledgments ............................................................................................. xli SECTION 1 ASTAXANTHIN FROM DIVERSE SOURCES LEADING TO INDUSTRIAL PRODUCTION Chapter 1: Synthesis of astaxanthin and its esters ................................................. 1 Stanislav Vitalievich Pechinskii, Anna Gurgenovna Kuregyan, and Eduard Tonikovich Oganesyan

1 Introduction............................................................................................................... 3 2 General characteristics of natural astaxanthin esters............................................... 4 2.1 Chemical synthesis of astaxanthin for industrial production ................................. 4 2.2 Composition of natural astaxanthin esters .............................................................. 5 2.3 The benefits of astaxanthin esters ........................................................................... 7 2.4 Direction modification of astaxanthin structure ..................................................... 7 3 Prospects for the use of biocatalysts in the synthesis of semisynthetic derivatives of astaxanthin......................................................................................... 8 3.1 Characterization of biocatalysts .............................................................................. 8 3.2 Difficulties when working with biocatalysts .......................................................... 8 3.3 Pharmacy biocatalysis ............................................................................................. 9 4 Synthesis of astaxanthin esters............................................................................... 10 4.1 Acid selection for esterification of astaxanthin .................................................... 10 4.2 The selection of conditions for the synthesis of astaxanthin esters ..................... 11 4.3 Synthesis of semisynthetic astaxanthin esters....................................................... 11 4.4 Confirmation of the structure of astaxanthin esters.............................................. 12 5 Conclusions............................................................................................................. 14 References................................................................................................................... 15

Chapter 2: The physiology of astaxanthin production by carotenogenic microalgae ...................................................................................................... 19 Alexei Solovchenko and Galina Minyuk

1 Introduction............................................................................................................. 19 2 Biosynthesis of astaxanthin and its coordination with lipid biosynthesis............. 21 ix

Contents

3 Triggering of astaxanthin biosynthesis and its regulation..................................... 24 4 Biological significance of astaxanthin accumulation ............................................ 26 4.1 Optical shielding of the cell .................................................................................. 26 4.2 Possible antioxidative function of astaxanthin ..................................................... 28 4.3 Suppression of ROS formation ............................................................................. 29 4.4 Sink for photosynthates and energy store ............................................................. 29 5 Concluding remarks: Perspectives for astaxanthin from microalgae .................... 29 Acknowledgment ........................................................................................................ 30 References................................................................................................................... 31

Chapter 3: Astaxanthin from Chromochloris zofingiensis: Feasibility analysis ...... 37 Jun-Hui Chen, Dong Wei, Ambati Ranga Rao, and Gokare A. Ravishankar

1 Introduction............................................................................................................. 38 2 Astaxanthin production by Chromochloris zofingiensis........................................ 39 2.1 Characteristics of Chromochloris zofingiensis...................................................... 39 2.2 Culture conditions of Chromochloris zofingiensis for astaxanthin production.... 40 3 Advantages and limitations of Chromochloris zofingiensis for astaxanthin production ............................................................................................................... 46 3.1 Advantages of Chromochloris zofingiensis-based astaxanthin production .......... 46 3.2 Limitations and challenges for Chromochloris zofingiensis application ............. 48 4 Engineering strategies for enhanced astaxanthin production ................................ 48 4.1 Genetic and metabolic engineering for strain improvement ................................ 48 4.2 Bioprocess engineering for enhanced astaxanthin production ............................. 50 4.3 Integrated microalgal biorefinery for low-cost astaxanthin production............... 52 5 Conclusions............................................................................................................. 55 Acknowledgments ...................................................................................................... 55 Conflict of interest...................................................................................................... 55 References................................................................................................................... 55

Chapter 4: Astaxanthin in microalgae Eustigmatophyceae .................................... 61 Clara B. Martins, Mariana F.G. Assunc¸a˜o, Joana D. Ferreira, and Lı´lia M.A. Santos

1 Introduction............................................................................................................. 61 2 Astaxanthin structure.............................................................................................. 62 3 Synthesis and accumulation of astaxanthin ........................................................... 62 4 Bioactivity of astaxanthin....................................................................................... 64 5 Current global market of astaxanthin..................................................................... 64 6 Astaxanthin in microalgae...................................................................................... 65 7 Astaxanthin in the Eustigmatophyceae .................................................................. 66 8 Conclusions............................................................................................................. 67 References................................................................................................................... 68

Chapter 5: Astaxanthin production by autotrophic cultivation of Haematococcus pluvialis: A success story .................................................................................. 71 Ignacio Niizawa, Brenda Y. Espinaco, Susana E. Zorrilla, and Guillermo A. Sihufe

1 Introduction............................................................................................................. 71 x

Contents

2 Physiological aspects of Haematococcus pluvialis................................................ 72 2.1 Taxonomy and life cycle....................................................................................... 72 2.2 Biochemical composition throughout Haematococcus pluvialis life cycle ................................................................................................................ 74 3 Astaxanthin biosynthesis in Haematococcus pluvialis .......................................... 74 3.1 Overall pathway for astaxanthin biosynthesis ...................................................... 74 3.2 Effects of light radiation on carotenoid genes...................................................... 76 3.3 Advantages of astaxanthin synthesized by Haematococcus pluvialis.................................................................................................................. 77 4 Astaxanthin market aspects .................................................................................... 78 5 Haematococcus pluvialis mass cultivation process for astaxanthin production ............................................................................................................... 79 5.1 Inoculum propagation............................................................................................ 79 5.2 Haematococcus pluvialis mass culture process .................................................... 80 5.3 Biomass harvesting................................................................................................ 83 5.4 Astaxanthin extraction from biomass.................................................................... 84 5.5 Astaxanthin final product formulation .................................................................. 84 6 Conclusions............................................................................................................. 85 Acknowledgments ...................................................................................................... 86 References................................................................................................................... 86

Chapter 6: Optimization of astaxanthin production processes from microalga Haematococcus.............................................................................................. 91 Xin Li, Xiaoqian Wang, Duanpeng Yang, Zhengquan Gao, and Jian Li

1 Introduction............................................................................................................. 91 2 Current industrial production ................................................................................. 93 2.1 Current production processes ................................................................................ 93 2.2 Current production facilities.................................................................................. 94 2.3 Current production cost estimation ....................................................................... 96 3 Strain selection and improvement.......................................................................... 97 3.1 Strain selection ...................................................................................................... 97 3.2 Strain improvement by mutagenesis ..................................................................... 98 3.3 Strain improvement by metabolic engineering................................................... 100 4 Optimizing vegetative growth for biomass amassment....................................... 102 4.1 Optimization of growth media ............................................................................ 102 4.2 Optimization of temperature, light intensity, and pH conditions............................................................................................................. 103 4.3 Optimization of aeration and CO2 addition levels ............................................. 103 4.4 Selection of PBRs for cultivation ....................................................................... 104 5 Optimizing stress conditions for AX accumulation............................................. 105 5.1 Light supply ......................................................................................................... 105 5.2 Temperature ......................................................................................................... 106 5.3 Nutrient depletion ................................................................................................ 106 5.4 Salinity and CO2 levels ....................................................................................... 107 5.5 Perspective on process optimization ................................................................... 107 xi

Contents

6 Developing novel cultivation methodologies ...................................................... 108 6.1 Sequential heterotrophic and phototrophic cultivation....................................... 108 6.2 One-step approach ............................................................................................... 108 6.3 Indoor artificial light cultivation ......................................................................... 109 6.4 Biofilm cultivation............................................................................................... 110 6.5 Mixotrophic cultivation ....................................................................................... 110 7 Conclusions........................................................................................................... 111 Acknowledgments .................................................................................................... 112 References................................................................................................................. 112

Chapter 7: Production of astaxanthin by Haematococcus pluvialis: Lab processes to scale up including the cost considerations ...................................................... 121 Guilherme Augusto Colusse, Maria Eug^enia Rabello Duarte, Julio Cesar de Carvalho, and Miguel Daniel Noseda

1 2 3 4 5

Introduction........................................................................................................... 121 Upstream laboratory process evaluation .............................................................. 122 Haematococcus pluvialis biomass and astaxanthin production cost................... 125 Astaxanthin extraction laboratory conditions ...................................................... 125 Astaxanthin producing Haematococcus pluvialis: Broadening horizon .................................................................................................................. 127 6 Conclusion ............................................................................................................ 127 Acknowledgments .................................................................................................... 128 References................................................................................................................. 128

Chapter 8: Astaxanthin-biological production and regulation for enhanced yields.............................................................................................. 131 Mingcan Wu, Zhenfan Chen, Jiayi He, and Jiangxin Wang

1 2 3 4

What is astaxanthin .............................................................................................. 132 The biological functions and applications of astaxanthin ................................... 132 Biosources of astaxanthin..................................................................................... 133 Astaxanthin biosynthesis pathways in H. pluvialis ............................................. 134 4.1 The astaxanthin biosynthesis pathways .............................................................. 135 4.2 Gene regulation of carotenoid biosynthesis........................................................ 138 4.3 The systems biology of H. pluvialis ................................................................... 138 5 Astaxanthin production improvement methods ................................................... 139 5.1 Light density and quality..................................................................................... 139 5.2 Temperature ......................................................................................................... 140 5.3 Metal ions ............................................................................................................ 140 5.4 Salinity ................................................................................................................. 142 6 Strain developments ............................................................................................. 142 6.1 Random mutagenesis for astaxanthin production ............................................... 142 6.2 Rational genetic engineering in H. pluvialis strains........................................... 144 7 Conclusion and perspectives ................................................................................ 144 References................................................................................................................. 145 xii

Contents

Chapter 9: Metabolic engineering of astaxanthin pathway and heterologous production in novel organisms.......................................................................... 151 Anila Narayanan, Daris P. Simon, Kathiresan Shanmugam, Sarada Ravi, Ambati Ranga Rao, and Gokare A. Ravishankar

1 Introduction........................................................................................................... 153 2 Astaxanthin biosynthetic pathway........................................................................ 154 3 Classical genetic approaches to improve astaxanthin yield (mutagenicity)....................................................................................................... 157 4 Metabolic pathway engineering ........................................................................... 159 4.1 Pathway engineering by genetic transformation................................................. 159 4.2 Carotenoid production through metabolic engineering ...................................... 162 5 Conclusion ............................................................................................................ 171 References................................................................................................................. 171

Chapter 10: Revealing mechanisms of algal astaxanthin production and bioengineering potential using multiomics.......................................................... 181 Tim L. Jeffers and Melissa S. Roth

1 Introduction: Astaxanthin, a carotenoid for human health and industry ............ 181 2 Evolution and distribution of astaxanthin accumulation in algae ....................... 185 3 Comparative genomics of H. pluvialis and C. zofingiensis................................. 186 3.1 Gene family expansions in astaxanthin biosynthesis.......................................... 187 4 Regulation of carotenoid biosynthesis for astaxanthin accumulation ................. 188 4.1 Strategies to accumulate astaxanthin .................................................................. 189 4.2 Integrating varied signals to lead to astaxanthin accumulation ......................... 190 4.3 Strategies to find candidate transcription factors that initiate astaxanthin accumulation........................................................................................................ 194 4.4 Posttranslational regulation of the carotenoid biosynthesis pathway................. 195 5 Biosynthesis, modification, and packaging of astaxanthin in algae.................... 196 5.1 Synthesizing astaxanthin precursors in the chloroplast ...................................... 196 5.2 Exporting astaxanthin precursors from the chloroplast ...................................... 197 5.3 Final steps of astaxanthin biosynthesis and esterification likely occur at the ER .............................................................................................................. 198 5.4 Formation of the astaxanthin-rich lipid droplet .................................................. 200 5.5 Carotenoid and fatty acid biosynthesis: Coordination or competition? ............. 200 5.6 What happens to astaxanthin when red cells regreen?....................................... 201 6 Conclusion: From mechanistic models to engineering strategies ....................... 201 Acknowledgments .................................................................................................... 202 References................................................................................................................. 202

Chapter 11: Astaxanthin production from Haematococcus pluvialis by using illuminated photobioreactor ............................................................................. 209 Yiu-Hang Ho, Yee-Keung Wong, and Ambati Ranga Rao

1 Introduction........................................................................................................... 209 2 Comparison between the closed and open system .............................................. 210 xiii

Contents

3 Physical and chemical configuration of the illuminated photobioreactors.................................................................................................... 210 3.1 Light conditions in the biomass and astaxanthin production period.................. 211 3.2 Design of the illuminance system for the photobioreactors............................... 212 3.3 Air mixing in the photobioreactors ..................................................................... 213 3.4 Temperature control in the photobioreactors...................................................... 213 3.5 Hydrodynamic and mass transfer in the photobioreactors ................................. 214 3.6 Chemicals factors of the illuminated photobioreactors ...................................... 214 4 Process for scaling up the production from laboratory to pilot and mass scale ...................................................................................................... 214 4.1 Selection of photobioreactors at different production stages ............................. 214 4.2 Mass culture of H. pluvialis in open versus closed systems .............................. 217 4.3 Astaxanthin production process at commercial scale......................................... 218 5 Conclusion ............................................................................................................ 221 Acknowledgment ...................................................................................................... 221 References................................................................................................................. 221

Chapter 12: Recent developments in astaxanthin production from Phaffia rhodozyma and its applications ...................................................................... 225 Yuan Zhuang and Ming-Jun Zhu

1 2 3 4

Introduction........................................................................................................... 226 Characteristics of P. rhodozyma as an astaxanthin source.................................. 227 Biosynthesis of astaxanthin from P. rhodozyma ................................................. 229 Accumulation strategies of astaxanthin from P. rhodozyma............................... 232 4.1 Strain improvement ............................................................................................. 232 4.2 Medium components ........................................................................................... 236 4.3 Cultivation conditions.......................................................................................... 238 4.4 Fermentation promoters....................................................................................... 241 5 Potential applications of astaxanthin from P. rhodozyma ................................... 242 6 Conclusions........................................................................................................... 243 Acknowledgments .................................................................................................... 244 References................................................................................................................. 244

Chapter 13: Turning leftover to treasure: An overview of astaxanthin from shrimp shell wastes ........................................................................................ 253 J.Y. Cheong and M. Muskhazli

1 2 3 4

Introduction......................................................................................................... 253 Properties of astaxanthin .................................................................................... 256 The color of astaxanthin..................................................................................... 257 Managing crustacean wastes and turning it into a gem..................................... 258 4.1 A better solution to manage crustacean waste.................................................. 258 4.2 The modern trend in managing crustacean wastes ........................................... 259 4.3 Mechanical methods .......................................................................................... 261

xiv

Contents

5 Extraction of astaxanthin.................................................................................... 261 6 Identification of astaxanthin............................................................................... 265 7 What comes next? .............................................................................................. 267 8 Contribution of astaxanthin to the economy...................................................... 268 9 Future prospects.................................................................................................. 272 10 Conclusion .......................................................................................................... 273 References................................................................................................................. 273

SECTION 2 ASTAXANTHIN: EXTRACTION, CHARACTERIZATION, AND DOWNSTREAM PROCESSING Chapter 14: Industrial perspective on downstream processing of Haematococcus pluvialis .............................................................................. 283 Thomas O. Butler and Ba´rbara Guimara˜es

1 Downstream processing of astaxanthin-derived Haematococcus pluvialis and the challenges to overcome ........................................................................... 283 2 Harvesting dilute cultures..................................................................................... 285 2.1 Batch, semicontinuous, and continuous harvesting approaches......................... 286 2.2 The commercial approach to harvesting............................................................. 288 2.3 Centrifugation ...................................................................................................... 289 2.4 Flocculation ......................................................................................................... 290 2.5 Future harvesting trends ...................................................................................... 292 3 Cell disruption and extraction .............................................................................. 292 3.1 The H. pluvialis aplanospore (hematocysts) challenge ...................................... 292 3.2 A dry or wet process ........................................................................................... 292 3.3 Cell disruption: Bead milling, the industry standard.......................................... 293 3.4 Drying and cell disruption................................................................................... 293 3.5 Extraction using ScCO2 ....................................................................................... 295 3.6 The wet approach bypassing drying ................................................................... 296 3.7 Alternative methods to bypass cell disruption and extraction ........................... 296 4 Astaxanthin delivery vehicles for humans: Nutraceutical and pharmaceutical ...................................................................................................... 297 4.1 Emulsification and encapsulation technology..................................................... 298 4.2 Challenges to address in encapsulation and formulation ................................... 301 5 Direct incorporation in feed and food.................................................................. 301 5.1 Aquaculture.......................................................................................................... 301 5.2 Livestock and pets ............................................................................................... 303 5.3 Functional food and fortifying agent for humans............................................... 303 6 Bioavailability, digestibility, stability, and scalability of astaxanthin........................................................................................................ 304 7 Conclusions and future direction ......................................................................... 305 References................................................................................................................. 305

xv

Contents

Chapter 15: Bioextraction of astaxanthin adopting varied techniques and downstream processing methodologies .............................................................. 313 Xiang Cheng and Mahfuzur Shah

1 Introduction........................................................................................................... 313 2 Cell wall structure and disruption techniques...................................................... 316 2.1 H. pluvialis cell morphology, life cycle, and cell wall structure ....................... 316 2.2 Cell wall disruption techniques of H. pluvialis .................................................. 319 3 Conventional and emerging astaxanthin extraction technologies ....................... 326 3.1 Chemical solvent extraction ................................................................................ 326 3.2 Vegetable oil-based extraction ............................................................................ 328 3.3 Microwave-assisted extraction ............................................................................ 328 3.4 Ultrasound-assisted extraction............................................................................. 329 3.5 ScCO2 extraction ................................................................................................. 329 3.6 Magnetic-assisted extraction ............................................................................... 331 3.7 IL-based extraction .............................................................................................. 331 3.8 Supramolecular solvent extraction ...................................................................... 332 4 Astaxanthin from other bioresources ................................................................... 332 5 Future prospect and conclusion............................................................................ 334 References................................................................................................................. 334

Chapter 16: Overview of extraction of astaxanthin from Haematococcus pluvialis using CO2 supercritical fluid extraction technology vis-a-vis quality demands ....................................................................................................... 341 Khem Chand Saini, Digvijay Singh Yadav, Sanjeet Mehariya, Parikshita Rathore, Bikash Kumar, Tiziana Marino, Gian Paolo Leone, Pradeep Verma, Dino Musmarra, and Antonio Molino

1 Introduction........................................................................................................... 342 2 Haematococcus pluvialis: The best suitable organism for the production of astaxanthin........................................................................................................ 344 3 Different extraction methods of astaxanthin from Haematococcus pluvialis................................................................................................................. 346 4 Supercritical CO2 fluid extraction (SFE-CO2) for extraction of astaxanthin ..... 346 5 Different influential parameters in astaxanthin extraction .................................. 348 5.1 Effect of temperature........................................................................................... 348 5.2 Effect of pressure................................................................................................. 349 5.3 Effect of CO2 flow rate ....................................................................................... 349 5.4 Effect of polar entrainer ...................................................................................... 350 6 Analysis of astaxanthin ........................................................................................ 350 6.1 HPLC analysis ..................................................................................................... 351 6.2 Antioxidant activity analysis ............................................................................... 351 7 Conclusions........................................................................................................... 351 Acknowledgments .................................................................................................... 352 Conflicts of interest .................................................................................................. 352 References................................................................................................................. 352 xvi

Contents

Chapter 17: High-pressure extraction of astaxanthin from Haematococcus pluvialis ....................................................................................................... 355 ´nchez M.D. Macias-Sa

1 2 3 4

Introduction........................................................................................................... 355 High-pressure extraction techniques .................................................................... 356 Haematococcus pluvialis-derived astaxanthin ..................................................... 357 Recovery of astaxanthin in red phase by high-pressure extraction techniques ............................................................................................................. 358 4.1 Drying step........................................................................................................... 359 4.2 Pretreatment step ................................................................................................. 360 4.3 Recovery step ...................................................................................................... 361 5 Current global market and market players of H. pluvialis astaxanthin ............................................................................................................ 366 6 Conclusion and perspectives ................................................................................ 367 Acknowledgments .................................................................................................... 369 References................................................................................................................. 369

Chapter 18: Astaxanthin extraction—Recent methods, developments and case studies............................................................................................. 375 Hamed Vatankhah and Hosahalli Ramasamy

1 2 3 4 5 6 7 8 9 10

Introduction......................................................................................................... 375 Solvent extraction ............................................................................................... 376 Microwave-assisted extraction (MAE) .............................................................. 376 Ultrasound-assisted extraction (UAE)................................................................ 376 Oil stripping method........................................................................................... 377 Ultrafiltration ...................................................................................................... 379 Supercritical fluid extraction (SFE) ................................................................... 379 Enzymatic hydrolysis method ............................................................................ 380 Magnetic field-assisted extraction (MF) ............................................................ 380 High-pressure extraction (HPE) ......................................................................... 381 10.1 HPE of astaxanthin: Case studies ................................................................... 383 11 Conclusions......................................................................................................... 385 References................................................................................................................. 385

SECTION 3 ASTAXANTHIN FOR FOOD, HEALTH, PHARMACEUTICALS—SAFETY AND REGULATORY ISSUES Chapter 19: Efficacy of astaxanthin from different sources: Reports on the suitability for human health and nutrition ......................................................... 391 Bob Capelli, Shawn Talbott, Lixin Ding, and Francis Capelli

1 Introduction......................................................................................................... 391 2 Chemical differences between forms of astaxanthin ......................................... 392 3 Differences in antioxidant activity between forms of astaxanthin.................... 395 xvii

Contents

4 Animal research shows superior efficacy and bioactivity of NAT-AX in comparison with SYN-AX and PH-AX......................................................... 397 5 Survival rates, stress resistance and growth rates in shrimp ............................. 398 6 Gastric ulcers in rats—Study #1 ........................................................................ 400 7 Gastric ulcers in rats—Study #2 ........................................................................ 400 8 Antioxidant activity and increase of lifespan in model organism for longevity in mammals .................................................................................. 400 9 Endurance in mice .............................................................................................. 402 10 Skin cancer, antioxidant activity, retinol levels and tyrosinase enzyme levels in rats........................................................................................................ 403 11 Summary of human clinical research................................................................. 406 12 Conclusions......................................................................................................... 406 Competing interests .................................................................................................. 408 Authors’ contributions .............................................................................................. 408 Acknowledgments and funding................................................................................ 408 Description of additional file ................................................................................... 408 References................................................................................................................. 408

Chapter 20: Oxidation products of astaxanthin: An overview.............................. 411 Takashi Maoka

1 Introduction........................................................................................................... 411 2 Apoastaxanthins as autooxidation products of astaxanthin ................................. 413 3 Astaxanthin epoxides and endperoxides as reaction products of astaxanthin with active oxygen species ............................................................... 413 4 Reaction products of astaxanthin with hypochlorous acid/hypochlorite (HOCl/OCl ) ........................................................................................................ 420 5 Reaction products of astaxanthin with active nitrogen reactive nitrogen species ................................................................................................................... 421 6 Apoastaxanthins as impurity of synthetic astaxanthin......................................... 423 7 Conclusion ............................................................................................................ 423 References................................................................................................................. 424

Chapter 21: Anticancer properties of astaxanthin: A molecule of great promise......................................................................................................... 427 Pinar Buket Demirel and Bilge Guvenc Tuna

1 Introduction........................................................................................................... 428 2 Cancer preventive mechanisms of ATX .............................................................. 429 2.1 Antioxidant activity ............................................................................................. 429 2.2 Immunomodulatory activity ................................................................................ 434 2.3 Antiinflammatory activity ................................................................................... 435 3 Effects of ATX on tumor progression ................................................................. 436 3.1 Proliferation ......................................................................................................... 436 3.2 Apoptosis ............................................................................................................. 437 3.3 Invasion and metastasis ....................................................................................... 438 4 Combination cancer treatment with ATX............................................................ 439 xviii

Contents

5 Conclusions........................................................................................................... 440 References................................................................................................................. 440

Chapter 22: Astaxanthin for improved muscle function and enhanced physical performance ...................................................................................... 447 ˚ ke Lignell Karen A. Hecht, Joerg Schnackenberg, Arun Nair, and A

1 Introduction........................................................................................................... 447 2 The impact of oxidative stress in skeletal muscle during exercise ..................... 449 3 The role of astaxanthin on mitochondrial function and oxidative stress during exercise............................................................................................ 450 4 Outcomes in strength and endurance training with astaxanthin.......................... 453 5 The function of astaxanthin in exercise-induced inflammation and muscle recovery .................................................................................................... 458 6 Conclusions........................................................................................................... 460 Acknowledgments .................................................................................................... 461 References................................................................................................................. 461

Chapter 23: Comprehensive integrated overview of the experimental and clinical neuroprotective effect of astaxanthin ..................................................... 469 Aya I. Abdelaziz, Amany M. Gad, and Samar S. Azab

1 Sources of astaxanthin.......................................................................................... 470 1.1 Natural ................................................................................................................. 470 1.2 Synthetic astaxanthin........................................................................................... 471 2 Chemical structure and structure-activity relationship of astaxanthin ................ 471 3 Bioavailability and pharmacokinetics .................................................................. 472 3.1 Bioavailability of astaxanthin.............................................................................. 472 3.2 Pharmacokinetics of astaxanthin ......................................................................... 472 4 Mechanism of action ............................................................................................ 472 5 Safety and dose administration ............................................................................ 473 6 Astaxanthin and central nervous system.............................................................. 473 6.1 Chemotherapy-induced neurotoxicity and neuroinflammation .......................... 474 6.2 The liver brain axis.............................................................................................. 476 6.3 The kidney-brain axis.......................................................................................... 477 6.4 Diabetes mellitus and insulin resistance associated neurotoxicity..................... 478 6.5 Gut-brain axis and neurodegenerative diseases .................................................. 480 6.6 Astaxanthin-experimental studies in neurological diseases................................ 482 6.7 Astaxanthin-clinical studies in neurological diseases......................................... 484 7 Conclusion ............................................................................................................ 486 References................................................................................................................. 486

Chapter 24: Rheological properties of astaxanthin oleoresins and their derived products ............................................................................................ 495 ´ lvarez, and Marjorie Ja ´uregui-Tirado Pedro Cerezal-Mezquita, Carolina Espinosa-A

1 Introduction........................................................................................................... 495 2 Rheological studies............................................................................................... 497 xix

Contents

3 Rheological models used for astaxanthin oleoresins and other derived products................................................................................................................. 498 3.1 Power law model ................................................................................................. 498 3.2 Herschel-Bulkley model ...................................................................................... 498 3.3 Bingham plastic model........................................................................................ 498 3.4 Casson model....................................................................................................... 499 3.5 Mizrahi-Berk model ............................................................................................ 499 4 Statistical tools for validation of rheological models.......................................... 499 5 Rheological properties of oleoresin and emulsions of astaxanthin ..................... 500 5.1 Astaxanthin oleoresin (5%) ................................................................................. 500 5.2 Astaxanthin oleoresin (10%) ............................................................................... 505 5.3 Astaxanthin oleoresin (oil-water macroemulsion) .............................................. 507 6 Red meal (1.5%)................................................................................................... 511 7 Conclusions........................................................................................................... 515 References................................................................................................................. 515

Chapter 25: Astaxanthin nanoparticles from fabrication to applications in food formulations including regulatory issues ................................................. 519 Elham Taghavi, Navideh Anarjan, Hoda Jafarizadeh-Malmiri, Ambati Ranga Rao, and Gokare A. Ravishankar

1 Introduction........................................................................................................... 519 2 General aspects ..................................................................................................... 520 2.1 Bioavailability of astaxanthin.............................................................................. 521 2.2 Solubility of astaxanthin...................................................................................... 522 2.3 Astaxanthin solubilization ................................................................................... 522 3 Size reduced-astaxanthin (astaxanthin microparticles and nanoparticles) .......... 523 3.1 Size reduction techniques.................................................................................... 525 3.2 Stabilizing mechanisms of astaxanthin nanoparticles (astaxanthin nanodispersions).............................................................................. 527 3.3 Morphology of astaxanthin nanoparticles........................................................... 530 3.4 Rheological characteristics of astaxanthin nanoparticles ................................... 531 3.5 Cellular uptake of astaxanthin nanodispersions.................................................. 531 3.6 Astaxanthin nanoparticles in food formulations ................................................. 532 3.7 Safety issues of astaxanthin nanoparticles.......................................................... 534 4 Conclusion ............................................................................................................ 534 Acknowledgments .................................................................................................... 535 References................................................................................................................. 535

Chapter 26: Stability of astaxanthin during food processing and methods of preservation .............................................................................................. 539 Reza Tahergorabi, Kelvin Adrah, and Mehdi Abdollahi

1 Introduction........................................................................................................... 539 2 Chemical structure of astaxanthin........................................................................ 540 3 Stability of astaxanthin......................................................................................... 541

xx

Contents

4 Effect of temperature, light, acids, and pH on astaxanthin ................................. 542 5 Effect of food processing on astaxanthin............................................................. 542 5.1 Effect of cooking ................................................................................................. 542 5.2 Effect of milling .................................................................................................. 543 5.3 Effect of extrusion ............................................................................................... 543 5.4 Effect of microwave heating ............................................................................... 544 5.5 Effect of salting and smoking ............................................................................. 544 5.6 Effect of high-pressure processing...................................................................... 545 6 Preventing degradation of astaxanthin in food products ..................................... 545 7 Preservation techniques of astaxanthin ................................................................ 547 7.1 Edible oils ............................................................................................................ 547 7.2 Encapsulation....................................................................................................... 547 7.3 Lipid-based carriers ............................................................................................. 549 7.4 Emulsions............................................................................................................. 551 8 Stability of astaxanthin during food storage ........................................................ 551 9 Conclusions........................................................................................................... 552 References................................................................................................................. 553

Chapter 27: Beneficial effects of astaxanthin in cosmeceuticals with focus on emerging market trends.............................................................................. 557 Evi Amelia Siahaan, Ratih Pangestuti, Idham Sumarto Pratama, Yanuariska Putra, and Se-Kwon Kim

1 Introduction........................................................................................................... 557 2 Cosmeceuticals potential of astaxanthin .............................................................. 559 2.1 UV radiation effect on skin................................................................................. 559 2.2 Skin photoprotective............................................................................................ 559 2.3 Skin moisturizer and antiwrinkle ........................................................................ 560 2.4 Skin wound healing ............................................................................................. 561 2.5 Currative treatment for dermatitis....................................................................... 562 3 Commercial available product from astaxanthin and its future perspective ............................................................................................................ 562 4 Conclusion ............................................................................................................ 564 References................................................................................................................. 565

Chapter 28: Safety assessment and pharmaceutical effects of astaxanthin: An overview .................................................................................................. 569 P. Madan Kumar, J. Naveen, R. Janani, and V. Baskaran

1 2 3 4 5 6 7

Introduction........................................................................................................... 569 Sources of ASX .................................................................................................... 570 Structure of ASX .................................................................................................. 571 Bioavailability of ASX ......................................................................................... 571 Metabolites of ASX.............................................................................................. 574 Clinical and safety aspects of ASX...................................................................... 574 Biological activities of ASX ................................................................................ 578

xxi

Contents 7.1 Hepatoprotective effects .................................................................................... 578 7.2 Skin protection................................................................................................... 578 7.3 Cardioprotective effects..................................................................................... 579 7.4 Ocular protection ............................................................................................... 579 7.5 Antidiabetic effects............................................................................................ 580 7.6 Antiobesity effects............................................................................................. 580 7.7 Antiinflammatory activity ................................................................................. 581 7.8 Antiangiogenic effects....................................................................................... 581 7.9 Anticancer effects .............................................................................................. 581 7.10 Pro- and antiapoptotic effects............................................................................ 582 8 Conclusion ............................................................................................................ 583 Acknowledgments .................................................................................................... 583 Author contributions................................................................................................. 583 Conflicts of interest .................................................................................................. 583 References................................................................................................................. 583

SECTION 4 GLOBAL-SCENARIO OF ASTAXANTHIN PRODUCTION Chapter 29: Astaxanthin production and technology in Vietnam and other Asian countries .............................................................................................. 595 Luu Thi Tam, Dang Diem Hong, Ambati Ranga Rao, and Gokare A. Ravishankar

1 2 3 4

Introduction......................................................................................................... 596 Characteristics and structure of astaxanthin ...................................................... 597 Biosynthesis pathways of astaxanthin................................................................ 598 Sources of astaxanthin........................................................................................ 599 4.1 Chemical synthetic source................................................................................. 599 4.2 Natural sources .................................................................................................. 600 5 Biochemistry of astaxanthin............................................................................... 603 6 Biological activities of astaxanthin and its applications ................................... 604 6.1 Toxicity of astaxanthin...................................................................................... 605 6.2 Astaxanthin in human health............................................................................. 605 6.3 Astaxanthin in aquaculture and poultry industry.............................................. 606 6.4 Astaxanthin in cosmetic industry ...................................................................... 606 7 Production of astaxanthin and technology in Vietnam and Asian countries.............................................................................................................. 607 7.1 Production of astaxanthin and technology in Vietnam..................................... 607 7.2 Production of astaxanthin and technology in other Asian countries............................................................................................................. 618 8 Extract methods, storage and stability of astaxanthin ....................................... 625 9 Commercial market, safety and challenges of astaxanthin ............................... 626 10 Conclusions......................................................................................................... 627 Acknowledgments .................................................................................................... 627 References................................................................................................................. 628 xxii

Contents

Chapter 30: Utilization of astaxanthin from Haematococcus for its use in aquaculture and poultry industries................................................................ 635 Gamze Turan

1 Introduction........................................................................................................... 635 2 Benefits of astaxanthin utilisation in aquaculture................................................ 636 2.1 Benefits of astaxanthin on reproductivity and egg quality of aquatic animals ................................................................................................................. 636 2.2 Benefits of astaxanthin on growth performance and survival............................ 637 2.3 Benefits of astaxanthin on stress tolerance and disease resistance .................... 638 2.4 Benefits of astaxanthin on pigmentation ............................................................ 640 3 Benefits of astaxanthin utilisation in poultry industry ........................................ 641 3.1 Benefits of astaxanthin utilisation on egg yolk pigmentation............................ 642 3.2 Benefits of astaxanthin on hen and breast muscle tissue improvement and higher feed efficiency................................................................................... 642 4 Conclusion and future directions ......................................................................... 642 Acknowledgments .................................................................................................... 643 References................................................................................................................. 643

Chapter 31: Astaxanthin from bacteria as a feed supplement for animals ............ 647 Osman N. Kanwugu, Ambati Ranga Rao, Gokare A. Ravishankar, Tatiana V. Glukhareva, and Elena G. Kovaleva

1 Introduction........................................................................................................... 647 2 Native bacterial producers of astaxanthin............................................................ 649 3 Bioengineered bacterial producers of astaxanthin ............................................... 650 4 Bacterial astaxanthin as a feed supplement ......................................................... 656 5 Bacterial astaxanthin in poultry farming ............................................................. 658 6 Bacterial astaxanthin in aquaculture .................................................................... 660 7 Bacterial astaxanthin in livestock farming........................................................... 662 8 Conclusion ............................................................................................................ 662 Acknowledgment ...................................................................................................... 663 References................................................................................................................. 663

SECTION 5 MISCELLANEOUS: CAROTENOIDS AND THEIR HEALTH BENEFITS; ASTAXANTHIN AS COLORANT IN FOODS Chapter 32: Carotenoid metabolic pathways and their functional role in health and diseases..................................................................................... 671 Marisiddaiah Raju, Poorigali Raghavendra-Rao Sowmya, Rudrappa Ambedkar, Bangalore Prabhashankar Arathi, and Rangaswamy Lakshminarayana

1 2 3 4 5

Introduction........................................................................................................... 671 Biosynthesis of carotenoids.................................................................................. 673 Absorption, distribution and disposition of carotenoids in humans.................... 675 Carotenoids distribution in cell membrane .......................................................... 677 Carotenoids health benefits .................................................................................. 677 xxiii

Contents

6 Formation and biological significance of carotenoid metabolites or oxidative derivatives ........................................................................................ 679 7 Nonenzymatic photooxidation or autooxidation or in vitro generation of carotenoid oxidation products.......................................................................... 683 8 Conclusion ............................................................................................................ 685 Acknowledgments .................................................................................................... 686 References................................................................................................................. 686

Chapter 33: Current knowledge on the health benefits of carotenoids: Focus on the scientific evidence ................................................................................. 693 Delia B. Rodriguez-Amaya

1 Introduction........................................................................................................... 693 2 Carotenoids and chronic diseases......................................................................... 693 2.1 Mode of action..................................................................................................... 694 2.2 Methods of investigation ..................................................................................... 694 2.3 Appraisal of the scientific evidence.................................................................... 695 2.4 Present and future challenges.............................................................................. 711 2.5 Final considerations............................................................................................. 712 References................................................................................................................. 713

Chapter 34: Recent updates on the neuroprotective role of carotenoids: Astaxanthin and beyond.................................................................................. 719 Sajad Fakhri, Sana Piri, Mohammad Hosein Farzaei, and Eduardo Sobarzo-Sa´nchez

1 Introduction........................................................................................................... 720 2 The neuroprotective effects of carotenoids.......................................................... 722 2.1 Antiapoptotic effects............................................................................................ 722 2.2 Antineuroinflammatory effects ........................................................................... 724 2.3 Antioxidant effects .............................................................................................. 726 3 Beneficial effects of carotenoids on neurodegenerative disorders ...................... 728 3.1 Carotenoids and Alzheimer’s disease/Parkinson’s disease................................. 728 3.2 Carotenoids and multiple sclerosis/depression ................................................... 730 3.3 Carotenoids and central nervous system (brain/spinal cord) injuries ................................................................................................................. 731 3.4 Carotenoids and aging/autism/ALS..................................................................... 732 4 Conclusion ............................................................................................................ 733 References................................................................................................................. 733

Chapter 35: Storage stability studies of astaxanthin, oleoresins and emulsions, in products developed for human consumption ................................................... 741 ´ lvarez, Jenifer Palma-Ramı´rez, Pedro Cerezal-Mezquita, Carolina Espinosa-A ´uregui-Tirado, and Faviola Past en-Angel, Francisca Salinas-Fuentes, Marjorie Ja Marı´a del Carmen Ruı´z-Domı´nguez

1 Introduction........................................................................................................... 741 2 Function and application of astaxanthin .............................................................. 742 xxiv

Contents

3 Liquid and semiliquid dairy products with addition of astaxanthin oleoresin................................................................................................................ 743 3.1 Milks pigmented with astaxanthin oleoresin ...................................................... 743 3.2 Yogurts pigmented with astaxanthin oleoresin................................................... 749 4 Nongreasy liquid products with addition from astaxanthin oleoresin emulsion................................................................................................................ 754 4.1 Isotonic beverages ............................................................................................... 754 5 Mayonnaise dressing ............................................................................................ 760 5.1 Introduction.......................................................................................................... 760 5.2 Goal...................................................................................................................... 760 5.3 Materials and methods......................................................................................... 760 5.4 Results and discussion......................................................................................... 761 6 Conclusion ............................................................................................................ 766 References................................................................................................................. 766

Subject Index................................................................................................. 773

xxv

This page intentionally left blank

Contributors Aya I. Abdelaziz Department of Pharmacology, National Organization for Drug Control and Research (NODCAR), Egyptian Drug Authority (EDA), Giza, Egypt; Center of Integrative Health (CIH), Department of Research and Development, Faculty of Pharmacy, Heliopolis University, Cairo, Egypt Mehdi Abdollahi Department of Biology and Biological Engineering, Food and Nutrition Science, Chalmers University of Technology, Gothenburg, Sweden Kelvin Adrah Food and Nutritional Sciences Program, North Carolina Agricultural and Technical State University, Greensboro, NC, United States Rudrappa Ambedkar Department of Microbiology and Biotechnology, Jnana Bharathi Campus, Bangalore University, Bengaluru, India Navideh Anarjan Department of Engineering, Tabriz Branch, Islamic Azad University, Tabriz, Iran Bangalore Prabhashankar Arathi Department of Microbiology and Biotechnology, Jnana Bharathi Campus, Bangalore University, Bengaluru, India Mariana F.G. Assunc¸ a˜o Coimbra Collection of Algae (ACOI), Department of Life Sciences, University of Coimbra, Coimbra, Portugal Samar S. Azab Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt V. Baskaran Department of Biochemistry, CSIR-CFTRI, Mysore, India Thomas O. Butler Synalgae, Achterweg 65, De Kwakel, The Netherlands Bob Capelli Algae Health Sciences, Irvine, CA, United States Francis Capelli FJC Design, Ocala, FL, United States Julio Cesar de Carvalho Bioprocess Engineering and Biotechnology Department, Federal University of Parana´, Curitiba, Parana´, Brazil Pedro Cerezal-Mezquita Laboratory for Microencapsulation of Bioactive Compounds (LAMICBA), Department of Food Sciences and Nutrition, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile Jun-Hui Chen School of Food Science and Engineering, South China University of Technology, Guangzhou, PR China Zhenfan Chen Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, Shenzhen Engineering Laboratory for Marine Algal Biotechnology, Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China Xiang Cheng Iconthin Biotech Corp., Myhal Centre for Engineering Innovation and Entrepreneurship; Department of Mechanical & Industrial Engineering and Institute for Sustainable Energy, University of Toronto, Toronto, ON, Canada

J.Y. Cheong Department of Biology, Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Guilherme Augusto Colusse Postgraduate Program in Bioprocess Engineering and Biotechnology; Biochemistry and Molecular Biology Department, Federal University of Parana´, Curitiba, Parana´, Brazil Pinar Buket Demirel Department of Medical Biology and Genetics, Faculty of Medicine, Maltepe University, Istanbul, Turkey Lixin Ding BGG, Irvine, CA, United States Maria Eug^enia Rabello Duarte Biochemistry and Molecular Biology Department, Federal University of Parana´, Curitiba, Parana´, Brazil Brenda Y. Espinaco Instituto de Desarrollo Tecnolo´gico para la Industria Quı´mica (INTEC), Consejo Nacional de Investigaciones Cientı´ficas y Tecnicas (CONICET)-Universidad Nacional del Litoral (UNL), Santa Fe, Argentina ´ lvarez Laboratory for Microencapsulation of Bioactive Compounds Carolina Espinosa-A (LAMICBA), Department of Food Sciences and Nutrition, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile Sajad Fakhri Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran Mohammad Hosein Farzaei Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran Joana D. Ferreira Coimbra Collection of Algae (ACOI), Department of Life Sciences, University of Coimbra, Coimbra, Portugal Amany M. Gad Department of Pharmacology, National Organization for Drug Control and Research (NODCAR), Egyptian Drug Authority (EDA), Giza, Egypt Zhengquan Gao School of Life Sciences, Shandong University of Technology, Zibo, PR China Tatiana V. Glukhareva Institute of Chemical Engineering, Ural Federal University named after the first President of Russia B.N. Yeltsin, Ekaterinburg, Russia Ba´rbara Guimara˜es Porto, Portugal Jiayi He Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, Shenzhen Engineering Laboratory for Marine Algal Biotechnology, Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China Karen A. Hecht, Ph.D. AstaReal Inc., Moses Lake, WA, United States Yiu-Hang Ho School of Engineering, The Hong Kong University of Science and Technology, Hong Kong, China Dang Diem Hong Institute of Biotechnology, Vietnam Academy of Science and Technology (VAST); Graduate University of Science and Technology, VAST; ThuyLoi University, Dong Da, Hanoi, Vietnam Hoda Jafarizadeh-Malmiri Department of Chemical Engineering, Sahand University of Technology, Tabriz, Iran R. Janani Department of Biochemistry, CSIR-CFTRI, Mysore, India Marjorie Ja´uregui-Tirado Laboratory for Microencapsulation of Bioactive Compounds (LAMICBA), Department of Food Sciences and Nutrition, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile Tim L. Jeffers Department of Plant and Microbial Biology, University of California, Berkeley, CA, United States

Osman N. Kanwugu Institute of Chemical Engineering, Ural Federal University named after the first President of Russia B.N. Yeltsin, Ekaterinburg, Russia Se-Kwon Kim Department of Marine Life Science, College of Ocean Science and Technology, Korea Maritime and Ocean University, Busan, Republic of Korea Elena G. Kovaleva Institute of Chemical Engineering, Ural Federal University named after the first President of Russia B.N. Yeltsin, Ekaterinburg, Russia Bikash Kumar Bioprocess and Bioenergy Laboratory, Department of Microbiology, Central University of Rajasthan, Ajmer, Rajasthan, India Anna Gurgenovna Kuregyan Department of Pharmaceutical Chemistry, Pyatigorsk Medical and Pharmaceutical Institute, Volgograd State Medical University, Pyatigorsk, Russia Rangaswamy Lakshminarayana Department of Microbiology and Biotechnology, Jnana Bharathi Campus, Bangalore University, Bengaluru, India Gian Paolo Leone ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Department of Sustainability—CR Casaccia, Rome, Italy Jian Li School of Biological and Chemical Engineering, Panzhihua University, Panzhihua, PR China Xin Li School of Biological and Chemical Engineering, Panzhihua University, Panzhihua, PR China ˚ ke Lignell, Ph.D. AstaReal AB, Nacka, Sweden A M.D. Macias-Sa´nchez Department of Chemical Engineering and Food Technology, Science Faculty, University of Ca´diz, Ca´diz, Spain P. Madan Kumar Department of Biochemistry, CSIR-CFTRI, Mysore, India Takashi Maoka Research Institute for Production Development, Kyoto, Japan Tiziana Marino Department of Engineering, University of Campania “L.Vanvitelli”, Real Casa dell’Annunziata, Aversa, CE, Italy Clara B. Martins Coimbra Collection of Algae (ACOI), Department of Life Sciences, University of Coimbra, Coimbra, Portugal Sanjeet Mehariya ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Department of Sustainability—CR Portici, Portici, NA; Department of Engineering, University of Campania “L.Vanvitelli”, Real Casa dell’Annunziata, Aversa, CE, Italy; Bioprocess and Bioenergy Laboratory, Department of Microbiology, Central University of Rajasthan, Ajmer, Rajasthan, India Galina Minyuk A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS, Sevastopol, Russia Antonio Molino ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Department of Sustainability—CR Portici, Portici, NA, Italy M. Muskhazli Department of Biology, Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Dino Musmarra Department of Engineering, University of Campania “L.Vanvitelli”, Real Casa dell’Annunziata, Aversa, CE, Italy Arun Nair, Ph.D. AstaReal Inc., Moses Lake, WA, United States Anila Narayanan Department of Botany, St. Xaviers College for Women, Aluva, Kerala, India

J. Naveen Department of Biochemistry, CSIR-CFTRI, Mysore, India Ignacio Niizawa Instituto de Desarrollo Tecnolo´gico para la Industria Quı´mica (INTEC), Consejo Nacional de Investigaciones Cientı´ficas y Tecnicas (CONICET)-Universidad Nacional del Litoral (UNL), Santa Fe, Argentina Miguel Daniel Noseda Biochemistry and Molecular Biology Department, Federal University of Parana´, Curitiba, Parana´, Brazil Eduard Tonikovich Oganesyan Department of Organic Chemistry, Pyatigorsk Medical and Pharmaceutical Institute, Volgograd State Medical University, Volgograd, Russia Jenifer Palma-Ramı´rez Laboratory for Microencapsulation of Bioactive Compounds (LAMICBA), Department of Food Sciences and Nutrition, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile Ratih Pangestuti Research and Development Division for Marine Bio-Industry, Indonesian Institute of Sciences (LIPI), West Nusa Tenggara; Research Center for Oceanography, Indonesian Institute of Sciences (LIPI), Jakarta, Republic of Indonesia Faviola Pasten-Angel Laboratory for Microencapsulation of Bioactive Compounds (LAMICBA), Department of Food Sciences and Nutrition, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile Stanislav Vitalievich Pechinskii Pyatigorsk Medical and Pharmaceutical Institute, Volgograd State Medical University, Pyatigorsk, Russia Sana Piri Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran Idham Sumarto Pratama Research and Development Division for Marine Bio-Industry, Indonesian Institute of Sciences (LIPI), West Nusa Tenggara, Republic of Indonesia; College of Sciences and Engineering, James Cook University, Douglas, QLD, Australia Yanuariska Putra Research and Development Division for Marine Bio-Industry, Indonesian Institute of Sciences (LIPI), West Nusa Tenggara, Republic of Indonesia Marisiddaiah Raju Department of Botany, St. Joseph’s College Autonomous, Bengaluru, India Hosahalli Ramasamy Department of Food Science and Agricultural Chemistry, McGill University, St-Anne-de-Bellevue, QC, Canada Ambati Ranga Rao Centre of Excellence, Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research (Deemed to be University), Vadlamudi, Guntur, Andhra Pradesh, India Parikshita Rathore Department of Biochemistry and Microbial Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bhatinda, Punjab, India Sarada Ravi Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore, Karnataka, India Gokare A. Ravishankar C.D. Sagar Centre for Life Sciences, Dayananda Sagar College of Engineering, Dayananda Sagar Institutions, Bangalore, Karnataka, India Delia B. Rodriguez-Amaya Faculty of Food Engineering, University of Campinas, Campinas, SP, Brazil Melissa S. Roth Department of Plant and Microbial Biology, University of California, Berkeley, CA, United States

Marı´a del Carmen Ruı´z-Domı´nguez Laboratory for Microencapsulation of Bioactive Compounds (LAMICBA), Department of Food Sciences and Nutrition, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile Khem Chand Saini Molecular Genetics Laboratory, Center for Biosciences, School of Basic and Applied Sciences, Central University of Punjab, Bhatinda, Punjab, India Francisca Salinas-Fuentes Laboratory for Microencapsulation of Bioactive Compounds (LAMICBA), Department of Food Sciences and Nutrition, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile Lı´lia M.A. Santos Coimbra Collection of Algae (ACOI), Department of Life Sciences, University of Coimbra, Coimbra, Portugal Joerg Schnackenberg, Ph.D. AstaReal Co. Ltd., Tokyo, Japan Mahfuzur Shah Excel Career College, Courtenay, BC, Canada Kathiresan Shanmugam Department of Molecular Biology, Madurai Kamaraj University, Madurai, Tamilnadu, India Evi Amelia Siahaan Research and Development Division for Marine Bio-Industry, Indonesian Institute of Sciences (LIPI), West Nusa Tenggara, Republic of Indonesia Guillermo A. Sihufe Instituto de Desarrollo Tecnolo´gico para la Industria Quı´mica (INTEC), Consejo Nacional de Investigaciones Cientı´ficas y Tecnicas (CONICET)-Universidad Nacional del Litoral (UNL), Santa Fe, Argentina Daris P. Simon Department of Chemistry, Biochemistry and Physics, University of Quebec at Trois-Rivieres, Trois-Rivieres, QC, Canada Eduardo Sobarzo-Sa´nchez Laboratory of Pharmaceutical Chemistry, Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain; Instituto de Investigacio´n e Innovacio´n en Salud, Facultad de Ciencias de la Salud, Universidad Central de Chile, Santiago, Chile Alexei Solovchenko Lomonosov Moscow State University, Moscow, Russia Poorigali Raghavendra-Rao Sowmya Department of Microbiology and Biotechnology, Jnana Bharathi Campus, Bangalore University, Bengaluru, India Elham Taghavi Faculty of Fisheries and Food Science, University Malaysia Terengganu, Kuala Nerus, Terengganu, Malaysia Reza Tahergorabi Food and Nutritional Sciences Program, North Carolina Agricultural and Technical State University, Greensboro, NC, United States Shawn Talbott Eqqil, Draper, UT, United States Luu Thi Tam Institute of Biotechnology, Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam Bilge Guvenc Tuna Department of Biophysics, Faculty of Medicine, Yeditepe University, Istanbul, Turkey Gamze Turan Ege University, Fisheries Faculty, Aquaculture Department, Izmir, Turkey Hamed Vatankhah Department of Food Science and Agricultural Chemistry, McGill University, St-Anne-de-Bellevue, QC, Canada Pradeep Verma Bioprocess and Bioenergy Laboratory, Department of Microbiology, Central University of Rajasthan, Ajmer, Rajasthan, India

Jiangxin Wang Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, Shenzhen Engineering Laboratory for Marine Algal Biotechnology, Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China Xiaoqian Wang School of Biological and Chemical Engineering, Panzhihua University, Panzhihua, PR China Dong Wei School of Food Science and Engineering, South China University of Technology, Guangzhou, PR China Yee-Keung Wong Episteme Company Limited, Hong Kong, China Mingcan Wu Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, Shenzhen Engineering Laboratory for Marine Algal Biotechnology, Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China Digvijay Singh Yadav Molecular Genetics Laboratory, Center for Biosciences, School of Basic and Applied Sciences, Central University of Punjab, Bhatinda, Punjab, India Duanpeng Yang School of Biological and Chemical Engineering, Panzhihua University, Panzhihua, PR China Ming-Jun Zhu Guangdong Provincial Engineering and Technology Research Center of Biopharmaceuticals, School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou, China; College of Life and Geographic Sciences; The Key Laboratory of Biological Resources and Ecology of Pamirs Plateau in Xinjiang Uygur Autonomous Region, Kashi University, Kashi, China Yuan Zhuang Guangdong Provincial Engineering and Technology Research Center of Biopharmaceuticals, School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou, China Susana E. Zorrilla Instituto de Desarrollo Tecnolo´gico para la Industria Quı´mica (INTEC), Consejo Nacional de Investigaciones Cientı´ficas y Tecnicas (CONICET)-Universidad Nacional del Litoral (UNL), Santa Fe, Argentina

About the editors Gokare A. Ravishankar, professor of biotechnology, is presently the vice president of R&D in life Sciences and biotechnology at Dayananda Sagar Institutions, Bengaluru, India. Earlier he had a distinguished research career of over 30 years at the Central Food Technological Research Institute (CFTRI), Mysore, and in the institutions of government of India. Served as chairman of board of studies in biotechnology at the Visvesvaraya Technological University, Belgavi, and Academic Council Member of Dayananda Sagar University. He has also been a member of the boards of eight universities. He is an internationally recognized expert in the areas of food science and technology, plant biotechnology, algal biotechnology, food biotechnology and postharvest technologies, plant secondary metabolites, functional foods, herbal products, genetic engineering, and biofuels and served as visiting professors to Universities in Japan, Korea, Taiwan, and Russia. Dr. Ravishankar holds a master’s and PhD degree from Maharaja Sayajirao University of Baroda. He mentored over 40 PhD students, 62 master’s students, 7 postdocs, and 8 international guest scientists and authored over 260 peer-reviewed research papers in International and national journals, 50 reviews, 55 patents in India and abroad, and edited 5 books, with a h-index of 63 and over 17,000 citations. He has presented over 220 lectures in various scientific meetings in India and abroad, including visits to over 25 countries. Dr. Ravishankar received several coveted honors and awards as follows: Young Scientist award (Botany) by the then Prime Minister of India in 1992; National Technology Day Award of Government of India in 2003; Laljee Goodhoo Smarak Nidhi Award for food biotechnology R&D of industrial relevance and the prestigious, professor V. Subramanyan Food Industrial Achievement Award; Professor S.S. Katiyar Endowment Lecture Award in New Biology by Indian Science Congress; Professor Vyas Memorial Award of Association of Microbiologists of India; Professor V.N. Raja Rao Endowment Lecture Award in Applied Botany, University of Madras; Lifetime achievement award by the Society of Applied Biotechnologists, Dr. Diwaker Patel Memorial award by Anand Agricultural University, Anand; Prof. C.S. Paulose Memorial

xxxiii

About the editors Oration Award by Society for Biotechnologists of India; Prof. Gadgil Memorial Lecture Award, Plant Tissue Culture Association of India, 2020. He is honored as fellow of several national organizations in India, namely, National Academy of Sciences, National Academy of Agricultural Sciences, Association of Microbiologists of India, Society of Agricultural Biochemists, Society of Applied Biotechnologists, Indian Botanical Society, and the Association of Food Scientists and Technologists of India. He is also an elected member of Plant Tissue Culture Association of India. He has held honorary positions of President in Society of Biological Chemists, Mysore Chapter and President of Association of Microbiologists of India, and Mysore and Bangalore Chapters. He is a lifetime member of Nutrition Society of India and several biotechnology societies including Society for Biotechnologists of India, Biotechnology Research Society of India, International Coffee Genome Network, American Society of Plant Biologists, and Global Harmonization Initiative. He is a consultant to World Bank projects in the domains of postharvest technologies, plant biotechnologies for value addition to crop plants, and food biotechnologies. He has also served as advisor and resource person in international conferences, seminars, workshops, short courses and has convened national and international seminars in biology, biotechnology, and food science and technology. He is an associate editor and reviewer in a large number of reputed research journals. Ambati Ranga Rao is a senior scientist and associate professor in the Department of Biotechnology at Vignan’s Foundation for Science, Technology, and Research (Deemed to be University), Andhra Pradesh, India. Dr. Ranga Rao holds Bachelors and master’s degree from Acharya Nagarjuna University, Andhra Pradesh, India, and PhD degree from University of Mysore. He started his research career in 2004 as research assistant at the Department of Plant Cell Biotechnology, Council of Scientific and Industrial Research (CSIR)-Central Food Technological Research Institute (CFTRI), Mysuru, India, under the supervision of Dr. G. A. Ravishankar and Dr. R. Sarada. He was awarded Senior Research Fellow of Indian Council of Medical Research (ICMR), New Delhi in the year 2007. His PhD work at CFTRI focused on production of astaxanthin from cultured green alga Haematococcus pluvialis and its biological activities. He worked extensively on process optimization of algal biomass production, mass culture of various algal species in raceway ponds and photobioreactors and downstream processing of algal metabolites and evaluation of their possible nutraceutical applications in in vitro and in vivo models. Further Dr. Ranga Rao worked as lead scientist in Algal Technologies, Carot Labs Pvt. Ltd-India; Postdoctoral Research Associate in Laboratory of Algal Research and Biotechnology, Arizona State University, USA, under the supervision of Prof.

xxxiv

About the editors Milton Sommerfeld and Prof. Qiang Hu; Visiting Assistant Professor in Food Science and Technology Program, Beijing Normal University and Hong Kong Baptist University, United International college, China under the supervision of Prof. Bo Lei; and Visiting Senior Research Fellow (Associate Professor Grade) in Institute of Ocean and Earth Sciences, University of Malaya, Malaysia under the guidance of Prof. Phang Siew-Moi. He is the author of more than 40 peer-reviewed publications, 50 International/national conferences/symposia/invited talks, and 10 chapters in books. His research citations exceed 2180 with h-index (16) and i10-index (20) as Google Scholar. He has delivered lectures at international/national conferences/symposia in the United States, Canada, Brazil, China, Malaysia, Indonesia, and Oman. He has edited two books (CRC Press, USA), as coeditor, namely, Handbook of Algal Technologies and Phytochemicals: Volume-I Food, Health, and Nutraceutical Applications and Handbook of Algal Technologies and Phytochemicals: Volume II Phycoremediation, Biofuels, and Global Biomass Production. He was selected for Junior Scientist of the Year Award (2015) by National Environmental Science Academy, New Delhi, India; honored TWAS-Young Affiliate (2014) by Regional Office of South East Asia and the Pacific Chinese Academy of Sciences (CAS), China; received Young Scientist Award (2014) at the World Food Congress by International Union of Food Science and Technology (IUFoST), Canada; Carl Storm International Diversity Fellowship Award (2010) by Gordon Research Conferences, USA. He is an associate fellow of Andhra Pradesh Akademi of Sciences (2019) Government of Andhra Pradesh, India, and also fellow of the Society of Applied Biotechnology (2013), India. He has received research grants and travel grant fellowship as both international and national awards, under Young Scientist schemes. He is also serving as editorial board member and reviewer for reputed international and national journals.

xxxv

This page intentionally left blank

Preface Algae are indispensable resources for food, bioactives, nutraceuticals, pigments, bioenergy molecules, biofertilizers, and agents for bioremediation. They are also the primary sources of nutrients to aquatic and semiaquatic organisms supporting a number of life forms. The present day challenges in meeting food security are compounded by issues of climate change, demanding sustainable sources of food and food ingredients. In this context the food additives from microalgae are of prime importance as they can be grown autotrophically in an ecofriendly manner. Algae are also sources of novel molecules of high economic value, namely, specialty lipids, carbohydrates, pigments, and secondary metabolites. Carotenoids assume importance as the major natural pigments from algae. Algal forms are rich sources of β carotene, which is the precursor of vitamin A. They also produce α-carotene, lycopene, β-cryptoxanthin, lutein, zeaxanthin, astaxanthin, and a range of esters. Carotenoids in general exhibit antioxidant properties, and the most potent of them all is astaxanthin. In view of the importance of astaxanthin as a pigment and health-promoting compound, it is one of the most highly researched molecule in the recent times. It is produced not only by microalgae but also by few microorganisms. Astaxanthin’s demand as a pigment source is mainly met by the synthetic chemistry processes; however, from the biological activity point of view, the astaxanthin esters are of greater value. The natural sources provide the much needed high quality astaxanthin and its derivatives; however, it is expensive to extract and purify, compared with its substitutes produced from synthetic chemistry route. Crustaceans are popular sources of these molecules by virtue of their ability to accumulate the pigment through dietary means by feeding on phytoplanktons rich in astaxanthin and its esters. This volume is intended to provide a holistic understanding of the biological sources of astaxanthin and the biomass production technologies coupled with downstream processing for isolating the pigment for various applications. It comprises 35 chapters written by 121 authors, from 22 countries, representing the global coverage of R&D and industrial scenario on this topic. The chapters are divided into five sections as detailed in the succeeding text.

xxxvii

Preface Section 1: Astaxanthin from diverse sources leading to industrial production This section provides various production processes of astaxanthin and its esters, namely, the synthetic and semisynthetic routes, microalgae-mediated production, microbial sources of these pigments, and through crustacean wastes. The production of biomass and induction of astaxanthin formation using innovative culture methods have been described in detail. Improvement of yields of astaxanthin and production in novel hosts using metabolic engineering approaches has been discussed. Thus the production of natural astaxanthin and esters has been presented using innovative cultures systems. Section 2: Astaxanthin: extraction, characterization, and downstream processing After the production of astaxanthin-rich biomass using culture methodologies, or through the primary processing of pigment-rich crustacean wastes, the next important step is the downstream processing to effectively recover the valuable pigment. Accordingly, efficient extraction of astaxanthin coupled with its purification and characterization has been dealt in detail. Section 3: Astaxanthin for food, health, pharmaceuticals; safety and regulatory issues The purified astaxanthin and their derivatives are being used as functional foods and for specific health benefits as well as for pharmaceutical applications. The potential applications, namely, antioxidant, anticancer agent, promotion of muscular health, treatment of neurological disorders, and skin care agent in cosmeceutics, have been elegantly presented. The safety aspects of astaxanthin are of prime importance in finding relevant applications for supporting this industry. Accordingly the studies on safety, undertaken globally, have been reviewed. Section 4: Global-scenario of astaxanthin production Production of astaxanthin in Asian countries has been reviewed. Utilization of biomass rich in astaxanthin, in algae and bacteria, as a pigmentation source in aquaculture and poultry industries has been presented in detail. These technologies have gained worldwide popularity, ever since its approval by FDA for the use of astaxanthin as a source of pigmentation in cultured fishes in aquaculture industry. Section 5: Miscellaneous applications: caroteinoids and their health benefits; astaxanthin as food colorant Often, astaxanthin is associated with certain carotenoids; therefore it is important to recognize the benefits of carotenoids in health applications. Hence, we have brought in the relevance of carotenoids in general with respect to health and food applications. Moreover the use of astaxanthin as food colorant has been reviewed since there is increasing consumer preference for natural pigments as opposed to the use of synthetic dyes for food or pharmaceutical applications.

xxxviii

Preface We sincerely hope that this volume will immensely attract research scientists, students, industrialists, regulators, health care professionals, food industries, aquaculture industries, and phyco- and mycobiotechnology enterprises interested in the production and utilization of astaxanthin in food, health, and pharmaceuticals. It would also cater to the needs of professionals such as food and agricultural scientists, food experts, biotechnologists, bioengineers, ecologists, and biomass specialists. Its global relevance and outreach have implications for economical and sustainable production of astaxanthin to meet the increasing demands of this valuable pigment. Gokare A. Ravishankar Ambati Ranga Rao

xxxix

This page intentionally left blank

Acknowledgments It gives us great pleasure to thank all the contributors representing the global scientific community working on the topic of this unique volume on astaxanthin. Every one of them offered their best support in bringing out this exhaustive book by sharing their research and long-standing experience for the benefit of the readers who are interested in this topic. We gratefully acknowledge their total support especially during the COVID-19 pandemic, which demonstrates their total commitment. We are grateful to Mr. Samuel Young and Team of Elsevier in working closely with the editors and contributors to bring out the volume in an elegant manner. We are thankful to our families for their constant encouragement in the difficult times, which involved management of uncertain situations mostly by themselves, during the pandemic, while we were devoting our time for editing this volume. G.A.R thanks his wife, Shyla; son, Prashanth; daughter-in-law, Vasudha; and daughter, Apoorva. A.R.R thanks his wife, Deepika; daughter, Jesvisree; parents, Venkateswaralu and Tulasidevi; brothers; sisters-in-law; sisters; and brothers-in-law. G.A.R. is thankful to Dr. Premachandra Sagar, Vice Chairman, Dayananda Sagar Institutions, and Pro-Chancellor of Dayananda Sagar University, Bengaluru, for granting permission to take this additional responsibility. A.R.R is thankful to Dr. L. Rathaiah, Chairman; Mr. L. Sri Krishnadevarayalu, Vice-Chairman (Member of Parliament), Prof. Dr. K. Ramamurthy Naidu, Chancellor; Dr. M.Y.S. Prasad, Vice-Chancellor; Dr. Kavi kishor, Scientific Advisor, Dr. Madhusudhan Rao, Director, Engineering and Management; Dean Academics, Dean R&D; and Head, Biotechnology Department of Vignan’s Foundation for Science, Technology and Research University for providing facility and support to fulfill this additional assignment. We thank the institutions of the Government of India, Department of Biotechnology, Department of Science and Technology, and Indian Council of Medical Research for the grant of financial support to our studies on astaxanthin at CSIR-Central Food Technological Research Institute (CFTRI), Mysuru, India. xli

Acknowledgments We thank the staff and students of the Plant Cell Biotechnology Department and collaborating departments at CSIR-CFTRI for the research done by our team, especially Dr. R. Sarada, Dr. V. Baskaran, Dr. Shylaja M. Dharmesh, Dr. Kadimi Udaya Sankar, and many nontechnical staff. Also, thanks to a number of research students and associates of G.A.R for their dedication to research and authorship in a number of publications on astaxanthin from Haematococcus. Gokare A. Ravishankar Ambati Ranga Rao

xlii

SECTION 1

Astaxanthin from diverse sources leading to industrial production

This page intentionally left blank

CHAPTER 1

Synthesis of astaxanthin and its esters Stanislav Vitalievich Pechinskiia, Anna Gurgenovna Kuregyanb, and Eduard Tonikovich Oganesyanc a

Pyatigorsk Medical and Pharmaceutical Institute, Volgograd State Medical University, Pyatigorsk, Russia Department of Pharmaceutical Chemistry, Pyatigorsk Medical and Pharmaceutical Institute, Volgograd State Medical University, Pyatigorsk, Russia cDepartment of Organic Chemistry, Pyatigorsk Medical and Pharmaceutical Institute, Volgograd State Medical University, Volgograd, Russia

b

Abbreviations 1

H NMR APCI C14:0 C16:0 C16:4 C18:4 C20:5 C20:6 C22:6 CDX-085 CID DDA DMSO-d6 FDA USA HPLC pH TMS WHO ω-fatty acids

proton nuclear magnetic resonance chemical ionization at atmospheric pressure 1-tetradecanoic acid hexadecanoic acid hexadecatetraenoic acid octadecatetraenoic acid eicosapentaenoic acid eicosahexaenoic acid docosahexaenoic acid synthetic compound of astaxanthin collision-induced dissociation disodium astaxanthin disuccinate dimethyl sulfoxide-d6 Food and Drug Administration of United States of America high-performance liquid chromatography hydrogen indicator tetramethylsilane World Health Organization omega fatty acids

1 Introduction Astaxanthin is a representative of natural terpenoids with a length of carbon skeleton C-40 and belongs to the family of oxygen—containing carotenoids and xanthophylls. The structure of astaxanthin has 13 conjugated double bonds distributed between the polyene chain and two β-ionon cycles. The latter contain two alcohol hydroxyl at position 3 and 30 chiral atoms and two ketogroups at positions 4 and 40 [1, 2]. Studies of the antioxidant function of natural astaxanthin and related carotenoids have shown that in this type of action, it is superior to α-tocopherol, α-carotene, lutein, lycopene, and Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00031-3 Copyright # 2021 Elsevier Inc. All rights reserved.

3

4

Chapter 1

β-carotene. Such a high antioxidant activity of astaxanthin is associated with the presence of β-ionon rings, ketogroups in the positions 4 and 40 , and 3 and 30 hydroxyl groups in chiral atoms [3–5]. In all experiments, it is important to take into account the structural features of the geometric and optical isomers of astaxanthin, because they exhibit different biological effects [6, 7]. Natural astaxanthin is approved for use as a component of food additives in the United States, Europe, and Japan [8, 9]. Due to the difference in the biological activity of optical isomers and the potential hazard of residual organic solvents used in its synthesis, all synthetic astaxanthin [10–13] is not approved by FDA USA for medical use. The use of synthetic astaxanthin is limited to aquaculture and animal studies [14–17].

2 General characteristics of natural astaxanthin esters 2.1 Chemical synthesis of astaxanthin for industrial production The industrial production of astaxanthin is carried out in two main directions: extraction of astaxanthin from natural sources and chemical synthesis. Natural astaxanthin is obtained biosynthetically using as rule yeast (Phaffia rhodozyma) [18] or microalgae (Haematococcus pluvialis). Moreover the proportion of free astaxanthin in the total astaxanthin pool of the final product is extremely low. Extraction of free natural astaxanthin requires additional manufacturing steps, which leads to a rise in the cost of a commercial product. However, this direction is important for the global astaxanthin market; therefore studies have been carried out on the economic optimization of the technology for producing natural astaxanthin from microalgae Haematococcus pluvialis, in particular the (3S, 30 S) astaxanthin isomer [5, 19]. Chemical synthesis of astaxanthin is more economically feasible [14]. The first synthesis of carotenoids was carried out in 1950 the schools of Karrer, Inhoffen, and Milas. One of the most important reactions for the synthesis of carotenoids is the Wittig reaction. The reaction was detected unexpectedly in 1953, and Wittig was awarded the Nobel Prize in 1979. The disadvantage of the reaction is the low stereoselectivity. This reaction led to the rapid development of industrial synthesis of carotenoids. The industrial production of carotenoids began in 1954, only 4 years after its first synthesis [20, 21]. The first companies to synthesize astaxanthin industrially were Hoffmann-La Roche and BASF. Currently, synthetic astaxanthin from Hoffmann-La Roche and BASF remains the dominant commercial product on the market. This product consists of (3S, 30 S), (3R, 30 S), (3S, 30 R), and (3R, 30 R) isomers in the ratio 1: 2: 2: 1 (meso). Stereoisomer astaxanthin in natural and synthetic mixtures is also extremely important in biochemical mechanisms, when creating pharmaceutical preparations.

Synthesis of astaxanthin and its esters 5 Roche is one of the established leaders in the production of carotenoids, including astaxanthin. Industrial synthesis astaxanthin by Roche can be obtained from canthaxanthin reaction with lithium diisopropylamide or sodium hexamethyldisilazane. Next the resulting compound can be oxidized directly to astaxanthin by reaction with l-phenylsulphonyl-2-phenyloxaziridine [22–24]. Synthetic astaxanthin has lower bioactivity compared with natural. To improve the properties of synthetic astaxanthin, new synthesis schemes are being developed, for example, using optically active chiral catalyst to stereoselectively reduce the diketone [25] or using carotene hydroxylase, which has an enzymatic activity for converting canthaxanthin into astaxanthin [26]. Cardax Pharmaceuticals produces synthetic astaxanthin, disodium astaxanthin disuccinate, (DDA) which contains a mixture of (3R, 30 R), (3R, 30 S), and (3S, 30 S) stereoisomers in 1:2:1 proportions. The same company patented the second synthetic compound of astaxanthin, CDX-085, which participated in animal experiments in the pathology of the liver and cardiovascular system [10–13]. In addition to the complete chemical synthesis of astaxanthin, data are published on a theoretical study of the mechanisms for producing semisynthetic astaxanthin. The starting materials for the synthesis are natural lutein esters, which are extracted from calendula (Tagetes erecta L.). They are subjected to alkaline hydrolysis followed by conversion to highly purified zeaxanthin (3R,30 S-zeaxanthin). Then, it is subjected to oxidation with iodine to astaxanthin. (3R,30 S-astaxanthin). Currently, these studies have not yet found their commercial application. [17]. The search for new, more effective synthetic routes of astaxanthin continues.

2.2 Composition of natural astaxanthin esters In nature, astaxanthin occurs in free form (1) and in the esterified state of one (2) or both hydroxyl groups (3) (Fig. 1.1). The variety of natural esters of astaxanthin can be explained by the fact that xanthophyll binds to the residue of the same acid on both hydroxylsor to the acid residues of two different acids [27–29]. For example, Wade et al. [30] reported that astaxanthin monoesters are formed by saturated fatty acids, whereas its diesters are formed by monounsaturated and polyunsaturated fatty acids. Rita Frassanito and coauthors published data that the monoester fraction of astaxanthin obtained from Tovellia sanguinea is represented by acids: C14:0, C16:0, C16:4, C18:4, C20:5, and C22:6 [31]. The diesters of To. sanguine include the following acids: C16: 0/C16:0, C14: 0/C14:0, C14:0/C16:0, C16:0/C20:6, and C16:0/C22:6. According to Coral-Hinostroza and Jerking [32] in red crab langostilla (Pleuroncodes planipes), astaxanthin diesters are formed by saturated and monounsaturated fatty acids (C16:0 and C18:1). The fraction of astaxanthin monoether also contains saturated fatty acids but in

6

Chapter 1 O OH

HO O

1 O R

O O HO O

2 O R

O O

O R

O O

3

Fig. 1.1 Astaxanthin and general structures of its natural esters: astaxanthin (1), astaxanthin monoesters (2), and diesters (3).

smaller amounts than the diester fraction. Astaxanthin monoesters are rich in polyunsaturated fatty acids, namely, C20:5 and C22:6 [32]. Thus astaxanthin, as a rule, forms natural esters with saturated fatty acids such as myristic (C14:0), palmitic (C16:0), stearic (C18:0), nondecanoic (C19:0), and eicosane (C20:0); with monounsaturated fatty acids such as myristoleic (C14:1), oleic (C18:1), and eicosenic (C20:1); and with polyunsaturated fatty acids such as octadecatrienoic (C18:3), octadecatetraenoic (18:4), eicosapentaenoic (C20:5), eicosahexaenoic (C20:6), and docosahexaenoic (C22:6) acids [29–34]. The fractional composition of free astaxanthin and its esters depends on the type of xanthophyll source, features of metabolism, life cycle stage, climatic conditions, and many other environmental factors. In addition, these same factors affect which acids acetylate astaxanthin, and, consequently, what will be the composition of astaxanthin esters [32, 35, 36]. In the algae H. pluvialis, the astaxanthin pool is represented by free astaxanthin (5%), monoesters (70%), and diesters (25%) [5]. The main forms of astaxanthin esters in H. lacustris are monoesters, which make up 79% of the total number of carotenoids [37]. In the algae T. sanguinea, the main carotenoid fraction is represented by free astaxanthin, nine monoesters, and five diesters of astaxanthin [33, 38]. After analysis of H. pluvialis by HPLC, 4 free carotenoids, 15 astaxanthin monoesters, 12 astaxanthin diesters, and 3 astacin monoesters—product of astaxanthin oxidation—were detected [34]. Lipid extract from

Synthesis of astaxanthin and its esters 7 shrimp waste Litopenaeus vannamei contained all-trans-astaxanthin and its 9-cis and 13-cis isomers, 5 monoesters, and 10 diesters of astaxanthin [27]. As demonstrated by the results of many experiments [27–38], the composition of natural esters of astaxanthin depends on the whole complex of individual characteristics of the source of its production. In this regard, it is still almost impossible to characterize unambiguously the dependence between the completeness of esterification of astaxanthin and the type of acids.

2.3 The benefits of astaxanthin esters An important distinguishing property of astaxanthin esters is that they are more stable during storage, heating, and oxidation [39]. As a result of astaxanthin oxidation, astacene is formed, but this process is slower for esterified forms of xanthophyll [40]. Another advantage of astaxanthin esters can be considered—their higher bioavailability [29, 41]. Numerous experiments on the use in aquaculture and animal husbandry confirm that the bioavailability of natural astaxanthin and its esters is much higher than the synthetic analog [5]. It should be noted that free astaxanthin and its esters differ in biological activity. For example, natural astaxanthin, derived from the microalgae H. pluvialis, is a mono- or diether (3S,30 S) of isomer, which leads to different results in experiments for the treatment of cardiovascular diseases [42]. It is shown that the degree of esterification and the nature of acids influence the antioxidant activity of xanthophylls [43]. In addition, astaxanthin esters are an additional source of ω-fatty acids [27]. Therefore astaxanthin in the form of esters has a number of advantages over free xanthophyll, while the main ones, in our opinion, are greater bioavailability and stability, which is important in the creation of pharmaceutical substances.

2.4 Direction modification of astaxanthin structure Carotenoids are characterized by selective accumulation in organs and tissues [44, 45], which is advisable to use for directed transport of pharmaceutical substances to the target organ. Targeted delivery of pharmaceutical substances to the target organ will allow to increase the therapeutic effect and, importantly, reduce the dose and side effects of the medical product. Modification of the carotenoid molecule, such as the synthesis of esters, will affect its pharmacokinetic characteristics. Along with this the remaining carotenoid fragment will allow to preserve the original properties of the basic structure. Significance of search for modified semisynthetic derivatives of carotenoids is that, in nature, these compounds occur in bound form, predominantly in the form of complex esters of organic acids [27–30]. Successful implementation of the idea of obtaining astaxanthin esters with pharmaceutical substances is possible only with theoretical justification and experimental confirmation of the principles of their synthesis. Features of the structure of astaxanthin and its physicochemical

8

Chapter 1

properties do not allow to conduct the esterification reaction under classical conditions. Another important problem is the obtaining of stereo-specific esters, since the probability of isomerization of astaxanthin and its derivatives in the synthesis process is extremely high [32]. The solution of these problems is facilitated by the use of enzymes as catalysts, in particular lipases [40].

3 Prospects for the use of biocatalysts in the synthesis of semisynthetic derivatives of astaxanthin 3.1 Characterization of biocatalysts The successful development of molecular biology, bioinformatics, and bioengineering over the past 20 years has led to an increase in the frequency of biocatalysts’ application. They are increasingly used in various industries: in the food industry [46], in the production of biofuels [47], in pharmaceutical preparation [48], and in agroindustry [49], because it makes production environmentally friendly (green) and cost-effective [50–52]. Enzymatic catalysis has a number of advantages over traditional chemical catalysts. Due to high stereoselectivity, enzymes provide an increase in the production capacity of synthesis or semisynthesis of individual compounds [53, 54]. The growing demand for biocatalysts as a replacement for traditional chemical catalysts determines the need for new improved enzymes with optimal catalytic and economic characteristics [55, 56]. The use of in silico technologies allows to modify directionally natural enzymes and obtain more effective enzymes [48]. Currently, lipase, transaminases, reductases, esterases, oxidases, aldolases, cytochrome P450monooxygenases, etc. are used as biocatalysts [57].

3.2 Difficulties when working with biocatalysts When working with biocatalysts, it is necessary to take into account that they are effective in their natural conditions at certain pH values, temperature, and solvent characteristics, and, as a rule, the range of these parameters is quite narrow. Changes in the environmental conditions under which the enzymes are active may be a cause of reduction in the life of industrial operation of the enzyme may lead to a decrease or loss of activity of the enzyme. This is typical for industrial processes where extreme pH, temperature, and artificial substrates are used [58]. An important economic problem of biocatalyst application is the solution of the problem of their reuse [59], which is achieved by immobilization of enzymes [60, 61].

Synthesis of astaxanthin and its esters 9 Enzyme immobilization is a process in which an enzyme is fixed to a carrier to create an insoluble environment-resistant complex for repeated use [62]. Currently the reuse of the enzyme is not the only purpose of immobilization. Immobilization can act as a powerful tool to optimize activity, selectivity, and resistance to inhibitors by changing the conformation of the enzyme [63]. For the immobilization of enzymes, the physical binding of the enzyme is often needed. The carrier selection and adsorption or chemical fixation due to the covalent binding of the enzyme and the carrier are common features of evaluation for maximal efficacy. Chemical binding is a more complex and time-consuming process, but it significantly prevents the enzyme from leaching from the carrier surface, so it is economically justified and allows the development of more environmentally friendly synthesis technologies (greener process) [64–66]. Thus immobilization of biocatalysts is an effective technique for improving the economic and environmental performance of the synthesis process by reusing the enzyme and increasing its activity and stability [67].

3.3 Pharmacy biocatalysis The use of biocatalysis in the production of medicinal products has a number of features. In nature, enzymes work at millimolar concentrations of reagent in the synthesis of products. In laboratory or production conditions, the concentration of initial substances and target products is many times higher, and this can affect the activity of the biocatalyst. From an economic point of view, the cost of the enzyme is extremely important for production. Immobilization of biocatalysts is one of the options for increasing the efficiency. Another characteristic feature of the pharmaceutical industry is the need to rationally and fully use the time of patent protection of the compound, since clinical trials take a long time [68]. This task can be solved by creating new effective processes for the development and industrial production of medicines, including the use of biocatalysis. A positive property of biocatalysis can be considered for the stereoselectivity of the target synthesis. In pharmaceutical sector the synthesis of products with special reference to optically active intermediates increased from 35% to 70% during the period from 2000 to 2010 [48, 69, 70]. The use of stereoselectivity of enzymes reduces the number of stages of the production process, because the stage of purification of the product from isomers is excluded or reduced. As a result, this approach to production increases the purity of the target product and most importantly minimizes the side effects of pharmaceutical substance. In this regard, in our opinion, the main advantages of enzymatic catalysis are high specificity

10

Chapter 1

and stereoselectivity, especially when it comes to substances that are initially optically active, such as astaxanthin. Close attention should be paid to industrial scaling of synthesis processes in pharmaceutical production using biocatalysis. There are two main problems when transferring synthesis technology from the laboratory to the plant. First, with respect to laboratory conditions, the time of synthesis increase proportionally with the scale of production. This can lead to degradation of both the enzyme and the carrier, hence increasing the amount of impurities. So the development of techniques of purity for the controlled production pharmaceutical substance should be a matter of concern. Secondly, in industrial reactors, the problems of large volumes lead to heterogeneity of mixing, which need to be addressed. To cope with such a task, as a rule, increasing the speed of mixing is often adopted, which could lead to degradation of the enzyme as well [69]. Thus the advantages of biocatalysts such as stereoselectivity, the possibility of reuse, the possibility of synthesis at low temperature, the organic medium of the substrate, and the cost-effectiveness can definitely be used to solve the difficulties associated with the synthesis of astaxanthin derivatives, in particular its esters.

4 Synthesis of astaxanthin esters 4.1 Acid selection for esterification of astaxanthin For esterification of astaxanthin, model compound acids, namely, benzoic, 4-methylbenzoic, and phenylglycolic (mandelic) acids, were selected for initial studies. Taking into account the necessity of gradual complication of acid structures and transition to structures of pharmaceutical substances, we opted for 2-hydroxybenzoic acid (salicylic acid), which has long been used as a pharmaceutical substance [71]. As a result of this selection of candidates, a list of acids with the closest structures was obtained. To expand the list of acids that could participate in the synthesis of astaxanthin esters, it was necessary to conduct an experiment with a heterocyclic compound. Due to the fact that a sufficient number of pharmaceutical substances are pyridine derivatives, nicotinic acid that is used in medical practice was included in the experiment [71]. Given that ibuprofen, as a nonsteroidal antiinflammatory medicinal product, is included in the WHO list of essential medicines and in the treatment standards of many nosologies [71], we decided to use this compound as a candidate acid for the synthesis of astaxanthin esters. Thus the experiment involved six compounds as acids: benzoic, 4-methylbenzoic and phenylglycolic (mandelic), 2-hydroxybenzoic (salicylic), nicotinic acid, and ibuprofen.

Synthesis of astaxanthin and its esters 11

4.2 The selection of conditions for the synthesis of astaxanthin esters The classical esterification reaction proceeds most fully and quickly in the presence of active metals, such as zinc, nickel, magnesium, and strong acids—sulfuric or hydrochloric. Another necessary condition is the heating of the reaction mixture, as a rule, to 100°С [72–74]. It is obvious that the esterification of xanthophylls, in particular astaxanthin, requires the adaptation of traditional conditions of its course, because astaxanthin is practically insoluble in water and soluble in nonpolar organic solvents [75]. An important factor is the temperature regime: the reaction at temperatures above 50°С will lead to the destruction of the main pharmacophore of astaxanthin—polyene chain. Another feature is the use of metals and acids as catalysts. It should be borne in mind that in addition to the main reaction, hydrogen is released. It is obvious that the presence of astaxanthin in the reaction system that will likely be accompanied by partial or complete hydrogenation of the polyene chain leading to changes in the carotenoid structure. Taking into account the described difficulties of carrying out the esterification reaction of astaxanthin under classical conditions, we used a more gentle mode of obtaining its esters with the use of biocatalysts, in particular lipases. Enzymatic catalysis using lipases allows to obtain esters at low temperatures and without the use of aggressive reagents. An important property of biocatalysts is that the active site of the enzyme has a hydrophobic character and therefore the esterification reactions with their participation take place in an environment of organic solvents. It is believed that hydrophobic alcohols are most suitable for esterification catalyzed by lipase. Due to the presence of this set of properties in lipases, we found it possible to carry out the esterification reaction of astaxanthin in the presence of the biocatalyst novozyme 435, which is a lipase Candida antarctica immobilized on acrylic resin [76]. The choice of this catalyst is due to the fact that it was previously used in the synthesis of retinol derivatives, a structure close to carotenoids [72], and meets all the requirements inherent in biocatalysts. The peculiarity of immobilized enzymes is associated with the fragility of the carrier particles, so you should choose the optimal number of revolutions of the agitator, because intensive mixing leads to a loss of enzyme activity and reducing the speed increases the duration of synthesis. However, the most important problem, in our opinion, is that there is not only the release of the enzyme but also the polymer components of the carrier in the reaction medium. This can certainly be one of the causes of contamination of the target product, which is extremely important when creating pharmaceutical preparations.

4.3 Synthesis of semisynthetic astaxanthin esters Astaxanthin for the synthesis of esters was obtained from the shells of shrimp according to the method developed by us and published [77–79]. The synthesis of esters was carried out in toluene/methanol (10:1), at a temperature of 37°С. It was found that after 6h the composition of the reaction mixture did not significantly change and astaxanthin esters are formed with a yield

12

Chapter 1

of about 60%. To separate the resulting mono- and diesters, as well as separating them from unreacted astaxanthin, the resulting mixture was passed through a column filled with aluminum oxide. After separation, astaxanthin esters were washed with 95% ethyl alcohol and water, dried in a vacuum (20–25mmHg.) at a temperature of 40°С for 2h. Thus six new semisynthetic esters of astaxanthin with benzoic (1), 4-methylbenzoic (2), nicotinic (3), phenylglycolic (mandelic) (4), 2-hydroxybenzoic (salicylic) (5) acids, and ibuprofen (6) were obtained (Fig. 1.2). On the example of salicylic, nicotinic acids, and ibuprofen, the principal possibility of obtaining esters of astaxanthin and pharmaceutical substances is shown.

4.4 Confirmation of the structure of astaxanthin esters The structure of the obtained compounds was confirmed by1HNMR and mass spectrometry. The 1H NMR spectra of synthesized esters 1–6 clearly trace the signals of astaxanthin and ester fragments. The absence of bands near 4.00 (2Н, Н3, and Н3’) indicates the formation of a complex ester bond. Н NMR spectra were recorded using a Bruker AMXIII-400 spectrometer at 400 MHz in DMSO-d6 with TMS as internal reference. Mass spectra were recorded using an Agilent 6420 mass spectrometer combined with an Agilent HPLC 1100 system by means of chemical ionization at atmospheric pressure (APCI), ion source temperature 120°C, helium as carrier gas, and CID energy 40eV. HPLC parameters include column Dionex Acclaim C30 (2504.6mm 5 μm); column temperature 25°С; mobile phases, methanol (phase A), tert-butyl methyl ether (phase B), and water (phase C); linear gradient, phases A 85, B 12, C 3% ! A 5, B 92C 3% during 45 min; sample volume 20 μL and mobile phase rate 0.5 mL/min; and injected volume 20 μL. Melting points were determined using a PTP (M) instrument. 1

4,40 -Dioxo-β,β-carotene-3,30 -diyl dibenzoate (1). 1Н NMR spectrum, δ, ppm (J, Hz): 1.13 s 0 0 0 (6Н, Ме17,17 ), 1.25 s (6Н, Ме16,16 ), 1.77 t (2Н, Н2, J ¼ 13.0), 2.00 s (6Н, Ме13,13 ), 2.01 s 0 0 0 0 0 (6Н, Ме9,9 ), 6.21 d (2Н, Н7,7 , J ¼ 16.0), 6.28–6.31 m (4Н, Н10,10 ,14,14 ), 6.41 d (2Н, Н8,8 , 0 0 0 0 0 J¼15.0), 6.67–6.76m (4Н, Н11,11 ,15,15 ), 7.46 d (4Н, Н24,24 ,26,26 , J¼ 9.0), 7.57t (2Н, Н25,25 , 0 J¼ 7.0), 8.03 d (2Н, Н23,23 , J¼8.0). Mass spectrum, m/z: 805.4436 [M+Н]+. С54Н60О6Н+. Мcalc 805.4463. 4,40 -Dioxo-β,β-carotene-3,30 -diyl di(4-methyl-benzoate) (2). 1Н NMR spectrum, δ, ppm (J, 0 0 0 Hz): 1.13s (6Н, Ме17,17 ), 1.25s (6Н, Ме16,16 ), 1.77t (2Н, Н2, J¼ 13.0), 2.00s (6Н, Ме13,13 ), 0 0 0 2.01 s (6Н, Ме9,9 ), 2.42 s (6Н, Ме25,25 ), 6.21 d (2Н, Н7,7 , J ¼ 16.0), 6.28–6.31 m 0 0 0 0 0 (4Н, Н10,10 ,14,14 ), 6.42 d (2Н, Н8,8 , J ¼ 15.0), 6.67–6.76 m (4Н, Н11,11 ,15,15 ), 7.42 d. d 0 0 0 0 (4Н, Н24,24 ,26,26 , J ¼ 8.0, 2.0), 7.74 d. d (4Н, Н23,23 ,27,27 , J ¼ 9.0, 2.0). Mass spectrum, m/z: 833.4758 [M + Н]+. С56Н64О6Н+. Мcalc 833.4776.

O O O

O O O

1 O O O

O O O

2 N

O O O

O O O

N

3 OH

O O O

O O O

OH

4 O O O

O

OH

O O OH

O

5

O O O

O O O

6

Fig. 1.2 Structures of semisynthetic astaxanthin esters: 4,40 -dioxo-β,β-carotene-3,30 -diyldibenzoate (1), 4,40 dioxo-β,β-carotene-3,30 -diyldi(4-methyl-benzoate) (2), 4,40 -dioxo-β,β-carotene-3,30 -diyldi(pyridine3-carboxylate) (3), 4,40 -dioxo-β,β-carotene-3,30 -diyldi(2-hydroxy-2-phenylethanoate) (4), 4,40 dioxo-β,β-carotene-3,30 -diyldi(2-hydroxy-benzoate) (5), and 4,40 -dioxo-β,β-carotene-3,30 -diyldi(2(4-isobutylphenyl)-propionate) (6).

14

Chapter 1

4,40 -Dioxo-β,β-carotene-3,30 -diyldi(pyridine-3-carboxylate) (3). 1Н NMR spectrum, δ, ppm 0 0 (J, Hz): 1.13 s (6Н, Ме17,17 ), 1.25 s (6Н, Ме16,16 ), 1.77 t (2Н, Н2, J ¼ 13.0), 2.00 s (6Н, 0 0 0 0 0 Ме13,13 ), 2.01s (6Н, Ме9,9 ), 6.21 d (2Н, Н7,7 , J¼ 16.0), 6.28–6.31m (4Н, Н10,10 ,14,14 ), 6.41 d 0 0 0 0 (2Н, Н8,8 , J¼15.0), 6.67–6.76m (4Н, Н11,11 ,15,15 ), 7.49 d. d (2Н, Н25,25 , J¼8.0, 5.0), 8.01 d. d 0 0 0 (2Н, Н26,26 , J¼8.0, 1.0), 8.57 d. d (2Н, Н24,24 , J¼4.0, 2.0), 8.78t (2Н, Н23,23 , J¼2.0). Mass spectrum, m/z: 807.4324 [M+ Н]+. С52Н58N2О6Н+. Мcalc 807.4368. 4,40 -Dioxo-β,β-carotene-3,30 -diyldi(2-hydroxy-2-phenylethanoate) (4). 1Н NMR 0 0 spectrum, δ, ppm (J, Hz): 1.13s (6Н, Ме17,17 ), 1.25s (6Н, Ме16,16 ), 1.77t (2Н, Н2, J¼13.0), 0 0 0 2.00 s (6Н, Ме13,13 ), 2.01 s (6Н, Ме9,9 ), 6.21 d (2Н, Н7,7 , J ¼ 16.0), 6.28–6.31 m 0 0 0 0 0 (4Н, Н10,10 ,14,14 ), 6.41 d (2Н, Н8,8 , J ¼ 15.0), 6.67–6.76 m (4Н, Н11,11 ,15,15 ), 7.33 t 0 0 0 0 0 (6Н, Н25,25 ,26,26 ,27,27 , J ¼ 7.0, 1.0), 7.40 d. d (4Н, Н24,24 ,28,28 , J ¼ 8.0). Mass spectrum, m/z: 865.4661 [M + Н]+. С56Н64О8Н+ Мcalc 865.4674. 4,40 -Dioxo-β,β-carotene-3,30 -diyldi(2-hydroxybenzoate) (5). 1Н NMR spectrum, δ, ppm 0 0 (J, Hz): 1.13 s (6Н, Ме17,17 ), 1.25 s (6Н, Ме16,16 ), 1.77 t (2Н, Н2, J ¼ 13.0), 2.00 s 0 0 0 0 0 (6Н, Ме13,13 ), 2.01s (6Н, Ме9,9 ), 6.21 d (2Н, Н7,7 , J ¼16.0), 6.28–6.31m (4Н, Н10,10 ,14,14 ), 0 0 0 0 0 6.42 d (2Н, Н8,8 , J ¼ 15.0), 6.67–6.76 m (4Н, Н11,11 ,15,15 ), 7.21 д. д (4Н, Н24,24 ,26,26 , 0 0 J ¼ 8.0, 1.0), 7.50 т (2Н, Н25,25 , J ¼ 8.0), 7.95 д. д (2Н, Н23,23 , J ¼ 8.0, 1.0). Mass spectrum, m/z: 837.4354 [M + Н]+. С54Н60О8Н+ Мcalc 837.4361. 4,40 -Dioxo-β,β-carotene-3,30 -diyl di(2-(4-isobutylphenyl)-propionate) (6). 1Н NMR 0 0 0 spectrum, δ, ppm (J, Hz): 0.82 д (12Н, Ме30,30 ,31,31 , J ¼ 7.0),1.13 s (6Н, Ме17,17 ), 1.25 s 0 0 0 (6Н, Ме16,16 ), 1.36 д (6Н, Ме23,23 , J ¼ 7.0), 1.77 t (2Н, Н2, J ¼ 13.0), 2.00 s (6Н, Ме13,13 ), 0 0 0 0 2.01 s (6Н, Ме9,9 ), 2.42 s (2Н, H28,28 ), 5.39 с (2Н, Н22, 22 ), 6.21 d (2Н, Н7,7 , J ¼ 16.0), 0 0 0 0 0 6.28–6.31 m (4Н, Н10,10 ,14,14 ), 6.42 d (2Н, Н8,8 , J ¼ 15.0), 6.67–6.76 m (4Н, Н11,11 ,15,15 ), 0 0 7.06 д (2Н, Н26,26 , J¼4.0), 7.21 д (2Н, Н25,25 , J¼4.0). Mass spectrum, m/z: 973.6332 [M+ Н]+. С66Н84О6Н+ Мcalc 973.6341.

5 Conclusions In this chapter, we wanted to highlight a few major promising points that follow from the presented experimental material. First the positive result in the synthesis of esters shows that there is a fundamental possibility of obtaining new semisynthetic derivatives of astaxanthin. The second point that deserves attention, in our opinion, is that the proposed conditions for the synthesis of astaxanthin esters can be used to work with other xanthophylls. Thirdly the expansion of research in the direction of obtaining individual esters of astaxanthin and other xanthophylls, and their further use in pharmacological experiments will allow to understand some of the relationships between the structure and selective accumulation of xanthophylls and their esters in tissues and organs.

Synthesis of astaxanthin and its esters 15 The fourth topical direction is due to the fact that a higher resistance of astaxanthin esters, to oxidation, will solve the problems of their stabilization. The fifth positive result of our research is that the stereoselectivity of biocatalysis provided high purity of semisynthetic esters of astaxanthin. Most likely, minimization of the number of isomers in the target product will increase the safety and effectiveness of both astaxanthin and its esters. From this point of view, the possibility of its use in the treatment of various diseases is expanding, and, at the same time, the prospect of targeted transport of some medicines opens up.

References [1] Subramanian B, Thibault MH, Djaoued Y, Pelletier С, Touaibiac M, Tchoukanova N. Chromatographic, NMR and vibrational spectroscopic investigations of astaxanthin esters: application to Astaxanthin-rich shrimp oil obtained from processing of Nordic shrimps. R Soc Chem 2015;1–11. https://doi.org/10.1039/c5an01261a. [2] Zhao L, Zhao G, Chen F, Wang Z, Wu J, Hu X. Different effects of microwave and ultrasound on the stability of (all-E)-astaxanthin. J Agric Food Chem 2006;54(21):8346–51. https://doi.org/10.1021/jf061876d. [3] Yousry MA. Naguib antioxidant activities of astaxanthin and related carotenoids. J Agric Food Chem 2000;48 (4):1150–4. https://doi.org/10.1021/jf991106k. [4] Terao J. Antioxidant activity of/I-carotene-related carotenoids in solution. Lipids 1989;24(7):659–61. https:// doi.org/10.1007/bf02535085. [5] Panis G, Rosales CG. Commercial astaxanthin production derived by green alga Haematococcus pluvialis: a microalgae process model and a techno-economic assessment all through production line Author links open overlay panel. Algal Res 2016;18:175–90. https://doi.org/10.1016/j.algal.2016.06.007. [6] Liu X, Osawa T. Cis-astaxanthin and especially 9-cisastaxanthin exhibits a higher antioxidant activity in vitro compared to the all-trans isomer. Biochem Biophys Res Commun 2007;357(1):187–93. https://doi.org/ 10.1016/j.bbrc.2007.03.120. [7] Liu X, Song M, Gao Z, Cai X, Dixon W, Chen X, et al. Stereoisomers of astaxanthin inhibit human colon cancer cell growth by inducing G2/M cell cycle arrest and apoptosis. J Agric Food Chem 2016;64:7750–9. https://doi. org/10.1021/acs.jafc.6b03636. [8] Lorenz RT, Cysewski GR. Commercial potential of Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol 2000;18:160–7. https://doi.org/10.1016/s0167-7799(00)01433-5. [9] Higuera-Ciapara I, F
elix-Valenzuela L, Goycoolea FM. Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr 2006;46(2):185–96. https://doi.org/10.1080/10408690590957188. [10] Fassett RG, Coombes JS. Astaxanthin: a potential therapeutic agent in cardiovascular disease. Mar Drugs 2011;9(3):447–65. https://doi.org/10.3390/md9030447. [11] Khan SK, Malinski T, Mason RP, Kubant R, Jacob RF, Fujioka K, et al. Novel astaxanthin prodrug (CDX-085) attenuates thrombosis in a mouse model. Thromb Res 2010;126:299–305. https://doi.org/10.1016/j. thromres.2010.07.003. [12] Gross GJ, Lockwood SF. Cardioprotection and myocardial salvage by a disodium disuccinate astaxanthin derivative (Cardax). Life Sci 2004;75:215–24. https://doi.org/10.1016/j.lfs.2003.12.006. [13] Gross GJ, Hazen SL, Lockwood SF. Seven day oral supplementation with Cardax (disodium disuccinate astaxanthin) provides significant cardioprotection and reduces oxidative stress in rats. Mol Cell Biochem 2006;283:23–30. https://doi.org/10.1007/s11010-006-2217-6. [14] Nguyen KD. Astaxanthin: a comparative case of synthetic vs. natural production. Chem Biomol Eng Publ Other Works 2013;1(1):1–11. [15] Dong H, Li X, Xue C, Mao X. Astaxanthin preparation by fermentation of esters from Haematococcus pluvialis algal extracts with Stenotrophomonas species. Biotechnol Prog 2016;32(3):649–56. https://doi.org/10.1002/ btpr.2258.

16

Chapter 1

[16] Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H. Astaxanthin, a carotenoid with potential in human health and nutrition. J Nat Prod 2006;69(3):443–9. https://doi.org/10.1021/np050354. [17] Rodrı´guez-deLeo´n E, Jim
enez-Halla OC, Ba´ez JE, Bah MM. A simple and efficient method for the partial synthesis of pure (3R,3’S)-astaxanthin from (3R,3’R,6’R)-lutein and lutein esters via (3R,3’S)-zeaxanthin and theoretical study of their formation mechanisms. Molecules 2019;24:1386–99. https://doi.org/10.3390/ molecules24071386. [18] slashed BFo, Christensen I, Larsen R, Johansen SR, Johnson EA. Patent 5712110А US. Publ. Date 27.01. [19] Boussiba S, Vonshak A, Cohen Z, Richmond A. Patent 6022701А US. Publ. Date 08.02. [20] Willig G, Pommer H. Patent 950552 Ger. Pat. Publ. Date 25.09. [21] Paust J. Technical syntheses. In: Britton G, Liaaen-Jensen S, Pfander H, editors. Carotenoids. vol. 2. Basel: Birkh€auser; 1996 361 p. [22] Moldt P. Patent 5166441А US. Publ. Date 24.11. [23] Moldt P. Patent 5225604А US. Publ. Date 067.07. [24] Bernhard K, Miller RK, Spruijtenburg R. Patent 4585885. Publ. Date 29.04. [25] Samuel F, Lockwood PCT, Nadolski G, Jackson HL, Fang Z, Du Y, Yang M, Geiss W, Williams R, Burdick D. Patent 7247752 B2 US. Publ. Date 24.07. [26] Linden H, Sandmann G. Patent 7041471 US. Publ. Date 09.05. ´ lvarez-Acero I, Montero P, Go´mez-Guill
en MC. Characterization and storage [27] Go´mez-Estaca J, Calvo MM, A stability of astaxanthin esters, fatty acid profile and a-tocopherol of lipid extract from shrimp (L. vannamei) waste with potential applications as food ingredient. Food Chem 2017;216:37–44. https://doi.org/10.1016/j. foodchem.2016.08.016. [28] Nu´n˜ez-Gast
eluma JA, Sa´nchez-Machadoa DI, Lo´pez-Cervantesa J, Rodrı´guez-Nu´n˜eza JR, Correa-Murrietaa MA, Sa´nchez-Duartea RG, et al. Astaxanthin and its esters in pigmented oil from fermented shrimp byproducts. J Aquat Food Prod Technol 2016;25:334–43. https://doi.org/ 10.1080/10498850.2013.851756. [29] Mariutti LRB, Mercadante AZ. Carotenoid esters analysis and occurrence: what do we know so far? Arch Biochem Biophys 2018;648:36–43. https://doi.org/10.1016/j.abb.2018.04.005. [30] Wade NM, Cheers S, Bourne N, Irvin S, Blyth D, Glencross BD. Dietary astaxanthin levels affect colour, growth, carotenoid digestibility and the accumulation of specific carotenoid esters in the Giant Tiger Shrimp. Penaeusmonodon Aquac Res 2017;48:395–406. https://doi.org/10.1111/are.12888. [31] Rita F, Giovanna F, Ines M, Graziano G. High production of unexpected carotenoids in Dinophyceae. Astaxanthin esters from the freshwater dinoflagellate T. sanguine. Biochem Syst Ecol 2006;34:843–53. https:// doi.org/10.1016/j.bse.2006.05.013. [32] Coral-Hinostroza GN, Bjerkeng B. Astaxanthin from the red crab langostilla (Pleuroncodesplanipes): optical R/S isomers and fatty acid moieties of astaxanthin esters. Comp Biochem Physiol B Biochem Mol Biol 2002;133(3):437–44. https://doi.org/10.1016/s1096-4959(02)00186-0. [33] Guillou A, Khalil M, Adambounou L. Effects of silage preservation on astaxanthin forms and fatty acid profiles of processed shrimp (Pandalus borealis) waste. Aquaculture 1995;130:351–60. [34] Fengping M, Dayan L, Yeguang L, Mingtao Z. Characterization of astaxanthin esters in Haematococcus pluvialis by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry. Anal Biochem 2006;352:176–81. https://doi.org/10.1016/j.ab.2006.03.006. [35] Shinichi T, Kumi M, Masahisa N, Mizuho M, Satoshi H. Fatty acids of astaxanthin esters in krill determined by mild mass spectrometry. Comp Biochem Physiol B Biochem Mol Biol 2003;136(2):317–22. https://doi.org/ 10.1016/S1096-4959(03)00209-4. [36] Toma´sˇ R, Linda N, Irena K, Karel S. LC–MS/APCI identification of glucoside esters and diesters of astaxanthin from the snow alga Chlamydomonas nivalis including their optical stereoisomers. Phytochemistry 2013;88:34–42. https://doi.org/10.1016/j.phytochem.2013.01.003. [37] Yuan JP, Gong XD, Chen F. Separation and identification of astaxanthin esters and chlorophylls in Haematococcus lacustris by HPLC. Biotechnol Tech 1996;10:655–60.

Synthesis of astaxanthin and its esters 17 [38] Qi Z, Huan Z, Zhengyu H, Guoxiang L. Blooms of the woloszynskioid dinoflagellate Tovelliadiexiensis sp. nov. (Dinophyceae) in Baishihai Lake at the eastern edge of Tibetan Plateau. Algae 2016;31(3):205–17. https:// doi.org/10.4490/algae.2016.31.9.7. [39] Miao F, Geng Y, Lu D, Zuo J, Li Y. Stability and changes in astaxanthin ester composition from Haematococcus pluvialis during storage. Chin J Oceanol Limnol 2013;31(6):1181–9. [40] Zhao Y, Guan F, Wang G, Miao L, Ding J, Guan G, et al. Astaxanthin preparation by lipase-catalyzed hydrolysis of its esters from Haematococcus pluvialis algal extracts. J Food Sci 2011;76(4):643–50. [41] Desmarchelier C, Borel P. Overview of carotenoid bioavailability determinants: from dietary factors to host genetic variations. Trends Food Sci Technol 2017;69:270–80. https://doi.org/10.1016/j.tifs.2017.03.002. [42] Visioli F, Artariac C. Astaxanthin in cardiovascular health and disease: mechanisms of action, therapeutic merits, and knowledge gaps. Food Funct 2017;8:39–63. https://doi.org/10.1039/c6fo01721e. [43] P
erez-Ga´ lvez A, Mı´nguez-Mosquera MI. Degradation of non-esterified and esterified xanthophylls by free radicals. Biochim Biophys Acta 2002;1569:31–4. https://doi.org/10.1016/s0304-4165(01)00229-x. [44] Horvath MP, George EW, Tran QT, Baumgardner K, Zharov G, Lee S, et al. Structure of the lutein-binding ˚ resolution and model of a complex with lutein. Acta Crystallogr F Struct domain of human StARD3 at 1.74 A Biol Commun 2016;72(8):609–18. https://doi.org/10.1107/S2053230X16010694. [45] Tanprasertsuk J, Li B, Bernstein PS, Vishwanathan R, Johnson MA, Poon L, et al. Correction: relationship between concentrations of lutein and StARD3 among pediatric and geriatric human brain tissue. PLoS One 2016;11(7):1–7. https://doi.org/10.1371/journal.pone.0159877. [46] Liu Y, Huang L, Fu Y, Zheng D, Ma J, et al. A novel process for phosphatidylserine production using a Pichiapastoris whole-cell biocatalyst with overexpression of phospholipase D from Streptomyces halstedii in a purely aqueous system. Food Chem 2019;274:535–42. https://doi.org/10.1016/j.foodchem.2018.08.105. [47] Babaki M, Yousefi M, Habibi Z, Mohammadi M, Yousefi P, et al. Enzymatic production of biodiesel using lipases immobilized on silica nanoparticles as highly reusable biocatalysts: effect of water, t-butanol and blue silica gel contents. Renew Energy 2016;9:196–206. https://doi.org/10.1016/j.renene.2016.01.053. [48] Rozzell D, Lalonde J. Enzymatic processes for the production of pharmaceutical intermediates. In: Yeh W-K, Yang H-C, McCarthy JR, editors. Enzyme technologies: metagenomics, evolution, biocatalysis, and biosynthesis. John Wiley & Sons, Inc.; 2010. p. 185–98 [49] Regner EL, Salvatierra HN, Baigorı´ MD, Pera LM. Biomass-bound biocatalysts for biodiesel production: tuning a lipolyticactivity from Aspergillus niger MYA 135 by submerged fermentation using agro-industrial raw materials and waste products. Biomass Bioenergy 2019;120:59–67. https://doi.org/10.1016/j. biombioe.2018.11.005. [50] Li G. The rise of enzyme engineering in China. Sheng Wu Gong Cheng Xue Bao 2015;31(6):805–19. [51] Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K. Engineering the third wave of biocatalysis. Nature 2012;485(7397):185–94. https://doi.org/10.1038/nature11117. [52] Bilal M, Iqbal HMN, Guo S, Hu H, Wang W, Zhang X. State-of-the-art protein engineering approaches using biological macromolecules: a review from immobilization to implementation view point. Int J Biol Macromol 2018;108:893–901. https://doi.org/10.1016/j.ijbiomac.2017.10.182. [53] Choi JM, Han SS, Kim HS. Industrial applications of enzyme biocatalysis: current status and future aspects. Biotechnol Adv 2015;33(7):1443–54. https://doi.org/10.1016/j.biotechadv.2015.02.014. [54] Tozlu C, Şahin E, Serencam H, Dertli E. Production of enantiomerically enriched chiral carbinols using Weissella paramesenteroides as a novel whole cell biocatalyst. Biocatal Biotransform 2019;37(5):388–98. https://doi.org/10.1080/10242422.2019.1568416. [55] Turner NJ. Directed evolution of enzymes for applied biocatalysis. Trends Biotechnol 2003;21(11):474–8. https://doi.org/10.1016/j.tibtech.2003.09.001. [56] Giorgi V, Chaves M, Men
endez P, Carnelli CG. Bioprospecting of whole-cell biocatalysts for cholesterol biotransformation. World J Microbiol Biotechnol 2019;35(1):12. https://doi.org/10.1007/s11274-018-2586-5. [57] Behrens GA, Hummel A, Padhi SK, Schаtzle S, Bornscheuer UT. Discovery and protein engineering of biocatalysts for organic synthesis. Adv Synth Catal 2011;353:2191–215. https://doi.org/10.1002/ adsc.201100446.

18

Chapter 1

[58] Bernala C, Rodrı´gueza K, Martı´nez R. Integrating enzyme immobilization and protein engineering: an alternativepath for the development of novel and improved industrial biocatalysts. Biotechnol Adv 2018;36 (5):1–10. https://doi.org/10.1016/j.biotechadv.2018.06.002. [59] Woodyer R, van der Donk WA, Zhao H. Optimizing a biocatalyst for improved NAD(P)H regeneration: directed evolution of Phosphite dehydrogenase. Comb Chem High Throughput Screen 2006;9:237–45. https:// doi.org/10.2174/138620706776843246. [60] Defaei M, Taheri-Kafrani A, Miroliaei M, Yaghmaei P. Improvement of stability and reusability of α-amylase immobilized on naringin functionalized magnetic nanoparticles: a robust nanobiocatalyst. Int J Biol Macromol 2018;113:354–60. https://doi.org/10.1016/j.ijbiomac.2018.02.147. [61] Homaei A, Saberi D. Immobilization of α-amylase on gold nanorods: an ideal system for starch processing. Process Biochem 2015;50(9):1394–9. https://doi.org/10.1016/j.procbio.2015.06.002. [62] Cao L. Immobilised enzymes: science or art? Curr Opin Chem Biol 2005;9(2):217–26. https://doi.org/10.1016/ j.cbpa.2005.02.014. [63] Secundo F. Conformational changes of enzymes upon immobilisation. Chem Soc Rev 2013;42(15):6250–61. https://doi.org/10.1039/c3cs35495d. [64] Del Arco J, P
erez E, Naitow H, Matsuura Y, Kunishima N, Ferna´ndez-Lucas J. Structural and functional characterization of thermostable biocatalysts for the synthesis of 6-aminopurine nucleoside-50 -monophospate analogues. Bioresour Technol 2019;276:244–52. https://doi.org/10.1016/j.biortech.2018.12.120. [65] Sheldon RA, Woodley JM. Role of biocatalysis in sustainable chemistry. Chem Rev 2018;118(2):801–38. https://doi.org/10.1021/acs.chemrev.7b00203. [66] Hosseini SM, Kim SM, Sayed M, Younesi H, Bahramifar N, Park JH, et al. Lipase-immobilized chitosan-crosslinked magnetic nanoparticle as a biocatalyst for ring opening esterification of itaconic anhydride. Biochem Eng J 2019;143:141–50. https://doi.org/10.1016/j.bej.2018.12.022. [67] Mohamad NR, Marzuki NHC, Buang NA, Huyop F, Wahab RA. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnol Biotechnol Equip 2015;29(2):205–20. https://doi.org/10.1080/13102818.2015.1008192. [68] Service RF. Surviving the Blockbuster syndrome. Science 2004;303(5665):1796–9. [69] Pollard DJ, Woodley JM. Biocatalysis for pharmaceutical intermediates: the future is now. Trends Biotechnol 2006;25(2):66–73. https://doi.org/10.1016/j.tibtech.2006.12.005. [70] Maureen Rouchi A. As pharmaceutical companies face bleak prospects, their suppliers diligently tend the fertile fields of chiral chemistry in varied ways. Chem Eng News 2002;80(23):43–50. https://doi.org/10.1021/ cen-v080n023.p043. [71] World Health Organization, n.d. Global website: https://www.who.int/selection_medicines/list/en. [72] Boaz NW, Clendennen SK. Patent 7566795 US. Publ. Date 28.07. [73] Koninklijke NV. Patent 2707193 US. Publ. Date 26.04. [74] Takahashi H. Patent 3673239 US. Publ. Date 27.07. [75] Britton G, Liaaen-Jensen S, Pfander H. Carotenoids handbook. Basel: Springer; 2004. [76] Sigma-Aldrich. https://www.sigmaaldrich.com. [77] Kuregyan AG, Pechinsky SV, Stepanova EF. Pat. of the Russian Federation 2648452. Bull. 2018. No.9. [78] Kuregyan AG, Pechinsky SV, Stepanova EF. Pat. of the Russian Federation 2659165. Bull. 2018. No. 19. [79] Kuregyan AG, Pechinsky SV. Production of carotenoids and their identification by spectroscopic methods in the IR and UV regions. Ques Biol Med Pharm Chem 2016;1:22–7.

CHAPTER 2

The physiology of astaxanthin production by carotenogenic microalgae Alexei Solovchenkoa and Galina Minyukb a

Lomonosov Moscow State University, Moscow, Russia bA.O. Kovalevsky Institute of Biology of the Southern Seas of RAS, Sevastopol, Russia

Abbreviation ROS reactive oxygen species

1 Introduction Recent years observed an explosive growth of interest in carotenogenic microalgae [1, 2]—the single-celled algae that accumulate, usually under adverse conditions, high amounts of secondary carotenoids. Carotenoids are an extensive group of terpenoid pigments present in all phototrophic organisms [3]. Until recently, researchers focused mostly on primary (photosynthetic) carotenoids associated with thylakoid membranes of chloroplasts performing light harvesting, protection from photooxidative damage, and maintenance of the structure of the photosynthetic apparatus—the functions vital for all autotrophs [3, 4]. Unlike primary carotenoids, secondary carotenoids exemplified by astaxanthin do not participate in photosynthesis and are not coupled to the photosynthetic apparatus either structurally or functionally [5, 6]. The functions of secondary carotenoids are still the subject of lively discussion. The postulated functions of these pigments are screening of excess light, utilization of the excess photosynthates, and suppression of formation and detoxification of reactive oxygen species (ROS). Accumulation of secondary carotenoids including astaxanthin is triggered by adverse environmental conditions—high light, extreme temperatures, salinity, drought, and combinations thereof. Secondary carotenogenesis is particularly characteristic of stress-tolerant species, such as “snow algae” dwelling on the surfaces of Antarctic glaciers and high-altitude snowfields, such as Chlorella nivalis. Such natural phenomena as “blood rain” and “blood snow” are associated with the mass growth of these microorganisms [7, 8]. Carotenogenic microalgae often dominate ecosystems with extreme living conditions due to their high resilience to the stressors conveyed by the accumulation of astaxanthin in their cells [9, 10]. Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00026-X Copyright # 2021 Elsevier Inc. All rights reserved.

19

20

Chapter 2

(A)

(B) Fig. 2.1 The typical secondary keto-carotenoid astaxanthin (A) and its precursor β-carotene (B).

Importantly, astaxanthin (Fig. 2.1) is very photostable (resistant to photobleaching) in situ [11]. In this regard the stress protection offered by astaxanthin is dramatically different from other photoprotective mechanisms such as nonphotochemical quenching of excited chlorophyll states and enzymatic detoxification of ROS, which require continuous investments of metabolites and energy [12, 13]. Therefore the protection based on astaxanthin accumulation is crucial for microalgae that have to endure long-term periods of adverse conditions, for example, for inhabitants of small drying reservoirs and “snow” algae [5, 7]. A striking example is the cosmopolitan carotenogenic microalgae Haematococcus pluvialis, distributed worldwide from deserts and mountains to the Arctic seas [14, 15]. Secondary carotenogenesis and, particularly, accumulation of astaxanthin by microalgae are also very important from a practical point of view [16–18] since astaxanthin exerts a plethora of beneficial effects in humans and animals [19–21]. Thus astaxanthin is the most efficient antioxidant, preventing the emergence and development of age-related degenerative and cancer diseases; they are widely used as safe natural colorants in the food industry and feed additives in aquaculture [2, 22, 23]. In this regard, microalgae producers of astaxanthin are important objects of biotechnology. The composition and ratio of primary carotenoids are quite conservative, since they are “hard coded” in the genomic program for building of the photosynthetic apparatus. In contrast the content of secondary carotenoids is less strictly controlled making possible their gross accumulation, so carotenogenic microalgae are more suitable for biotechnological production of natural astaxanthin. In the succeeding text, we cover the biochemical pathway of astaxanthin biosynthesis in carotenogenic microalgae and its regulation as the biological basis of currently widespread twostage cultivation approach. The role of the controlled stress as the inducing factor of astaxanthin accumulation will be discussed. Current limitations and prospects of the biotechnological production of natural astaxanthin from microalgae will be considered in conclusion.

The physiology of astaxanthin production by carotenogenic microalgae 21

2 Biosynthesis of astaxanthin and its coordination with lipid biosynthesis The first stages of astaxanthin biosynthesis—the assembly of the carbon skeleton desaturation and cyclization—are identical to those of primary carotenoid biosynthesis. A brief description of these stages is given in the succeeding text. For detailed description of the corresponding reactions, enzymes, and genes, the reader is referred to the excellent reviews [17, 24–26]. In green microalgae, as in higher plants, carotenoids are formed from the common precursor, isopentenyl pyrophosphate (IPP, C5), synthesized either via the mevalonate, as in Euglenophyceae, or nonmevalonate pathway [17, 27, 28] as in H. pluvialis [29]. In a reversible reaction catalyzed by IPP isomerase, IPP is converted to its allyl isomer, dimethylallyl pyrophosphate (DMAPP), the first activated substrate for the synthesis of polyisoprenoid chains. This enzyme is an important point of carotenoid biosynthesis control limiting the precursor inflow to this pathway [24]. As a result of three consecutive IPP linking to a DMAPP molecule, a geranylgeranyl pyrophosphate (GGPP, C20) molecule is formed with the participation of GGPP synthase. Two GGPP molecules form a symmetrical phytoene molecule in a reaction catalyzed by phytoene synthase (PSY). The phytoene molecule undergoes four successively desaturation reactions catalyzed by phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) to form first phytofluene, then ζ-carotene, neurosporene, and finally lycopene containing 5, 7, 9, and 11 conjugated double bonds, respectively [24]. As a result of elongation of the conjugated double bond system, the colorless precursors turn into colored (starting from ζ-carotene) compounds. Desaturation reactions are also among the limiting stage of astaxanthin biosynthesis [26]. An important cofactor of the microalgal desaturases is the plastid terminal oxidase (PTOX) oxidizing plastoquinol and reducing dioxygen to water in chlororespiration [30]. Wang et al. [31] found in the transcriptome of H. pluvialis two forms of PTOX—ptox1 and ptox2 involved in the biosynthesis of astaxanthin. The importance of this enzyme for protection against oxidative stress has been shown [32–34]. The membrane-bound β-lycopene cyclase catalyzes the formation of dicyclic rings containing two β-ionone rings from a symmetrical linear lycopene molecule [24]. All the reactions mentioned earlier occur in the chloroplast. The precursor of astaxanthin, β-carotene is exported from chloroplast, most likely with participation of protein carriers from the lipocalin family, so the remaining stages of astaxanthin biosynthesis take place in the membranes of cytoplasmic lipid globules—oleosomes (the role of these structures in astaxanthin biosynthesis is elaborated in the succeeding text). As a result of hydroxylation at the position 3 of the α- and β-carotene rings, zeaxanthin and lutein are formed, respectively; the addition of a ketogroup at the position 4 to one or both rings leads to the formation of orange-red ketocarotenoids, echinenon and canthaxanthin, precursors of astaxanthin. The pathway of astaxanthin biosynthesis at his stage has been studied in detail

22

Chapter 2 Common pathway of carotenoid biosynthesis

CRTO, BKT

β,β-Carotene

CRTR-b

Echinenone β-Cryptoxanthin

CRTR-b

CRTO, BKT

CRTO

CRTO, BKT

CRTR-b 3'-Hydroxyechinenone Violaxanthin

ZEP1 VDE1

Canthaxanthin Zeaxanthin CRTO, BKT

CRTR-b

CRTO, BKT

CRTR-b

Adonirubin CRTR-b

Adonixanthin CRTO, BKT Astaxanthin

Fig. 2.2 Pathways potentially involved in the formation of secondary carotenoids exemplified by the biosynthesis of astaxanthin in Haematococcus pluvialis. Highly expressed routes are indicated with bold arrows. Thin arrows indicate putative and/or weakly expressed branches. CRTR-b, 3,30 -hydroxylase; BKT, CRTO, 4,40 -ketolase; VDE1, violaxanthin deepoxidase; ZEP1, zeaxanthin epoxidase. Adapted from Grunewald K, Hirschberg J, Hagen C. Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis. J Biol Chem 2001;276(8):6023–9; Lemoine Y, Schoefs B. Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress. Photosynth Res 2010;106(1):155–77. https://doi.org/10.1007/s11120-010-9583-3.

(Fig. 2.2), and many associated genes have been isolated and cloned [35, 36]. Thus, to form astaxanthin, two hydroxyl groups (at the positions 3 and 30 ) and two ketogroups (at the positions 4 and 40 ) must be introduced into the β-carotene molecule. Different strains of H. pluvialis have different isoforms of the BKT gene, Bkt1-Bkt3 differentially upregulated under stress [37]. The β-carotene oxygenation reaction is catalyzed by β-C-4-oxygenase (ketolase, CRTO, or BKT) encoded in H. pluvialis by the crtO or bkt gene; different strains of H. pluvialis have up to three BKT genes encoding functionally similar enzymes BKT1-BKT3 [24, 35, 36]. These enzymes belong to an extensive family of hydrophobic membrane-bound Fe-containing oxygenases [35]. This enzyme is unable to use the dihydroxy xanthophyll zeaxanthin as a substrate; therefore, in the process of astaxanthin biosynthesis, the introduction of ketogroups occurs before hydroxylation [26]. Thus astaxanthin formation proceeds via echinenone

The physiology of astaxanthin production by carotenogenic microalgae 23 (one ketogroup), canthaxanthin (two keto groups), and adonirubin (two keto and one hydroxy group). The importance of CRTO for the carotenogenic response is also evidenced by the fact that the activity of this enzyme and accumulation of astaxanthin take place in parallel. The presence of active CRTO in H. pluvialis not only in chloroplast but also in cytoplasmic lipid globules [38] was established by inhibitory analysis and immunocytochemistry. In contrast to primary carotenoids, localized exclusively in the thylakoid membranes of the chloroplasts, secondary carotenoids are accumulated only outside the thylakoids since the pigment composition of the photosynthetic apparatus is strictly conserved. Indeed, attempts to embed astaxanthin in the chloroplast membranes of transgenic tobacco plants led to their destabilization [39]. The accumulation of free astaxanthin in the cytoplasm is also impossible due to hydrophobicity of this pigment. Therefore molecules of astaxanthin must be decorated by fatty acid molecules to form less polar esters before deposition in the hydrophobic environment of the oleosomes. Thus, in astaxanthin-producing strains of H. pluvialis and Chromochloris zofingiensis, more than 95% of this pigment is converted into fatty acid esters at the final stages of the stress-induced haematocyst (“red” cell) formation [10]. Interestingly the images obtained by nonlinear optical microscopy showed that astaxanthin molecules deposited in H. pluvialis oleosomes is characterized by ordered isotropic packaging, while in the aggregates of synthetic astaxanthin molecules are packed anisotropically [40, 41]. This fact supports the participation of (multi) enzyme complexes, likely assembled in rafts floating in the oleosome membrane in the final stages of astaxanthin biosynthesis. Further details on the metabolic rearrangements of the microalgal cell accompanying stress-induced massive accumulation of astaxanthin can be found in our recent review [42, 43]. Induction of astaxanthin biosynthesis in H. pluvialis and other carotenogenic microalgae is accompanied by an intensive accumulation of neutral reserve lipids triacylglycerides. At the ultrastructural level, this is manifested as the formation of large oleosomes in the cytoplasm and reduction of chloroplasts [15, 44]. Membrane polar lipids make up about 5% of total oleosome lipids, surrounded by a single-layer membrane. They also include phosphatidylethanol, characteristic of extraplastidial membranes, and diacylglyceryltrimethylhomoserine—the major polar lipid of the oleosome membranes [45]. The monolayer membranes of oleosomes in H. pluvialis are stabilized by the protein HOGP (Haematococcus oil globule protein), which expression is synchronized with the rate of oleosome biogenesis [29]. Inhibition of neutral lipid biosynthesis suppressed astaxanthin accumulation, whereas inhibition of this pigment biosynthesis did not affect neutral lipid accumulation and oleosome formation [46]. It is assumed that the formation of oleosomes, the hydrophobic structures capable of accommodating large amounts of astaxanthin esters, creates a potent sink for this pigment and shifts the chemical equilibrium in the direction favorable for the synthesis of astaxanthin. More specific mechanisms, for example, lifting the inhibition of the astaxanthin biosynthesis enzymes by the terminal product by its sequestration to the oleosomes, are also

24

Chapter 2

possible [46, 47]. Collectively, these findings suggest a close relationship between the biosynthesis of astaxanthin and neutral lipid accumulation, which was proved experimentally. This close relationship is also important for the biotechnological production of astaxanthin. Thus, for hyperproduction of primary carotenoids (such as lutein or fucoxanthin), very significant changes would have to be made to the microalgae genome, in particular to create an extrathylakoid depot offering a sink for these pigments, whereas in the case of secondary carotenoids, for example, astaxanthin such a depot already exists in the form of oleosomes.

3 Triggering of astaxanthin biosynthesis and its regulation Accumulation of astaxanthin is observed under the influence of various stressors that slow down cell division and photosynthesis [6, 48], often in a dose-dependent manner (Fig. 2.3). It was found that stressors triggering astaxanthin accumulation increase the expression of approximately the same set of carotenoid biosynthesis genes. Thus the action of high-intensity light combined with increased salinity enhances the transcription of genes that control a number of steps from IPP biosynthesis to β-carotene hydroxylation [49, 50]. The level of transcripts of the CrtR-b gene was linearly correlated with the accumulation of astaxanthin in wild-type H. pluvialis and in the astaxanthin hyperaccumulator mutant MT 2877, indicating the participation of this gene in the transcriptional control of astaxanthin biosynthesis [34, 51]. On the other hand, parallel accumulation of the transcript of gene (s) encoding BKT and the corresponding enzyme (s) was recorded only at the initial stages of stress-induced astaxanthin biosynthesis. Later the protein accumulation outpaced the transcript accumulation, suggesting translational and/or posttranslational regulation of β-carotene ketolation [38]. Most of the genes that control carotenoid biosynthesis is upregulated at the transcription level by high-intensity light, excess iron sulfate, and sodium acetate in a synergistic manner [34, 52]. It is assumed that carotenogenic microalgae possess both constitutive and stress-responsive mechanisms of astaxanthin accumulation, which result in development of a similar phenotype [53, 54]. Stress sensing in photosynthetic organisms occurs via reduction of the plastoquinone pool, cell membrane fluidity, and stationary concentrations of ROS [55, 56] and so-called “hub metabolites” [57]. Similar sensing mechanisms, especially those mediated by ROS, are involved in the induction of astaxanthin biosynthesis [38, 58, 59]. Thus treatment with ROS generator compounds such as hydrogen peroxide, peroxynitrite, and sodium hypochlorite triggers the accumulation of astaxanthin even in the dark, mimicking the environmental stress effect in this regard [60]. One cannot rule out that the ROS formed under stress also act as secondary messengers activating astaxanthin biosynthesis [61, 62]. In the case of Haematococcus, it is believed that plastoquinone pool is the main redox sensor in control of astaxanthin biosynthesis: its reduction is correlated with astaxanthin accumulation under stress [63, 64]. Indeed, treatment with a plastoquinone reduction inhibitor diuron inhibits

The physiology of astaxanthin production by carotenogenic microalgae 25

Stress exposure Absolute photsynthesis

Antioxidative enzymes

Chlorophyll+ photsynthetic carotenoids Respiration

Neutral lipids

Astaxanthin

Cell division rate Starch

GREEN (motile vegetative cells)

BROWN (immotile cellswith intermediate Car/Chl)

RED (resting cells, haematocysts)

Fig. 2.3 A hypothetic scenario of the physiological changes accompanying the induction of astaxanthin accumulation in the stressed cells of H. pluvialis. At the initial stages of the astaxanthin accumulation, the cell retains a significant amount of photosynthetic pigments and photosynthetic activity driving accumulation of starch. At this stage the upregulated antioxidative enzymes protect the cell. Later the starch synthesis is followed by its degradation, a rise of respiration rate and induction of fatty acid and astaxanthin biosynthesis resulting in the appearance of red lipid droplets in the cell. Decline in photosynthetic pigments proceeds (although the specific photosynthesis rate might be preserved), the antioxidative enzyme activity reverts to the basal level. Reprinted from Solovchenko AE. Recent breakthroughs in the biology of astaxanthin accumulation by microalgal cell. Photosynth Res 2015;125(3): 437–49. https://doi.org/10.1007/s11120-015-0156-3 with kind permission from Springer.

the activation of astaxanthin biosynthesis genes under high light [63, 64]. In the succeeding text, specific stressor effects on astaxanthin biosynthesis are considered in more detail. There is a large body of evidence in the literature supporting the stimulating effect of high light on the biosynthesis of astaxanthin in microalgae (see Refs. [42, 43] and references therein). Still, astaxanthin can be accumulated also in the dark in the presence of an organic carbon source (sodium acetate) and/or ROS generators (see aforementioned). Interestingly, treatment with a cytostatic agent vinblastine promotes astaxanthin accumulation even in the absence of

26

Chapter 2

stress. Mineral nutrient shortage especially that of nitrogen and phosphorus also facilitates the accumulation of astaxanthin slowing down cell division. Moreover, herbicide inhibitors of glutamine synthase that disrupt nitrogen uptake by the cell induce astaxanthin accumulation in H. pluvialis like nitrogen starvation. Osmotic (salinity) and extreme temperature stress efficiently trigger the accumulation of astaxanthin as well. Availability of an organic carbon source is an important factor enhancing the accumulation of astaxanthin in carotenogenic microalgae capable of heterotrophic or photoheterotrophic growth [60]. Acetate and sugars added to the culture medium can downregulate photosynthetic carbon fixation on the feedback principle and increase the accumulation of astaxanthin [53, 59]. It is essential that glucose analogues that cannot be directly metabolized do not show such effect, so it cannot be attributed to the osmotic activity of the sugars. It is possible that “glucose sensors” such as hexokinase can trigger astaxanthin accumulation and changes in the redox status of the cytoplasm and electron transport chains in mitochondria and chloroplasts [26, 65, 66]. An interesting avenue of research on astaxanthin biosynthesis regulation is represented by studies of phytohormone effect(s) [65, 66]. Thus treatment of H. pluvialis cells with gibberellin A3 (GA3) and epibrassinolide (EBR) differentially upregulated the genes involved in carotenoid biosynthesis genes in a concentration-dependent manner. These results suggest that exogenous GA3 and EBR hormone application is a promising approach to increasing the productivity astaxanthin-producing carotenogenic microalgae.

4 Biological significance of astaxanthin accumulation The effects of astaxanthin on the microalgal cell stress resilience have been in the focus of vigorous research for more than 30 years [26, 34, 48, 67–73], but the functions of secondary carotenoids including astaxanthin in microalgae remained are still actively debated. On one extremity is the opinion that astaxanthin is a metabolic dead end without a significant role. However, many recent studies (for reviews, see Refs. [42, 43]) strongly support a plethora of protective functions of astaxanthin implemented in the microalgal cell by diverse mechanisms including photoprotection. Potential mechanisms augmenting stress tolerance of the organisms expressing the CR include (i) optical shielding by the secondary car, (ii) providing the sink for excessive photosynthates, (iii) consumption of O2, and (iv) a local antioxidant effect (see Fig. 2.4). In this section, we consider these functions in more detail.

4.1 Optical shielding of the cell Photosynthetic organisms require light energy to drive photosynthesis although excessive light, which cannot be utilized in photochemical reactions, brings about a risk of photooxidative damage to the cell [74, 75]. Apart of “classical” photoprotective mechanisms based on light harvesting regulation, thermal dissipation of the absorbed light energy, and enzymatic

Structural lipids assembly

Primary carotemoids

RuBP

Fatty acid synthesis Pyruvate PEP

Acetyl-CoA

β-Carotene

CO2

3-PGA

IPP G-3-P

Phytoene

– e

OEC

H2O

PS II

O2

PS I

– e

RC

PQ

– e

b6f

–e

e–

PC

O2

H2O

FNR

LHC

LHC

RC

H+

3 Starch synthesis and degradation

Storage lipid (TAG) assembly

Astaxanthin FA esters

Starch

Fatty acids Astaxanthin

Export from the chloroplast 6

Carotenoid synthesis

ADP-Glucose

4

5

Malate

TCA cycle

Fatty acid synthesis

Acetyl-CoA β-carotene

GABA

RuBP

Glycolysis

Pyruvate

PEP 3-PGA

IPP

3-PGA

ζ-carotene Phytoene

NPQ

He

at

– e

H2O

2

O2

RC

H+ NADP+ NADPH ATP ADP PS I FNR

PS 1II LHC

Calvin cycle

ATP, NADPH,NADH

PTOX H2O

CO2

G-3-P

Glucose

PDS

ΔpH

Thylakoid membrane

Endoplasmic reticulum

Lipid droplet

–e

– e

LHC PQ

b6 f

PC

ATPase

He at

H+ NADP+ NADPH ATP ADP

PTOX

PDS NPQ

ATP, NADPH, NADH

ATPase

ζ-Carotene

Thylakoid membrane

ΔpH

H+

Fig. 2.4 The schematic view of major changes in photosynthetic electron flow and carbon partitioning patterns of (A) the “green” cells of H. pluvialis in the course of the stress-induced accumulation of astaxanthin (B) with an emphasis on lipid and carotenoid synthesis. In the “green” cells, linear electron flow in the electron transport chain of chloroplast dominates and the fixed carbon mainly Continued

28

Chapter 2

elimination of ROS [74], certain organisms engage special pigments for optical attenuation (screening) of the excessive light. The latter mechanism operates proactively preventing the absorption of light by chlorophyll and other photosensitizers [76]. Indeed, astaxanthin was shown to be able to intercept in vivo a considerable part of light otherwise reaching chlorophyll, and the degree of its protection is tightly related with the ratio of the screen (astaxanthin) and the photosensitizer (chlorophyll) in the cell [11, 71, 77, 78]. Furthermore the significance of light screening by the lipid droplets (LD) containing astaxanthin was further supported by the phenomenon of quick migration of the LD to the periphery of cell in the H. pluvialis cells exposed to high light [79].

4.2 Possible antioxidative function of astaxanthin The astaxanthin-enriched lipid droplets are believed to form an antioxidant barrier around the nucleus and the chloroplast and protecting these structures from ROS attacks [48, 70, 72]. Antioxidative effect of astaxanthin in vivo has been debated [53, 54] since it is unlikely that astaxanthin acts as antioxidant in the thylakoid membranes because it is localized apart from these membranes in cytoplasmic lipid droplets. On the other hand, astaxanthin can bind to the photosynthetic pigment-protein complexes in transgenic plants [39]. The role of astaxanthin as antioxidant is more significant in the lipid droplets where astaxanthin protects the lipids prone to peroxidation by ROS.

Fig.2.4, cont’d partitioned between structural components of the cell and photosynthetic pigments. The onset of carotenogenesis is accompanied by (1) reduction of photosynthetic apparatus and (2) increase of the cyclic electron flow contribution. The photosynthetically fixed carbon and metabolites repartitioned from other metabolic pathways (such as (3) starch turnover and (4) tricarbonic acid cycle) are converted mainly to astaxanthin (5) and (6) fatty acids consumed for the assembly of neutral lipids and esterification of the astaxanthin molecules. Compiled from Gu W, Li H, Zhao P, Yu R, Pan G, Gao S, et al. Quantitative proteomic analysis of thylakoid from two microalgae (Haematococcus pluvialis and Dunaliella salina) reveals two different high light-responsive strategies. Sci Rep 2014;4. doi:https://doi.org/ 10.1038/srep06661; Gwak Y, Hwang Y-S, Wang B, Kim M, Jeong J, Lee C-G, et al. Comparative analyses of lipidomes and transcriptomes reveal a concerted action of multiple defensive systems against photooxidative stress in Haematococcus pluvialis. J Exp Bot 2014;65(15):4317–34. doi:https://doi.org/10.1093/jxb/eru206; Recht L, Zarka A, Boussiba S. Patterns of carbohydrate and fatty acid changes under nitrogen starvation in the microalgae Haematococcus pluvialis and Nannochloropsis sp. Appl Microbiol Biotechnol 2012;94(6):1495–503. doi: https://doi.org/10.1007/s00253-012-3940-4; Scholz MJ, Weiss TL, Jinkerson RE, Jing J, Roth R, Goodenough U, et al. Ultrastructure and composition of the Nannochloropsis gaditana cell wall. Eukaryot Cell 2014;13 (11):1450–64. Reprinted from Solovchenko AE, Chivkunova OB, Maslova IP. Pigment composition, optical properties, and resistance to photodamage of the microalga Haematococcus pluvialis cultivated under high light. Russ J Plant Physiol 2011;58(1):9–17. https://doi.org/10.1134/S1021443710061056 with kind permission from Springer.

The physiology of astaxanthin production by carotenogenic microalgae 29

4.3 Suppression of ROS formation The risk of photooxidative damage of photosynthetic organisms under stress is directly related with stationary concentration of O2 and hence that of ROS [74, 80]. Therefore the metabolic processes consuming O2 can, in principle, decrease the rate ROS generation and alleviate the risk of photodamage. The biosynthesis of astaxanthin consumes a lot of O2 as the cosubstrate of plastid terminal oxidase (PTOX)—a plastoquinol oxidase serving as a cofactor of the phytoene desaturases and the key oxidase in chlororespiration [30] and reducing O2 to H2O [32–34]; see also Fig. 2.4. Additional amounts of O2 are consumed by the different oxygenases and oxidases participating the oxygenation of β-carotene in the course of its conversion to secondary xanthophylls [24]. The relative decline in steady-state O2 concentration in the cell during vigorous astaxanthin accumulation is estimated to be above 10% of its stationary concentration, which may be significant under adverse conditions [34].

4.4 Sink for photosynthates and energy store Vigorously dividing microalgal cells feature a high demand for energy and reducing power normally satisfied by photosynthesis. Stresses slow down cell division and drive off balance the rates of photosynthate generation and consumption in the cell [57, 81, 82]. Under such conditions the electron carriers in the electron transport chain of chloroplast end up in an overreduced state increasing the probability of electrons leak to O2 and hence the ROS formation. This risk is mitigated by photoprotective mechanisms (see aforementioned) and/or by channeling the excessive photosynthates to the biosynthesis of carbon- and energy-rich reserve compounds such as carbohydrates (starch), lipids (triacylglycerols), and astaxanthin [83]. Accordingly, induction of massive astaxanthin accumulation provides a potent sink for photosynthates via two processes. One is astaxanthin synthesis itself, another one the biosynthesis of neutral lipids forming lipid globules the depot necessary for astaxanthin deposition (see aforementioned; Refs. [44, 47, 84]). Importantly the cells that failed to accumulate large amounts of astaxanthin and lipids can be damaged by abrupt and/or prolonged stress; this is not the case in the cells with a high astaxanthin content [11, 69]. This observation is important for biotechnological production of astaxanthin from microalgae since a high cell mortality due to a low stress resilience might decline severely the astaxanthin productivity of microalgal cultures [85].

5 Concluding remarks: Perspectives for astaxanthin from microalgae Microalgae have distinct advantages as a source of natural astaxanthin, since they (i) selectively accumulate it (up to ca. 95% of the total carotenoids) in high amounts (6% of cell dry weight) by far exceeding other organisms, (ii) rapidly accumulate biomass, and (iii) do not compete with crops for arable land [86]. However, the cost efficiency of current microalgal biotechnologies for production of astaxanthin from microalgae is severely limited by productivity of known strains [2]. Currently

30

Chapter 2

the chlorophyte H. pluvialis is the most biotechnologically significant microalga producer of astaxanthin despite its intrinsic shortcomings. The latter include slow growth, low biomass yield, and high risk of contamination at the “green” (vegetative cell) stage. A promising alternative is represented by C. zofingiensis [50, 87] featuring a faster growth and culture robustness [77, 88]. Productivity of astaxanthin-producing microalgal cultures can be increased by optimization of cultivation conditions. In frame of the conventional two-stage cultivation approach [2, 15, 18, 89, 90], the optimization includes increasing the biomass productivity at the “green” (vegetative cell stage) and optimization of the nutrient supply, illumination, and temperature conditions to decline cell mortality. However, this already highly explored and exploited direction [86, 87] is unlikely to produce a revolutionary increase in astaxanthin productivity. A novel attracting way of boosting astaxanthin accumulation beyond 5% cell dry weight (the threshold that should be surpassed for natural astaxanthin from microalgae to be competitive with the synthetic pigment [89]) is microalgal strain engineering. One approach is rewiring the regulatory networks in the cell to remove metabolic bottlenecks of astaxanthin biosynthesis by increasing the metabolic flux toward the biosynthesis of the pigment of interest without significant disturbance to the rest of the metabolic network of the cell [25]. However, it is important to avoid, for example, competition between secondary and primary carotenoid biosynthesis, which will most like result in growth inhibition. Possible targets for engineering include the rate-limiting enzymes of astaxanthin biosynthesis catalyzing phytoene desaturation, lycopene cyclization [26], and ketolation of β-carotene [17]. Still, metabolic pathways in microalgae are complex, with numerous regulatory inputs and interplay with other pathways, so it is difficult to find the best manipulation target(s) and predict the outcome with confidence [25, 91]. In the case of astaxanthin, this approach is complicated at least by (i) distribution of the enzymes of astaxanthin biosynthesis between compartments (chloroplast and LD) and (ii) a potential bottleneck associated with the export of β-carotene from the chloroplast to the LD that mechanism remains so far elusive. Remarkably a sufficiently developed transformation method and genetic toolbox were developed recently [92]. Further hints for target selection are expected to come from recent studies in system biology of microalgae. More sophisticated strategies based on fine-tuned transgene expression and thermodynamic and kinetic models will be then necessary to balance the metabolic fluxes in the entire astaxanthin biosynthesis pathway.

Acknowledgment Financial support of Russian Ministry of Science and Higher Education (Contract # 075-15-2019-1719/ RFMEFI60419X0213) is greatly appreciated.

The physiology of astaxanthin production by carotenogenic microalgae 31

References [1] Guedes AC, Amaro HM, Malcata FX. Microalgae as sources of carotenoids. Mar Drugs 2011;9(4):625–44. [2] Lorenz RT, Cysewski GR. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol 2000;18(4):160–7. [3] Sun T, Yuan H, Cao H, Yazdani M, Tadmor Y, Li L. Carotenoid metabolism in plants: the role of plastids. Mol Plant 2018;11(1):58–74. https://doi.org/10.1016/j.molp.2017.09.010. [4] Takaichi S. Tetraterpenes: carotenoids. In: Ramawat KG, M
erillon J-M, editors. Natural products. Berlin Heidelberg: Springer; 2013. p. 3251–83. [5] Remias D, Pichrtova M, Pangratz M, Lutz C, Holzinger A. Ecophysiology, secondary pigments and ultrastructure of Chlainomonas sp. (Chlorophyta) from the European Alps compared with Chlamydomonas nivalis forming red snow. FEMS Microbiol Ecol 2016;92(4):fiw030. https://doi.org/10.1093/femsec/fiw030. [6] Solovchenko A. Physiology and adaptive significance of secondary carotenogenesis in green microalgae. Russ J Plant Physiol 2013;60(1):1–13. [7] Czygan F. Blood-rain and blood-snow: nitrogen-deficient cells of Haematococcus pluvialis and Chlamydomonas nivalis. Arch Mikrobiol 1970;74(1):69. [8] Varshney P, Mikulic P, Vonshak A, Beardall J, Wangikar PP. Extremophilic micro-algae and their potential contribution in biotechnology. Bioresour Technol 2014;184:363–72. [9] Bidigare R, Ondrusek M, Kennicutt M, Iturriaga R, Harvey H, Hoham R, Macko S. Evidence a photoprotective for secondary carotenoids of snow algae. J Phycol 1993;29(4):427–34. [10] Sussela M, Toppo K. Haematococcus pluvialis—a green alga, richest natural source of astaxanthin. Curr Sci 2006;90(12):1602–3. [11] Solovchenko AE, Chivkunova OB, Maslova IP. Pigment composition, optical properties, and resistance to photodamage of the microalga Haematococcus pluvialis cultivated under high light. Russ J Plant Physiol 2011;58(1):9–17. https://doi.org/10.1134/s1021443710061056. [12] Horton P. Developments in research on non-photochemical fluorescence quenching: emergence of key ideas, theories and experimental approaches. In: Demmig-Adams B, Garab G, Adams III W, Govindjee, editors. Non-photochemical quenching and energy dissipation in plants, algae and cyanobacteria. vol. 40. Springer Netherlands; 2014. p. 73–95. [13] Tikhonov A. Induction events and short-term regulation of electron transport in chloroplasts: an overview. Photosynth Res 2015;1–30. https://doi.org/10.1007/s11120-015-0094-0. [14] Klochkova TA, Kwak MS, Han JW, Motomura T, Nagasato C, Kim GH. Cold-tolerant strain of Haematococcus pluvialis (Haematococcaceae, Chlorophyta) from Blomstrandhalvøya (Svalbard). Algae 2013;28(2):185–92. [15] Solovchenko A, Chekanov K. Production of carotenoids using microalgae cultivated in photobioreactors. In: Paek K-Y, Murthy HN, Zhong J-J, editors. Production of biomass and bioactive compounds using bioreactor technology. Springer Netherlands; 2014. p. 63–91. [16] Grung M, D’Souza FM, Borowitzka M, Liaaen-Jensen S. Algal carotenoids 51. Secondary carotenoids 2. Haematococcus pluvialis aplanospores as a source of (3S, 30 S)-astaxanthin esters. J Appl Phycol 1992;4 (2):165–71. [17] Han D, Li Y, Hu Q. Astaxanthin in microalgae: pathways, functions and biotechnological implications. Algae 2013;28(2):131–47. [18] Han D, Li Y, Hu Q. Biology and commercial aspects of Haematococcus pluvialis. In: Richmond A, Hu Q, editors. Handbook of microalgal culture: applied phycology and biotechnology. 2nd ed. Blackwell; 2013. p. 388–405. [19] Ambati RR, Phang S-M, Ravi S, Aswathanarayana RG. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications—a review. Mar Drugs 2014;12(1):128–52. [20] Choi CI. Astaxanthin as a peroxisome proliferator-activated receptor (PPAR) modulator: its therapeutic implications. Mar Drugs 2019;17(4); https://doi.org/10.3390/md17040242. [21] Lim KC, Yusoff FM, Shariff M, Kamarudin MS. Astaxanthin as feed supplement in aquatic animals. Rev Aquac 2017; https://doi.org/10.1111/raq.12200.

32

Chapter 2

[22] Dhankhar J, Kadian SS, Sharma A. Astaxanthin: a potential carotenoid. Int J Pharm Sci Res 2012;3 (5):1246–59. [23] Nishino H, Murakoshi M, Tokuda H, Satomi Y. Cancer prevention by carotenoids. Arch Biochem Biophys 2009;483(2):165–8. [24] Cunningham Jr. F, Gantt E. Genes and enzymes of carotenoid biosynthesis in plants. Annu Rev Plant Biol 1998;49(1):557–83. [25] Farr
e G, Blancquaert D, Capell T, Van Der Straeten D, Christou P, Zhu C. Engineering complex metabolic pathways in plants. Annu Rev Plant Biol 2014;65:187–223. [26] Lemoine Y, Schoefs B. Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress. Photosynth Res 2010;106(1):155–77. https://doi.org/10.1007/s11120-010-9583-3. [27] Lichtenthaler HK. The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Biol 1999;50(1):47–65. [28] Zhao L, Chang W-c, Xiao Y, Liu H-w, Liu P. Methylerythritol phosphate pathway of isoprenoid biosynthesis. Annu Rev Biochem 2013;82(1):497–530. https://doi.org/10.1146/annurev-biochem-052010-100934. [29] Gwak Y, Hwang Y-s, Wang B, Kim M, Jeong J, Lee C-G, Jin E. Comparative analyses of lipidomes and transcriptomes reveal a concerted action of multiple defensive systems against photooxidative stress in Haematococcus pluvialis. J Exp Bot 2014;65(15):4317–34. https://doi.org/10.1093/jxb/eru206. [30] Nawrocki WJ, Tourasse NJ, Taly A, Rappaport F, Wollman F-A. The plastid terminal oxidase: its elusive function points to multiple contributions to plastid physiology. Plant Biol 2015;66:14.11–26. [31] Wang J, Sommerfeld M, Hu Q. Occurrence and environmental stress responses of two plastid terminal oxidases in Haematococcus pluvialis (Chlorophyceae). Planta 2009;230(1):191–203. [32] Bennoun P. Chlororespiration and the process of carotenoid biosynthesis. BBA-Bioenergetics 2001;1506 (2):133–42. [33] Bennoun P. The present model for chlororespiration. Photosynth Res 2002;73(1):273–7. [34] Li Y, Sommerfeld M, Chen F, Hu Q. Consumption of oxygen by astaxanthin biosynthesis: a protective mechanism against oxidative stress in Haematococcus pluvialis (Chlorophyceae). J Plant Physiol 2008;165 (17):1783–97. https://doi.org/10.1016/j.jplph.2007.12.007. [35] Fraser PD, Miura Y, Misawa N. In vitro characterization of astaxanthin biosynthetic enzymes. J Biol Chem 1997;272(10):6128–35. https://doi.org/10.1074/jbc.272.10.6128. [36] Fraser PD, Shimada H, Misawa N. Enzymic confirmation of reactions involved in routes to astaxanthin formation, elucidated using a direct substrate in vitro assay. Eur J Biochem 1998;252(2):229–36. [37] Huang J-C, Wang Y, Sandmann G, Chen F. Isolation and characterization of a carotenoid oxygenase gene from Chlorella zofingiensis (Chlorophyta). Appl Microbiol Biotechnol 2006;71(4):473–9. [38] Grunewald K, Hirschberg J, Hagen C. Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis. J Biol Chem 2001;276(8):6023–9. [39] R€oding A, Dietzel L, Schlicke H, Grimm B, Sandmann G, B€ uchel C. Production of ketocarotenoids in tobacco alters the photosynthetic efficiency by reducing photosystem II supercomplex and LHCII trimer stability. Photosynth Res 2015;123(2):157–65. https://doi.org/10.1007/s11120-014-0055-z. [40] Ota S, Kawano S. Three-dimensional ultrastructure and hyperspectral imaging of metabolite accumulation and dynamics in Haematococcus and Chlorella. Microscopy (Oxf ) 2018; https://doi.org/10.1093/jmicro/dfy142. [41] Tokarz D, Cisek R, El-Ansari O, Espie GS, Fekl U, Barzda V. Organization of astaxanthin within oil bodies of Haematococcus pluvialis studied with polarization-dependent harmonic generation microscopy. PLoS One 2014;9(9) e107804. [42] Solovchenko A, Neverov K. Carotenogenic response in photosynthetic organisms: a colorful story. Photosynth Res 2017; https://doi.org/10.1007/s11120-017-0358-y. [43] Solovchenko AE. Recent breakthroughs in the biology of astaxanthin accumulation by microalgal cell. Photosynth Res 2015;125(3):437–49. https://doi.org/10.1007/s11120-015-0156-3. [44] Zhekisheva M, Boussiba S, Khozin-Goldberg I, Zarka A, Cohen Z. Accumulation of oleic acid in Haematococcus pluvialis (Chlorophyceae) under nitrogen starvation or high light is correlated with that of astaxanthin esters. J Phycol 2002;38(2):325–31. https://doi.org/10.1046/j.1529-8817.2002.01107.x.

The physiology of astaxanthin production by carotenogenic microalgae 33 [45] K€unzler K, Eichenberger W. Betaine lipids and zwitterionic phospholipids in plants and fungi, Phytochemistry 1997;46(5):883–92. Retrieved from: http://www.sciencedirect.com/science/article/pii/ S0031942297812745. [46] Zhekisheva M, Zarka A, Khozin-Goldberg I, Cohen Z, Boussiba S. Inhibition of astaxanthin synthesis under high irradiance does not abolish triacylglycerol accumulation in the green alga Haematococcus pluvialis (Chlorophyceae). J Phycol 2005;41(4):819–26. https://doi.org/10.1111/j.0022-3646.2005.05015.x. [47] Rabbani S, Beyer P, Lintig J, Hugueney P, Kleinig H. Induced b-carotene synthesis driven by triacylglycerol deposition in the unicellular alga Dunaliella bardawil. Plant Physiol 1998;116(4):1239–48. [48] Boussiba S. Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Physiol Plant 2000;108(2):111–7. https://doi.org/10.1034/j.1399-3054.2000.108002111.x. [49] Gao Z, Meng C, Chen Y, Ahmed F, Mangott A, Schenk P, Li Y. Comparison of astaxanthin accumulation and biosynthesis gene expression of three Haematococcus pluvialis strains upon salinity stress. J Appl Phycol 2014;1–8. https://doi.org/10.1007/s10811-014-0491-3. [50] Mulders KJ, Weesepoel Y, Bodenes P, Lamers PP, Vincken J-P, Martens DE, … Wijffels RH. Nitrogen-depleted Chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: a carotenoid metabolism study. J Appl Phycol 2015;27:125–40. [51] Li Y, Sommerfeld M, Chen F, Hu Q. Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae). J Appl Phycol 2010;22(3):253–63. [52] Li Y, Han D, Sommerfeld M, Hu Q. Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp.(Chlorophyceae) under nitrogen-limited conditions. Bioresour Technol 2011;102(1):123–9. [53] Kobayashi M. In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis. Appl Microbiol Biotechnol 2000;54(4):550–5. [54] Kobayashi M, Kakizono T, Nishio N, Nagai S, Kurimura Y, Tsuji Y. Antioxidant role of astaxanthin in the green alga Haematococcus pluvialis. Appl Microbiol Biotechnol 1997;48(3):351–6. [55] Foyer CH, Ruban AV, Noctor G. Viewing oxidative stress through the lens of oxidative signalling rather than damage. Biochem J 2017;474(6):877–83. https://doi.org/10.1042/BCJ20160814. [56] Noctor G, Reichheld JP, Foyer CH. ROS-related redox regulation and signaling in plants. Semin Cell Dev Biol 2017; https://doi.org/10.1016/j.semcdb.2017.07.013. [57] Huner N, Dahal K, Hollis L, Bode R, Rosso D, Krol M, Ivanov AG. Chloroplast redox imbalance governs phenotypic plasticity: the “grand design of photosynthesis” revisited. Front Plant Sci 2012;3: https://doi.org/ 10.3389/fpls.2012.00255. [58] Jaspers P, Kangasj€arvi J. Reactive oxygen species in abiotic stress signaling. Physiol Plant 2010;138 (4):405–13. [59] Kobayashi M, Kakizono T, Nagai S. Enhanced carotenoid biosynthesis by oxidative stress in acetate-induced cyst cells of a green unicellular alga, Haematococcus pluvialis. Appl Environ Microbiol 1993;59(3):867–73. [60] Ip P, Chen F. Employment of reactive oxygen species to enhance astaxanthin formation in Chlorella zofingiensis in heterotrophic culture. Process Biochem 2005;40(11):3491–6. [61] Fan L, Vonshak A, Zarka A, Boussiba S. Does astaxanthin protect Haematococcus against light damage? Z Naturforsch C: Biosci 1998;53(1–2):93. [62] Yong YYR, Lee YK. Do carotenoids play a photoprotective role in the cytoplasm of Haematococcus lacustris (Chlorophyta)? Phycologia 1991;30(3):257–61. https://doi.org/10.2216/i0031-8884-30-3-257.1. [63] Steinbrenner J, Linden H. Regulation of two carotenoid biosynthesis genes coding for phytoene synthase and carotenoid hydroxylase during stress-induced astaxanthin formation in the green alga Haematococcus pluvialis. Plant Physiol 2001;125(2):810–7. [64] Steinbrenner J, Linden H. Light induction of carotenoid biosynthesis genes in the green alga Haematococcus pluvialis: regulation by photosynthetic redox control. Plant Mol Biol 2003;52(2):343–56. [65] Gao Z, Meng C, Gao H, Li Y, Zhang X, Xu D, Ye N. Carotenoid genes transcriptional regulation for astaxanthin accumulation in fresh water unicellular alga Haematococcus pluvialis by gibberellin A3 (GA3). Indian J Biochem Biophys 2013;50(6):548–53.

34

Chapter 2

[66] Gao Z, Meng C, Gao H, Zhang X, Xu D, Su Y, Ye N. Analysis of mRNA expression profiles of carotenogenesis and astaxanthin production of Haematococcus pluvialis under exogenous 2,4-epibrassinolide (EBR). Biol Res 2013;46(2):201–6. [67] Ben-Amotz A, Shaish A, Avron M. Mode of action of the massively accumulated β-carotene of Dunaliella bardawil in protecting the alga against damage by excess irradiation. Plant Physiol 1989;86:1286–91. [68] Gu W, Li H, Zhao P, Yu R, Pan G, Gao S, Wang G. Quantitative proteomic analysis of thylakoid from two microalgae (Haematococcus pluvialis and Dunaliella salina) reveals two different high light-responsive strategies. Sci Rep 2014;4: https://doi.org/10.1038/srep06661. [69] Gu W, Xie X, Gao S, Zhou W, Pan G, Wang G. Comparison of different cells of Haematococcus pluvialis reveals an extensive acclimation mechanism during its aging process: from a perspective of photosynthesis. PLoS One 2013;8(7)e67028. [70] Hagen C, Braune W, Birckner E, Nuske J. Functional aspects of secondary carotenoids in Haematococcus lacustris (Girod) Rostafinski (Volvocales). I. The accumulation period as an active metabolic process. New Phytol 1993;125(3):625–33. https://doi.org/10.1111/j.1469-8137.1993.tb03912.x. [71] Hagen C, Braune W, Bj€orn L. Functional aspects of secondary carotenoids in Haematococcus lacustris (Volvocales). III. Action as a sunshade. J Phycol 1994;30(2):241–8. [72] Hagen C, Braune W, Greulich F. Functional aspects of secondary carotenoids in Haematococcus lacustris [Girod] Rostafinski (Volvocales). IV: protection from photodynamic damage. J Photochem Photobiol B: Biol 1993;20(2–3):153–60. [73] Wang B, Zarka A, Trebst A, Boussiba S. Astaxanthin accumulation in Haematococcus pluvialis (Chlorophyceae) as an active photoprotective process under high irradiance. J Phycol 2003;39(6):1116–24. https://doi.org/10.1111/j.0022-3646.2003.03-043.x. [74] Erickson E, Wakao S, Niyogi KK. Light stress and photoprotection in Chlamydomonas reinhardtii. Plant J 2015;82(3):449–65. https://doi.org/10.1111/tpj.12825. [75] Ort D. When there is too much light. Plant Physiol 2001;125(1):29–32. [76] Kar M, Streb P, Hertwig B, Feierabend J. Sensitivity to photodamage increases during senescence in excised leaves (of Secale cereale). J Plant Physiol 1993;141:538–44. [77] Han D, Wang J, Sommerfeld M, Hu Q. Susceptibility and protective mechanisms of motile and non motile cells of Haematococcus pluvialis (Chlorophyceae) to photooxidative stress. J Phycol 2012;48(3):693–705. [78] Solovchenko A, Aflalo C, Lukyanov A, Boussiba S. Nondestructive monitoring of carotenogenesis in Haematococcus pluvialis via whole-cell optical density spectra. Appl Microbiol Biotechnol 2013;97 (10):4533–41. https://doi.org/10.1007/s00253-012-4677-9. [79] Peled E, Pick U, Zarka A, Shimoni E, Leu S, Boussiba S. Light-induced oil globule migration in Haematococcus pluvialis (Chlorophyceae). J Phycol 2012;48(5):1209–19. https://doi.org/10.1111/j.15298817.2012.01210.x. [80] Schmitt F-J, Renger G, Friedrich T, Kreslavski VD, Zharmukhamedov SK, Los DA, … Allakhverdiev SI. Reactive oxygen species: re-evaluation of generation, monitoring and role in stress-signaling in phototrophic organisms. Biochim Biophys Acta 2014;1837(6):835–48. https://doi.org/10.1016/j.bbabio.2014.02.005. [81] Ensminger I, Busch F, Huner N. Photostasis and cold acclimation: sensing low temperature through photosynthesis. Physiol Plant 2006;126(1):28–44. € [82] Oquist G, Huner NP. Photosynthesis of overwintering evergreen plants. Annu Rev Plant Biol 2003;54 (1):329–55. [83] Solovchenko A. Physiological role of neutral lipid accumulation in eukaryotic microalgae under stresses. Russ J Plant Physiol 2012;59(2):167–76. [84] Mendoza H, Martel A, Jimenez del Rio M, Garcia Reina G. Oleic acid is the main fatty acid related with carotenogenesis in Dunaliella salina. J Appl Phycol 1999;11(1):15–9. [85] Wang B, Zhang Z, Hu Q, Sommerfeld M, Lu Y, Han D. Cellular capacities for high-light acclimation and changing lipid profiles across life cycle stages of the green alga Haematococcus pluvialis. PLoS One 2014;9(9) e106679.

The physiology of astaxanthin production by carotenogenic microalgae 35 [86] Mulders KJM, Lamers PP, Martens DE, Wijffels RH. Phototrophic pigment production with microalgae: biological constraints and opportunities. J Phycol 2014;50(2):229–42. https://doi.org/10.1111/jpy.12173. [87] Mulders KJ, Janssen JH, Martens DE, Wijffels RH, Lamers PP. Effect of biomass concentration on secondary carotenoids and triacylglycerol (TAG) accumulation in nitrogen-depleted Chlorella zofingiensis. Algal Res 2014;6:8–16. [88] Liu J, Sun Z, Gerken H, Liu Z, Jiang Y, Chen F. Chlorella zofingiensis as an alternative microalgal producer of astaxanthin: biology and industrial potential. Mar Drugs 2014;12(6):3487–515. [89] Del Campo J, Garcı´a-Gonza´lez M, Guerrero M. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol 2007;74(6):1163–74. https://doi.org/10.1007/ s00253-007-0844-9. [90] Leu S, Boussiba S. Advances in the production of high-value products by microalgae. Ind Biotechnol 2014;10 (3):169–83. [91] Farr
e G, Twyman RM, Christou P, Capell T, Zhu C. Knowledge-driven approaches for engineering complex metabolic pathways in plants. Curr Opin Biotechnol 2015;32(0):54–60. https://doi.org/10.1016/j. copbio.2014.11.004. [92] Sharon-Gojman R, Maimon E, Leu S, Zarka A, Boussiba S. Advanced methods for genetic engineering of Haematococcus pluvialis (Chlorophyceae, Volvocales). Algal Res 2015;10:8–15.

This page intentionally left blank

CHAPTER 3

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis Jun-Hui Chena, Dong Weia, Ambati Ranga Raob, and Gokare A. Ravishankarc a

School of Food Science and Engineering, South China University of Technology, Guangzhou, PR China Centre of Excellence, Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research (Deemed to be University), Vadlamudi, Guntur, Andhra Pradesh, India cC.D. Sagar Centre for Life Sciences, Dayananda Sagar College of Engineering, Dayananda Sagar Institutions, Bangalore, Karnataka, India

b

Abbreviations ABA ACC Acetyl-CoA Acyl-ACP ADP ATP BKT CAT CO2 DCMU DGAT DSP FA Fe3O4-PEI GA GGPP GSH-Px H2O IPP LCYB LDs LED mRNA NAA NADPH O2 PBR PDS PEP

abscisic acid cytokinin, 1-aminocyclopropane-1-carboxylic acid acetyl coenzyme A acyl carrier protein adenosine diphosphate adenosine triphosphate β-carotene oxygenase catalase carbon dioxide 3-(3,4-dichlorophenyl)-1,1-dimethylurea diacylglycerol acyltransferase downstream processing fatty acid superparamagnetic iron oxide-polyethyleneimine gibberellic acid geranylgeranyl diphosphate glutathione peroxidase hydrogen peroxide isopentenyl diphosphate lycopene β-cyclase lipid droplets light-emitting diode messenger ribonucleic acid 1-naphthylacetic acid nicotinamide adenine dinucleotide phosphate oxygen photobioreactor phytoene desaturase phosphoenolpyruvate

Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00008-8 Copyright # 2021 Elsevier Inc. All rights reserved.

37

38

Chapter 3

PQ PQH2 PSBR PSY PUFAs RFP ROS SOD TAG TALEs TCA TCA TFA UV ZFNs

plastoquinone plastoquinol porous substrate photobioreactor phytoene synthase polyunsaturated fatty acids energy-free rotating floating photobioreactor reactive oxygen species superoxide dismutase triglyceride transcription activator-like effector nucleases tricarboxylic acid tricarboxylic acid cycle total fatty acid ultraviolet zinc-finger nucleases

1 Introduction Astaxanthin is a valuable red keto-carotenoid with the highest antioxidative activity and most widespread applications in various areas in comparison with other natural carotenoids such as beta-carotene, lutein, lycopene, and zeaxanthin [1, 2]. Astaxanthin has been approved to have various medical functions, especially in free radical scavenging, lipid peroxidation inhibition, and prevention of various cardiovascular and neurological diseases [3]. Currently, astaxanthin is widely accepted by consumers who are much interested in uptaking biological active compounds for the therapy and prevention of chronic diseases such as diabetes and its complications [2]. Until date, astaxanthin has been developed not only as high-quality functional food and dietary supplements but also as cosmetics and pharmaceuticals due to their effective medical functions [4]. Currently, large-scale commercial production of natural astaxanthin by Haematococcus pluvialis still faces the challenges and limitations such as high production cost, low and unstable productivity, and easy contamination. Chromochloris zofingiensis has the capability of growing fast under favorable conditions and accumulating astaxanthin and lipids to a large extent under adverse conditions. Therefore C. zofingiensis is recognized as an alternative industrial producer for natural astaxanthin, though its cellular astaxanthin content is still comparatively low compared with that in the current producer H. pluvialis. In recent years, various studies have been conducted to evaluate the feasibility of C. zofingiensis for astaxanthin production, and different strategies in bioprocess and induction stress are applied to enhance astaxanthin accumulation in C. zofingiensis. These achievements have greatly promoted the feasibility of utilizing C. zofingiensis for astaxanthin production in the future. Therefore it is worth of reviewing recent advances in strategies for astaxanthin enhancement in C. zofingiensis to guide the further research and conduct in-depth studies.

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis 39

2 Astaxanthin production by Chromochloris zofingiensis 2.1 Characteristics of Chromochloris zofingiensis Chromochloris zofingiensis is a unique green microalga belonging to the genus Chromochloris and has many inherited characteristics similar to that of its close relatives Chlorella and Chlamydomonas despite the evolutionary distance [5]. Generally, C. zofingiensis is in the form of unicellular, haploid, and coccoid cells with a diameter of 2–15μm. The proliferation of this microalga is mediation by asexually reproducing autospores with the same size from parental cells, which involving cell growth, multiple fission, and cell division at uncertain division timing. The outstanding superiority of C. zofingiensis over other microalgae and plants is the special capability of biosynthesizing secondary carotenoids and lipids (especially triacylglycerols) to resist adverse conditions [5]. Intriguingly, under specific conditions, these valuable carotenoids in C. zofingiensis including canthaxanthin, ketolutein, astaxanthin, and its esters are simultaneously induced to biosynthesize in concert with lipid accumulation, which act as the roles of antioxidant filters and hydrophobic layers to reduce photodamage and photooxidation [6]. The accumulated level of lipids in C. zofingiensis is relatively higher than that of other previously estimated microalgae such as Chlorella species. Additionally, the cell wall of Chlorella species is two-layered and mainly composed of sporopollenin, mannose, and chitin-like polysaccharide, which is comparatively easy for cell wall disruption and intracellular target-bioproduct extraction when compared with the thick multilayer cell wall (1.8–2.2 μm) of mature red cysts of H. pluvialis [7]. These features warrant the utilization of C. zofingiensis replacing current astaxanthin producers for economical astaxanthin production. In recent years, with the rapid development of systems biology and genomics, the nuclear and organelle genomes of C. zofingiensis have already been sequenced and then investigated to deepen the understanding of biological features and cellular metabolic mechanisms of C. zofingiensis with the aim to provide guidance for future research [5]. It was found that C. zofingiensis has a compact 58-Mbp nuclear genome with more than 15,000 genes uniformly distributed over 19 chromosomes, and meanwhile, several crucial genes such as beta-ketolase, cytochrome P450 enzymes, and acyltransferase were identified to be indispensable for astaxanthin biosynthesis [5]. Referring to another astaxanthin-producing microalga H. pluvialis with a 669-Mb genome containing 18,000 genes, C. zofingiensis has a smaller genome size, fewer protein-coding genes, and lower content of repeat sequences but a higher fraction (ca. 39%) of protein sequence [5, 8]. Besides the mitochondrial and chloroplast genomes of C. zofingiensis were also analyzed, and it was found that RNA expression of genes from these organelle genomes accounted for a relatively high percentage of total mRNA, representing approximate 31% and 7%, respectively. The dramatical expression of organellar genes during the algal culture indicated the crucial roles and importance of organelles for metabolic and regulatory mechanisms of C. zofingiensis, particularly in the case of chloroplast that is the largest organelle (ca. 40%–60% of cell volume) with its own genome. There have

40

Chapter 3

already been numerous studies focusing on genetic modification of microalgal chloroplast for producing valuable compounds such as carotenoids and terpenoids [9]. Especially, it was reported previously that, through chloroplast genetic engineering, the overexpression of the H. pluvialis pds gene was realized and resulted in overaccumulation of cellular astaxanthin [10], which provide us new clues and may be worthy of our special attention. Overall the nuclear and organellar genomic information would facilitate the elucidation of metabolic and regulatory mechanisms limiting high-yield astaxanthin accumulation in C. zofingiensis and then provide valuable insights into genetic and metabolic manipulation strategies for future strain improvement.

2.2 Culture conditions of Chromochloris zofingiensis for astaxanthin production Commercial microalgae-based astaxanthin production process is generally contained culturing, harvesting, and downstream processing. Among these procedures, microalgae cultivation constitutes the core aspect of the production process since it accounts for the majority of total production costs. To date, the low-cost and high-yield astaxanthin production by microalgae is still facing challenges forward. High costs of microalgae cultivation are mainly due to the algae-specific characteristics, such as relatively slow growth rate, discrepancies of cell proliferation and astaxanthin accumulation, long cultivation period, easy contamination, large consumption of freshwater, and high capital costs of cultivation systems. In the following subsections a concise overview of culture conditions and factors influencing astaxanthin accumulation, as well as different approaches and strategies improving astaxanthin production levels by C. zofingiensis, was presented and summarized to evaluate the potential feasibility of this microalga for astaxanthin production (Table 3.1). 2.2.1 Trophic mode C. zofingiensis has the flexible metabolism to utilize various carbon sources and grow under three trophic modes (i.e., photoautotrophy, heterotrophy, and mixotrophy) and exhibits distinct physiological performance in the cell growth and cellular metabolite accumulation. Generally, C. zofingiensis could use CO2 and light to grow photoautotrophically with a slow rate but grow fast heterotrophically with organic carbon sources in the dark [25]. However, compared with photoautotrophic and heterotrophic cultivations, mixotrophic cultivation using glucose is regarded as the best culture strategy for C. zofingiensis cultivation since glucose could coordinate cellular metabolisms of photosynthesis and aerobic respiration in favor of cell growth and astaxanthin accumulation in mixotrophic C. zofingiensis [25]. In addition, Roth et al. [26,27] observed the glucose-dependent chloroplast morphogenesis associated with cellular metabolic shifts during trophic transitions of C. zofingiensis and clarified that the regulation molecular mechanisms underlying the eukaryotic glucose responses were mediated by hexokinase, which is an important regulator of photosynthesis and cellular metabolism in cells. These studies provided useful information about photosynthesis and metabolism of

Table 3.1: Highest production level of astaxanthin and lipids by C. zofingiensis in the published literature. Biomass

Strains

Culture mode

ATCC30412 ATCC30412 ATCC30412 ATCC30412

P, batch M, batch M, batch P, batch

ATCC30412 ATCC30412 ATCC30412 ATCC30412 UTEX32 UTEX B32 UTEX B32 ATCC30412 a

ATCC30412 ATCC30412 a

Culture system

50-mL flasks MCS 50-mL flasks Tubular reactor P, batch Tubular PBR M, batch 100-mL flasks H, two-step RFP M, batch MCS P, semicontinuous Glass column P, batch 250-mL flasks P, batch Airlift-loop PBR M, batch 250-mL flasks H, fed-batch Fermentor H, 1L bioreactor semicontinuous

Astaxanthin

Carbon source

Conc. (g L21)

Prod. (g L21 day21)

Cont. (mg g21)

Yield (mg L21)

Prod. (mg L21 day21)

References

Glucose Glucose Glucose CO2

2.2 8.3 9.5 1.2

1.10 0.69 0.68 0.12

0.4 10.8 1.9 3.9

0.9 89.9 18.0 4.5

0.5 7.5 1.3 0.6

[11] [12] [13] [14]

CO2 Glucose

1.7–1.9 4.6

0.43–0.48 0.38

4.8 1.2

9.1 5.5

2.3 0.5

[15] [16]

Glucose Glucose CO2 CO2

98.4 6.0 N/A 4.6

7.03 0.50 1.04 0.33

0.7 6.5 3.2 1.4

73.3 38.9 N/A 6.5

5.3 3.2 3.3 0.46

[17] [18] [19] [20]

CO2

8.2

0.58

2.4

N/A

1.4

[21]

Glucose

11.9

0.59

2.2

25.8

1.3

[22]

Glucose CO2

45.6 12.9

4.46 1.55

1.2 1.1

56.1 13.6

5.6 1.7

[23] [24]

Mutants. P, photoautotrophic culture; M, mixotrophic culture; H, heterotrophic culture; MCS, microplate-based culture system; RFP, an energy-free rotating floating photobioreactor; Cult, cultivation; Conc, concentration; Cont, content; Prod, productivity; , estimated value; N/A, data not available.

42

Chapter 3

C. zofingiensis and facilitated the regulation and manipulation of its cellular metabolism toward valuable carotenoids and lipids. Currently, various types of cultivation systems, for example, shaking flask, fermenter, MCS, column PBR, airlift PBR, and rotating floating photobioreactor (RFP), are established in practice for C. zofingiensis cultivation [17–19,28,29]. Maximum biomass yield (98.4 g/L) and the astaxanthin yield (73.3 mg/L) were reported in the cultivation of algae using RFP integrated with high light irradiation and nitrogen limitation [17]. The microplate-based cultivation system was most suitable for astaxanthin accumulation due to its lowest light attenuation among these conventional culture apparatuses [18]. Although considerable progresses have been made in the cultivation of C. zofingiensis in recent years, the majority of them are still conducted at the laboratory scale, which needs more efforts to develop suitable systems for mass cultivation of C. zofingiensis. 2.2.2 Light quality and intensity The mixotrophic culture mode facilitates the efficient astaxanthin production by C. zofingiensis, in which light is the most essential culture condition and also the important requisite for inducing astaxanthin biosynthesis. As to each light with specific spectrum, there might be a saturation value of light intensity in regard to astaxanthin accumulation, which varies in accordance with cultivation systems and biomass densities [30]. White light is widely used for microalgae cultivation, and it was reported that C. zofingiensis is able to grow fast even under high light irradiation as high as 1000 μmol m 2 s 1 [28]. Comparatively, blue LED light was more favorable for astaxanthin accumulation and able to regulate the accumulation levels of astaxanthin and canthaxanthin since it could upregulate the transcript expression of certain carotenoid genes in spite of its high energy and deep penetration [31]. However, when light intensity exceeds the saturation value of 150 μmol m 2 s 1, astaxanthin accumulation in C. zofingiensis cells could be inhibited due to the severe photooxidative damage [18]. Besides, light was reported to be able to directly regulate the metabolism of starch and lipids, which provided new insights into the manipulation of light supply for C. zofingiensis culture [32]. 2.2.3 Culture medium nutrients As three most important nutrients, carbon, nitrogen, and phosphorus exert distinct influences on cell growth and astaxanthin production of C. zofingiensis. As to the essential nutrient carbon required for algal cultivation, it was proved that C. zofingiensis could utilize diverse carbon sources such as carbon dioxide, acetate, sucrose, glucose, fructose, and mannose, among which glucose is comparatively most favorable for cell growth and astaxanthin accumulation by C. zofingiensis [33]. Besides, nitrate is the commonly used exogenous nitrogen source for C. zofingiensis cultivation instead of other forms of ammonia and urea because of its convenience for use without causing the medium acidification and cell growth inhibition [23]. Numerous studies demonstrated that different nitrogen sources exerted significant effects on

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis 43 the cell growth of C. zofingiensis and found that nitrate limitation is capable of inducing algal cells to biosynthesize astaxanthin and/or lipids to a large extent. However, recent studies reported that not only nitrogen sources in the culture media (e.g., nitrate, ammonia, and urea) correlate closely with algal cell growth but also the endogenous nitrogen (i.e., chlorophylls and proteins) plays vital roles in inducing C. zofingiensis cells to accumulate astaxanthin and lipids [23,34]. Therefore particular efforts should be paid to the catabolism of endogenous nitrogen and its interactions with other crucial metabolisms, which may offer new strategies for enhanced production of astaxanthin and lipids by C. zofingiensis. Furthermore the metabolism of C. zofingiensis could also be shifted and regulated by the phosphorus-limiting condition. Our previous study demonstrated that the uptake of phosphate as well as algal cell growth was inhibited under high light conditions, indicating the close correlation between phosphorus and C. zofingiensis biomass [18]. It might be worth mentioning that the nutrient cost during microalgae cultivation is the largest hurdle for economical astaxanthin production. A potentially practical and feasible measure may be the substitution of nutrients with cheaper sources such as nitrogen- or phosphorus-rich wastewater for the economics of C. zofingiensis cultivation for biofuels and bioproducts [35]. 2.2.4 Temperature, pH, and dissolved oxygen The suitable temperature for C. zofingiensis cultivation is mainly at the range of 20–28°C, above which microalgal cell growth and astaxanthin biosynthesis tend to be inhibited seriously since the activities of various metabolic enzymes are temperature-dependent. Similarly, pH also is related to the enzymatic activity and exerts significant influences on microalgal cells and astaxanthin accumulation within the range of 5.5–8.5 [29]. Dissolved oxygen is another crucial operational factor influencing astaxanthin production by C. zofingiensis especially at the large scale. Imaizumi et al. [28] found that active photosynthesis caused by high light irradiation could increase the level of dissolved oxygen but considerably decrease the biomass production rate of C. zofingiensis cells [28]. Similarly, during H. pluvialis cultivation, dissolved oxygen at the relatively high concentration could not facilitate astaxanthin production but inhibit cell growth and cause oxidative damage [36]. 2.2.5 Abiotic stress conditions Besides the aforementioned environmental factors influencing astaxanthin production, chemical-induced abiotic stress conditions could also trigger the significant accumulation of astaxanthin in microalgal cells. Currently, tremendous studies have demonstrated that the additions of exogenous chemical additives could significantly improve astaxanthin production by C. zofingiensis (Fig. 3.1). These stimulating chemicals belong to four categories based on the effect mechanisms, namely, phytohormones, precursors, metabolic regulators (e.g., glyphosate, cerulenin, and DCMU) and oxidative stress inducers (e.g., high light, salt stress, and nutrient stress) [37]. In terms of underlying regulatory mechanisms of these chemicals, phytohormones and ROS are two typical well-known signaling molecules that directly interact with each other

Fig. 3.1 Hypothetical mechanisms underlying the effects of different chemicals and phytohormones on astaxanthin and lipid accumulation in C. zofingiensis. Red solid arrows represent stimulatory effects on downstream targets, and green solid arrows represent inhibition of targeted pathways.

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis 45 in signal transduction cascades and subsequently involve in the regulation of astaxanthin biosynthesis for resisting exogenous stresses. Until now, numerous phytohormones have been demonstrated to be stimulatory for astaxanthin biosynthesis via activating different signal transduction cascades, among which GA and ABA work antagonistically in signal transduction pathways but both enable to trigger astaxanthin biosynthesis through GA-ABA cross talk at signaling and transport levels [12]. Oxidative stress inducers, as well as certain metabolic regulators, stimulate astaxanthin biosynthesis mainly via the generation of the typical signaling molecule ROS in chloroplasts and the cytosol, which subsequently activates specific signal transduction cascades to upregulate the transcriptional expression of crucial astaxanthin biosynthetic genes. Among these chemical additives, phytohormones are the most effective in triggering astaxanthin accumulation and are able to improve astaxanthin accumulation level by several folds especially when in combination with high light and nitrogen deprivation. Certain chemicals (ACC and NAA) stimulating astaxanthin in other astaxanthin-producing organisms fail to enhance astaxanthin production by C. zofingiensis [38]. It was assumed that the discrepancies might be caused by the special physiological characteristics of C. zofingiensis and the species-specific stimulating effects of exogenous phytohormones [12]. Other chemical additives with low costs were also reported to be able to enhance astaxanthin production, though their stimulating effects were a little lower in contrast to phytohormones. However, for the economics of the large-scale astaxanthin production, not only the enhancement of the astaxanthin accumulation level but also the extra costs of inducing chemicals should be taken into consideration in which the cost analysis should be conducted to assess their industrial viability. Besides the cultivation of C. zofingiensis, harvesting and extraction are also important phases in astaxanthin production that account for appropriate 20%–30% of the overall astaxanthin production costs. Centrifugation is the widely used method for microalgal biomass harvesting in laboratory- and large-scale production. While at the pilot scale, additional preconcentration procedures before biomass harvesting are required to considerably reduce energy costs. The obtained wet biomass is further spray-dried for preservation or directly used for the extraction of cellular valuable components [39]. Conventional organic solvents such as methanol, dimethyl sulfoxide, and hexane widely used in lipid extraction are not suitable for astaxanthin extraction since the existence of poisonous solvent residues is not permitted in the production of foods and pharmaceuticals for human consumption. Thus supercritical fluids such as CO2 without any safety risks are regarded as the most suitable and green solvent for astaxanthin extraction process. To further enhance the efficiency, several cosolvents such as vegetable oils and animal fats are considered to be added to facilitate the extraction to a large extent [39]. To date, no economical and efficient methods are developed for harvesting and extraction of astaxanthin from C. zofingiensis, indicating more research efforts are required for the economics of commercial astaxanthin production.

46

Chapter 3

3 Advantages and limitations of Chromochloris zofingiensis for astaxanthin production 3.1 Advantages of Chromochloris zofingiensis-based astaxanthin production Recently, C. zofingiensis belonging to the genus of Chlorella has attracted worldwide interests due to its superior features favoring economical astaxanthin production. The first unique feature distinguishing C. zofingiensis from other microalgal species is that it could be cultivated under different environmental conditions and reach maximum cell density of up to 98.4 g/L under favorable conditions [17]. The high cell density helps to increase astaxanthin productivity and lower the overall production cost. Additionally, astaxanthin content is also a most important factor for assessing the feasibility of one organism species for astaxanthin production. Although the current astaxanthin production level by C. zofingiensis is ordinarily below 1% of cell dry weight, unable to fulfill the requirements of commercial production, some algal strains have the potential to significantly biosynthesize more astaxanthin above 1% under certain stress conditions or other external interferences [12]. Combining the fast-growing property of C. zofingiensis and its high potential of largely accumulating astaxanthin, C. zofingiensis is warranted to be developed as a promising astaxanthin producer with comparable performance to that of H. pluvialis. Also, C. zofingiensis has superior characteristics of accumulating multiple bioproducts (e.g., lipids, amino acids, and carbohydrates) to a large extent apart from astaxanthin production (Table 3.2). Through detailed analyses of pigments in C. zofingiensis cells, it was found that several other carotenoids, such as, lutein, zeaxanthin, beta-carotene, canthaxanthin, and adonixanthin, were also produced. Huang et al. [40] succeeded in genetically modifying C. zofingiensis by chemical mutagenesis and enabled the mutant to accumulate large amounts of essential zeaxanthin (7.0 mg/g), lutein (13.8 mg/g), and beta-carotene (7.2 mg/g) simultaneously [40]. Lipids and fatty acids commonly accounting for 20%–60% of algal biomass are another important types of potential bioproducts produced from C. zofingiensis, which could be developed as renewable feedstocks for biofuel production. Carbohydrates and proteins represent approximately 10%–30% of total C. zofingiensis biomass, which require our special attention and more efforts from the biorefinery perspective to make full use of cellular components for extra profits. Besides the special composition, C. zofingiensis cell wall consisting of sporopollenin, mannose and other polysaccharides like chitin, is at the maximum thickness of 25 nm and comparatively easy to be disrupted for the efficient recovery of astaxanthin and other desired compounds [7]. Thus the cost of astaxanthin recovery from C. zofingiensis biomass could be significantly lowered and then reduce the overall astaxanthin production cost, which facilitates the promotion of astaxanthin application in various industries and fields.

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis 47 Table 3.2: Production of other bioproducts besides astaxanthin in C. zofingiensis reported in recent published literature. Content (% dry weight)

References

M, batch culture M, batch culture P, batch culture P/H/M, batch culture P, semicontinuous culture H, batch culture P, batch culture P, batch culture

29.1–64.5 8.0–23.0 6.0–27.2 (TAG) 10.0–18.0 (TFA)

[12] [13] [14] [25]

25.0–58.0

[19]

14.9–30.5 (TFA) 6.5–24.5 (TFA) 19.5–33.5 (TAG)

[41] [42] [21]

P/H/M, batch culture P, batch culture P, batch culture

2.9–37.0

[25]

0.1–0.4 11/20

[43] [34]

M, batch culture P, batch culture P, batch culture P, batch culture P, batch culture

10–50 31.7–47.7 4.5–43.4 30–45 13–33

[32] [44] [42] [45] [34]

P, batch culture P, batch culture M, batch culture

16.6–33.2 10–25 9.1–18.2

[44] [45] [46]

Types

Cultivation systems

Conditions

Lipid

MCS 50-mL flasks Tubular PBR 500-mL flasks 100mL column PBR

Starch/ carbohydrate

Protein

Exopolysaccharides

500-mL flasks Airlift PBR Flat-panel and airlift-loop PBR 500-mL flasks 1 L reagent bottle 1.75-L flat-panel and airlift-loop PBR 250/500-mL flasks Airlift PBR Airlift PBR 240-L flat plate PBR 1.75-L flat-panel and airlift-loop PBR Airlift PBR 240-L flat plate PBR 1-L cultivation system

P, photoautotrophic culture; M, mixotrophic culture; H, heterotrophic culture; MCS, microplate-based culture system; PBR, photobioreactor.

Last but most importantly, C. zofingiensis has the superior capability of being cultivated robustly in various modern cultivation systems utilizing various organic carbon sources for rapid cell growth and substantially biosynthesizing various high-value components within cells when turned to adverse conditions [29]. Current commercial mass culture of H. pluvialis is generally carried out in horizontal tubular PBRs for cell growth and then in raceway pond complex for astaxanthin accumulation [36]. Comparatively, C. zofingiensis could be successfully cultivated in current industrial fermenter systems for large-scale bioproduct production without huge equipment upgrade. The enclosed culture systems are more able to keep process stability and product quality without the influence of weather and climate. Moreover the cultivation of C. zofingiensis could be carried out in various types of lands with less consumption of freshwater and would not compete agricultural lands and nutrients with crops for food production.

48

Chapter 3

3.2 Limitations and challenges for Chromochloris zofingiensis application Although considerable progresses have been made for astaxanthin production by C. zofingiensis at the laboratory scale recently, the current astaxanthin production level is comparatively far low to compete with other producing sources and then could not meet commercial demands with economical costs and benefits for future commercial mass production. Among these limitations the major bottleneck of C. zofingiensis-based microalgae cultivation is the low level of astaxanthin accumulation in algal cells. Given the level of below 1% astaxanthin in C. zofingiensis biomass, it is obviously unreasonable to utilize C. zofingiensis as astaxanthin producer since the low recovery efficiency will be a hurdle toward commercial astaxanthin production. Furthermore, more efforts are still required for the development of ideal economical culture systems for microalgae, and limited information is acquired about the corresponding strategies toward further improving the current economics of astaxanthin production. Various types of PBRs at laboratory scale have been developed for C. zofingiensis cultivation but fail to fulfill the requirements of commercial production at the industrial scale. The current production cost of 1kg of C. zofingiensis biomass in PBRs is several times higher than that of astaxanthin-rich H. pluvialis (approximately 14 US dollars per kilogram) [36], which is obviously uncompetitive in cost control. Henceforth, considering these limitations toward the economical astaxanthin production by C. zofingiensis at industrial large scale, more efforts need to be addressed in microalgal strain improvement, process development in culture systems, and multibioproduct production as discussed in the following section.

4 Engineering strategies for enhanced astaxanthin production 4.1 Genetic and metabolic engineering for strain improvement The utilization of C. zofingiensis for astaxanthin production will be of great research value and bright application prospects, while, for commercial production, the microalgal strain has to be significantly improved for enhanced astaxanthin production with competitive cost [5]. Although conventional random mutagenesis techniques such as chemical mutagens, UV-radiation, γ-radiation, and plasma mutagenesis are reported to succeed in developing desired strains with high astaxanthin content [47], these approaches are time-consuming and low in efficiency since the mutagenesis is not rationally controlled and theoretically predicted in most occasions. To overcome these bottlenecks and limitations of these conventional approaches, a thorough exploration and understanding of global metabolic regulation of astaxanthin biosynthesis and responses to exogenous environmental factors is required. Omics-based systems biology is an emerging interdisciplinary research field that deepens our knowledge of the whole-genome and cellular metabolites of microbes and their complicated interactions at a system level [48]. Its significant value is to guide the genomic design of

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis 49 organism species with desirable performance and further be developed as cell factories for valuable bioproduct production at the industrial scale. Roth et al. first systematically investigated the chromosome-level nuclear and organellar genomes of C. zofingiensis and primarily identified 15,000 protein-coding genes uniformly distributing in 19 nuclear chromosomes particularly those involving in astaxanthin and lipid biosynthesis, which provided valuable genetic information for astaxanthin and lipid biosynthesis [5]. Huang et al. [49] investigated glucose response-related genetic expressions of C. zofingiensis by comparative transcriptome analysis and revealed that a variety of important genes participate in the biosynthesis of fatty acids, lipids, and carotenoids [49]. Additionally, Liu et al. [50] carried out the multiomics analysis to unravel multiple metabolic processes related to lipid biosynthesis and the underlying mechanisms by integrating transcriptome analysis and metabolomic profiling analysis [50]. On the other hand, Want et al. focused on proteomic analysis of lipid droplets (LDs) in C. zofingiensis to decipher the mechanisms of LD biogenesis and lipid synthesis under nitrogen limitation, with an aim to facilitate strain improvement for lipid production by genetic engineering approaches [51]. These latest studies provided new insights into genetic information of crucial rate-limiting steps or pathways of astaxanthin biosynthesis in C. zofingiensis cells and laid a solid foundation for the strain and process improvement of algal species as efficient and cost-effective cell factories for high-value bioproducts. Thanks to the dramatical development of systems biology leading to more rational genetic and metabolic engineering approaches for strain improvement of plants and microorganisms. Generally, to significantly enhance the productivity of astaxanthin by C. zofingiensis, two main strategies have been adopted. Firstly to overcome the critical bottlenecks for astaxanthin overproduction by optimizing rate-limiting steps of astaxanthin biosynthetic pathways and secondly, by reducing the feedback inhibition of overproduced astaxanthin. In order to optimize carotenogenic pathways, various genes coding for crucial metabolic enzymes such as LCYB, PDS, PSY, BKT, and DGAT have been studied [52–55]. Overexpression of these enzymes not only could significantly improve astaxanthin content but also enable these microorganisms (e.g., bacteria, algae, and plants) incapable of biosynthesizing astaxanthin to biosynthesize astaxanthin [56]. However, the astaxanthin biosynthesis involves a series of regulatory enzymes and multiple metabolic pathways, the simple manipulation of certain crucial biosynthetic enzyme may not significantly enhance the productivity of microalgal astaxanthin [57]. This indicated that the whole metabolic pathways and correspondingly multiple participating enzymes should be coordinated and manipulated for highly-efficient astaxanthin accumulation. Additionally, Galarza et al. [10] reported that genetically modified H. pluvialis cells by chloroplast genetic engineering was able to overaccumulate astaxanthin. Similar approach could be followed to engineer C. zofingiensis for valuable carotenoid production [10].

50

Chapter 3

Another metabolic engineering strategy for astaxanthin overproduction by C. zofingiensis would be mostly to avoid the inhibition feedback on the activities of specific biosynthetic enzymes and also form sinks for the overaccumulation of astaxanthin through coordinating transport mechanisms [57]. To achieve these goals, more efforts are required to thoroughly investigate the mechanisms of astaxanthin transportation and storage as well as the feedback inhibition in carotenogenesis of C. zofingiensis. As a crucial precursor of astaxanthin, beta-carotene is biosynthesized from phytoene in cellular chloroplasts and then converted to astaxanthin and astaxanthin esters in cytoplasmic lipid droplets of algal cells. This indicated that astaxanthin biosynthesis is highly dependent on the formation of lipid droplets since the latter serves as a metabolic sink for alleviating feedback inhibition of overproduced astaxanthin on carotenogenesis of the green alga H. pluvialis [58]. This provides useful informative clues for genetic manipulation of C. zofingiensis cells for astaxanthin overaccumulation. Additionally, upon the formation of astaxanthin sinks, more efforts are required to optimize the metabolic flux and realize the maximum conversion of precursor substances into astaxanthin. Until now, various approaches and strategies have been constructed and applied to fluxome analysis of microalgae such as isotope labeling analysis, nuclear magnetic resonance-based analysis, mass spectrometry-based analysis, in silico flux stimulation, and computational modeling [48]. With considerable progresses in systems biology and associated biotechnological approaches and strategies such as CRISPR-Cas systems, TALEs, and ZFN-based methods [59,60], desired novel strains of C. zofingiensis would be successfully developed and utilized for commercial astaxanthin production with the competitive advantages in cost and production efficiency. It is worth mentioning that these innovative engineering approaches seem to offer the energetic and economic possibility of high astaxanthin yields. More comprehensive efforts are required to address environmental safety and sustainability issues hindering the future utilization of C. zofingiensis for commercial astaxanthin production. Overall, tremendous advances and progresses have been made in unraveling the underlying global-scale regulation mechanisms of astaxanthin biosynthesis in C. zofingiensis and will surely accelerate the exploration and development of C. zofingiensis for the future commercial production of natural astaxanthin and biofuels.

4.2 Bioprocess engineering for enhanced astaxanthin production 4.2.1 Cultivation systems and strategies for microalgal astaxanthin production Currently the most suitable and favorable cultivation systems for mass culture of microalgae are PBRs coupled with associated bioprocess strategies (e.g., batch/fed-batch, continuous/ semicontinuous, perfusion, and multistage cultivation associated with different feeding schemes). There have been numerous types of PBRs developed to address these issues, including flat plate, tubular/bubble/airlift column, membrane, and hybrid PBRs with different capabilities of boosting microalgal biofuel and bioproduct production [61–64]. In the particular case of astaxanthin

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis 51 production, the conventional PBRs have their intrinsic demerits of low light and carbon utilization efficiencies and microalgal productivity. In recent years, novel types of efficient PBRs (e.g., vacuum airlift, flat plate gas lift, twin-layer biofilm, and suspended solid phase) have been developed to maximize microalgal production, particularly the twin-layer PSBR, which is significantly superior to conventional suspended cultivation for microalgal astaxanthin production in terms of saving energy, time, and raw materials [65–67]. As to the design and selection of cultivation systems, more efforts should be based on the specific features of microalgae and the targeted products. These design and improvement methodologies for cultivation systems enhanced the carotenogenesis performance of microalgal cultivation and therefore provide insights into the measures for C. zofingiensis cultivation for enhanced astaxanthin production. Thus more efforts are required to overcome the bottlenecks of conventional PBRs, for example, severe light attenuation in cultivation systems and the failure to supply required light spectra and intensity [30]. Although the short light path designs of PBRs integrating with novel artificial light sources (i.e., LEDs and fluorescent or phosphorescent material light,) and light delivery systems were demonstrated to facilitate lab-scale algal production, efficient and cost-effective PBRs at pilot-plant scale have not been developed [63,68–70]. Therefore further development of these novel-efficient PBRs for immobilized cultivation of C. zofingiensis may be necessarily required for the low-cost and energy-efficient astaxanthin production. 4.2.2 Bioprocess engineering principles of Chromochloris zofingiensis cultivation To fulfill these specific needs of C. zofingiensis for targeted compound production, cultivation systems and operation strategies should be accordingly selected and is dependent on certain principles. Considering the noncoordinated production of biomass and astaxanthin, C. zofingiensis cultivation should be divided into two stages for maximizing the separate production of biomass and astaxanthin. The heterotrophic-phototrophic two-stage cultivation using PBRs coupling with conventional fermenters is regarded as the most suitable culture strategy for C. zofingiensis cultivation [29]. Similarly a two-step cultivation integrated with the fermenter and outdoor rotating floating PBR was developed and applied to astaxanthin production by C. zofingiensis, achieving the maximum values of biomass (98.4 g/L) and astaxanthin yield (73.3 mg/L) [17]. This principle could overcome the discrepancy of cell growth and astaxanthin biosynthesis during the astaxanthin production by C. zofingiensis and then would substantially improve the astaxanthin accumulation level for commercial production. The economic performance of microalgal cultivation is considerably hampered by capital expenditures of PBRs and the associated operational costs such as labor, utilities, raw materials, and energy sources. Particularly the capital investment and energy requirement of PBRs is several orders of magnitude higher in the production costs than open cultivation systems [71]. Thus, compared with conventional cultivation systems, innovative cultivation systems, for example, immobilized algal culture systems, are required for C. zofingiensis cultivation due to

52

Chapter 3

their low requirements of energy and water resources [65–67,71]. More efforts in the development of immobilized algae cultivation may bring the economical, efficient, and large-scale astaxanthin production by C. zofingiensis to reality in the near future. Furthermore the environmental principle of largely reducing the negative environmental impacts during the production process requires to be abided with for commercial astaxanthin production by C. zofingiensis. Land use, water management, nutrient supply, and pollution are the crucial environmental factors that should be monitored and examined for mitigating environmental impacts [72–74]. Similar to other microalgae, C. zofingiensis should be cultivated in inarable and marginal lands of low-latitude regions for higher light irradiation and less temperature variation [36]. Particularly, considering that algal cell growth requires a variety of nutrients particularly carbon sources, one feasible proposal is utilizing carbon dioxide from flue gas and fermentable sugars from lignocellulosic wastes for microalgal cultivation. Residual nutrient recycling integrated with wastewater treatment could also be applied to lower negative environmental impacts and avoid the eutrophication of ecosystems [74,75]. In all the possible culture schemes of C. zofingiensis, the environmental impacts are demonstrated to play vital roles in commercial large-scale production, and thus more necessary efforts are required to fulfill the ongoing requirements of the green and sustainable astaxanthin production by C. zofingiensis.

4.3 Integrated microalgal biorefinery for low-cost astaxanthin production Currently, there have already been numerous studies investigating the economic performance of microalgal biorefinery by evaluating total biorefinery costs and multiple product revenues. The cost analysis demonstrated that the revenues of microalgal multiproduct biorefinery are definitely higher than its overall process costs [76]. Currently the minimum cost of astaxanthin production could be reduced to half as much as $7 to 8/kg and that of H. pluvialis biomass is as high as $14/kg for the best case scenario [36]. The production cost is still much higher for commercial application in competition with synthetic astaxanthin. According to previous technoeconomic evaluations of microalgal biorefinery, the microalgal biomass production should be expected at the cost of about $3.3/kg (€3/kg) as well as the DSP cost, which represents about 50% of the total microalgal biorefinery cost [77]. Therefore large improvements on DSP associating with the rational selection and appropriate integration of advanced techniques from the perspective of the economics of microalgal biorefinery are necessarily required to fractionate valuable components for the industrial coproduction of multiple products or commodities. In this section, we will focus on the latest advanced biorefinery techniques particularly DSP and their rational selection principles toward specific component or product extraction besides astaxanthin from microalgal biomass, providing new insights into the biorefinery of C. zofingiensis for commercial astaxanthin production.

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis 53 Generally the DSP of microalgal biorefinery is divided into harvesting and extraction phases, which can be further classified as four main technological process groups: mechanical, chemical, biochemical, and thermochemical processes. For the harvesting phase and associated process technologies, gravity sedimentation and centrifugation are the widely used approaches for the harvesting of microalgal biomass. Comparatively, flocculation and microfiltration are two concentration technologies with promising potentials in microalgal biorefinery although the existence of their drawbacks such as high initial capital investments, nonfood-grade flocculant residues and long operational times [78]. Presently, Gerulova et al. successfully utilized the Fe3O4-PEI (polyethyleneimine) nanocomposites for the enhanced harvesting of different microalgal strains including C. zofingiensis with the efficiencies of as high as 95%–97%. Additionally, Mayers et al. developed a two-step process, that is, alkaline flocculation and sedimentation prior to centrifugation, for the harvesting of C. zofingiensis biomass and significantly reduced the biomass production cost to 0.52 € kg DW 1 [79]. These studies considerably contributed to the reduction of harvesting cost and facilitated the economic production of C. zofingiensis biomass. For the fractionation of complete valuable components from microalgal biomass, various physical, chemical, and biological measures should be selected and utilized for cell disruption, and then different extraction solvents and methods are arranged in succession to extract targeted cellular components of interest. Mechanical processes consisting of bead beating, milling, grinding, microwaving, ultrasonication, and high-pressure homogenization are the favorable means for microalgal cell disruption, while organic solvents, supercritical fluids, and ionic liquids are widely utilized in the multiple product biorefinery of microalgae associated with corresponding processing steps [80,81]. With the rapid development of innovative techniques and novel materials, huge progresses and advances in DSP processes will emerge subsequently and then dramatically reduce the production cost of microalgal biofuels and bioproducts. Toward C. zofingiensis-based biorefinery techniques for multiproducts (Fig. 3.2), the following two principles and rules have to be complied with to improve the biorefinery economics. The first and most important principle is to first extract high-value products with high prices (e.g., secondary carotenoids, PUFAs, and lipids) and medium-value products with large market volume (e.g., proteins and carbohydrates) and then make a whole utilization of the residue biomass to provide additional value. Generally, pigments, lipids, and carbohydrates generally accounted for appropriate 85%–95% of C. zofingiensis biomass, while the residue biomass primarily consists of proteins and nucleic acids in spite of different algal species and their variation in cellular components [80,82]. Thus, according to this principle, carotenoids and lipids with high values in C. zofingiensis should be extracted first following by medium-value carbohydrates, and corresponding DSP should be selected and optimized to boost overall efficiency.

54

Chapter 3 C. zofingiensis culture

Sunlight

CO2, H2O, nutrients

Harvesting

Algal biomass

Purification

Bioethanol

Carotenoids

Fermentation

Fractionation

Nutrition

Extraction

Proteins

Nutrition

Carbohydrates

Whole cell biomass

Lipids

Food and feed

Transesterification

Purification

Biofuels

Edible algal oils

Thermochemical

Biogas and conversion syngas Anaerobic digestion

Residual biomass

Biofertilizer and feed

Fig. 3.2 Simplified overview of C. zofingiensis-based biorefinery processes for the full utilization of valuable components in algal biomass.

Furthermore the major DSP and associated unit operations should be integrated in simplified and consecutive fractionation steps instead of the complex and high-cost processes [76], and the overdosage use of external chemicals and auxiliary materials should be avoided in the extraction phase separation as well. Cascade extraction is the current most favored approach in integrating harvesting and extraction processes and providing a theoretically possible route toward economical biorefinery up to date [83]. For example, the innovative ionic liquid-based aqueous biphasic system using organic salts for bioproduct downstream processes at room temperature was reported to be capable of carrying out the fractionation of wet microalgal biomass and combining these processes into an integrated and simplified process [84]. The protein-lipid-carbohydrates sequential extraction is proved to be the best scenario of multiproduct cascade extraction for microalgal biorefinery [85], which may be also suitable for the biorefinery of C. zofingiensis. Additionally, it is required to consider the priority of the cascade approaches based on the compound fragility and the preferable utilization of mild and low-cost techniques [76]. Currently, in the downstream processes of astaxanthin-producing microalgal biomass, organic solvents and concentrated acids and alkalis are the widely used chemicals for improving the extraction efficiency, while they have the potential toxic effects on human health. Therefore it is necessary to use external fields or other mechanical approaches to replace the use of auxiliary chemicals without sacrificing efficiency and simplicity. Nowadays, pulsed electric field and ultrasound treatment are widely investigated and applied as alternative means to replace the use of auxiliary materials

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis 55 and chemicals [80,83]. However, whether these approaches are able to simultaneously extract carotenoids, lipids, and carbohydrates from harvested fresh C. zofingiensis, biomass is still lacking in-depth study. More efforts are required to better conduct the research and evaluate their applications in multibioproduct biorefinery. Presently there is lack of studies systematically investigating the economics of C. zofingiensisbased biorefinery. It is believed that multibioproducts integrated biorefinery of C. zofingiensis will be theoretically and practically feasible along with considerable technology improvements in DSP and eventually provide an economical and eco-friendly approach for technoeconomic multiple-product biorefinery of various commodities from microalgae.

5 Conclusions Nowadays the economical cultivation of C. zofingiensis for astaxanthin production has attracted great interests due to its potential for the production of multiple valuable bioproducts and biofuels by modern fermentation technology. Additionally, engineering strategies for enhanced astaxanthin production, as well as new insights into integrated microalgal biorefinery for economically commercial astaxanthin production, are discussed and explored the potential of C. zofingiensis as astaxanthin producer. In conclusion, this review demonstrates that C. zofingiensis is a promising alternative astaxanthin producer with strong competition and bright future.

Acknowledgments The work was supported by Guangdong Basic and Applied Basic Research Foundation (Grant Nos. 2019A1515110591, 2019B1515120002) and SinoPec Technology Development Program (218017-1, 36100002-19FW2099-0035). This work was partly supported by the 111 Project, China (Grant No. B17018). Dr. ARR acknowledge Vignan’s Foundation for Science, Technology and Research University for providing facility for this work.

Conflict of interest The authors have declared no conflict of interest.

References [1] Ranga Rao A, Raghunath Reddy RL, Baskaran V, Sarada R, Ravishankar GA. Characterization of microalgal carotenoids by mass spectrometry and their bioavailability and antioxidant properties elucidated in rat model. J Agric Food Chem 2010;58:8553–9. [2] Rao AR, Sindhuja HN, Dharmesh SM, Sankar KU, Sarada R, Ravishankar GA. Effective inhibition of skin cancer, tyrosinase, and antioxidative properties by astaxanthin and astaxanthin esters from the green alga Haematococcus pluvialis. J Agric Food Chem 2013;61:3842–51. [3] Ambati RR, Phang SM, Ravi S, Aswathanarayana RG. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications-a review. Mar Drugs 2014;12:128–52.

56

Chapter 3

[4] Ambati RR, Gogisetty D, Gokare R, Ravi S, Bikkina PN, Bo L, Yuepeng S. Industrial potential of carotenoid pigments from microalgae: current trends and future prospects. Crit Rev Food Sci Nutr 2019;59:1880–902. [5] Roth MS, Cokus SJ, Gallaher SD, Walter A, Lopez D, Erickson E, Endelman B, Westcott D, Larabell CA, Merchant SS, Pellegrini M, Niyogi KK. Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production. Proc Natl Acad Sci U S A 2017;114:4296–305. [6] Focsan AL, Polyakov NE, Kispert LD. Photo protection of Haematococcus pluvialis algae by astaxanthin: unique properties of astaxanthin deduced by EPR, optical and electrochemical studies. Antioxidants 2017;6:80. [7] Kim D-Y, Vijayan D, Praveenkumar R, Han J-I, Lee K, Park J-Y, Chang W-S, Lee J-S, Oha Y-K. Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus. Bioresour Technol 2016;199:300–10. [8] Luo QL, Bian C, Tao M, Huang Y, Zheng YH, Lv YY, Li J, Wang CG, You XX, Jia B, Xu JM, Li JC, Li Z, Shi Q, Hu ZL. Genome and transcriptome sequencing of the astaxanthin-producing green microalga, Haematococcus pluvialis. Genome Biol Evol 2019;11:166–73. [9] Kwon YM, Kim KW, Choi TY, Kim SY, Kim JYH. Manipulation of the microalgal chloroplast by genetic engineering for biotechnological utilization as a green biofactory. World J Microbiol Biotechnol 2018;34:183. [10] Galarza JI, Gimpel JA, Rojas V, Arredondo-Vega BO, Henriquez V. Over-accumulation of astaxanthin in Haematococcus pluvialis through chloroplast genetic engineering. Algal Res 2018;31:291–7. [11] Zhang Z, Sun DZ, Zhang Y, Chen F. Chloroplast morphogenesis in Chromochloris zofingiensis in the dark. Algal Res 2020;45:101742. [12] Chen JH, Wei D, Lim PE. Enhanced coproduction of astaxanthin and lipids by the green microalga Chromochloris zofingiensis: selected phytohormones as positive stimulators. Bioresour Technol 2020;295:122242. [13] Zhang CW, Zhang FL, Dong SJ, He YY, Xu XM, Peng J, Yuan JP. The discrepancy of fatty acid composition of astaxanthin esters and total fatty acids in photoautotrophic and heterotrophic Chlorella zofingiensis. J Am Oil Chem Soc 2019;96:891–901. [14] Mao XM, Wu T, Sun DZ, Zhang Z, Chen F. Differential responses of the green microalga Chlorella zofingiensis to the starvation of various nutrients for oil and astaxanthin production. Bioresour Technol 2018;249:791–8. [15] Zhang Z, Sun DZ, Cheng KW, Chen F. Inhibition of autophagy modulates astaxanthin and total fatty acid biosynthesis in Chlorella zofingiensis under nitrogen starvation. Bioresour Technol 2018;247:610–5. [16] Jiang XY, Liu L, Chen JH, Wei D. Effects of Xanthophyllomyces dendrorhous on cell growth, lipid, and astaxanthin production of Chromochloris zofingiensis by mixed culture strategy. J Appl Phycol 2018;30:3009–15. [17] Zhang Z, Huang JJ, Sun DZ, Lee Y, Chen F. Two-step cultivation for production of astaxanthin in Chlorella zofingiensis using a patented energy-free rotating floating photobioreactor (RFP). Bioresour Technol 2017;224:515–22. [18] Chen J-H, Liu L, Wei D. Enhanced production of astaxanthin by Chromochloris zofingiensis in a microplate-based culture system under high light irradiation. Bioresour Technol 2017;245:518–29. [19] Liu J, Mao X, Zhou W, Guarnieri MT. Simultaneous production of triacylglycerol and high-value carotenoids by the astaxanthin-producing oleaginous green microalga Chlorella zofingiensis. Bioresour Technol 2016;214:319–27. [20] Mulders KJM, Weesepoel Y, Bodenes P, Lamers PP, Vincken J-P, Martens DE, Gruppen H, Wijffels RH. Nitrogen-depleted Chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: a carotenoid metabolism study. J Appl Phycol 2015;27:125–40. [21] Mulders KJM, Janssen JH, Martens DE, Wijffels RH, Lamers PP. Effect of biomass concentration on secondary carotenoids and triacylglycerol (TAG) accumulation in nitrogen-depleted Chlorella zofingiensis. Algal Res 2014;6:8–16. [22] Wang Y, Liu Z, Qin S. Effects of iron on fatty acid and astaxanthin accumulation in mixotrophic Chromochloris zofingiensis. Biotechnol Lett 2013;35:351–7.

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis 57 [23] Liu J, Sun Z, Zhong Y, Gerken H, Huang J, Chen F. Utilization of cane molasses towards cost-saving astaxanthin production by a Chlorella zofingiensis mutant. J Appl Phycol 2013;25:1447–56. [24] Liu J, Huang J, Jiang Y, Chen F. Molasses-based growth and production of oil and astaxanthin by Chlorella zofingiensis. Bioresour Technol 2012;107:393–8. [25] Zhang Z, Sun DZ, Wu T, Li YL, Lee YK, Liu J, Chen F. The synergistic energy and carbon metabolism under mixotrophic cultivation reveals the coordination between photosynthesis and aerobic respiration in Chlorella zofingiensis. Algal Res 2017;25:109–16. [26] Roth MS, Westcott DJ, Iwai M, Niyogi KK. Hexokinase is necessary for glucose-mediated photosynthesis repression and lipid accumulation in a green alga. Commun Biol 2019;2(1):1–10. [27] Roth MS, Gallaher SD, Westcott DJ, Iwai M, Louie KB, Mueller M, et al. Regulation of oxygenic photosynthesis during trophic transitions in the green alga Chromochloris zofingiensis. Plant Cell 2019;31 (3):579–601. [28] Imaizumi Y, Nagao N, Yusoff FM, Kurosawa N, Kawasaki N, Toda T. Lumostatic operation controlled by the optimum light intensity per dry weight for the effective production of Chlorella zofingiensis in the high cell density continuous culture. Algal Res 2016;20:110–7. [29] Liu J, Sun Z, Gerken H, Liu Z, Jiang Y, Chen F. Chlorella zofingiensis as an alternative microalgal producer of astaxanthin: biology and industrial potential. Mar Drugs 2014;12:3487–515. [30] Del Campo JA, Garcia-Gonzalez M, Guerrero MG. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol 2007;74:1163–74. [31] Bohne F, Linden H. Regulation of carotenoid biosynthesis genes in response to light in Chlamydomonas reinhardtii. Biochim Biophys Acta Gene Struct Expr 2002;1579:26–34. [32] Chen T, Liu J, Guo B, Ma X, Sun P, Liu B, Chen F. Light attenuates lipid accumulation while enhancing cell proliferation and starch synthesis in the glucose-fed oleaginous microalga Chlorella zofingiensis. Sci Rep 2015;5:14936. [33] Sun N, Wang Y, Li YT, Huang JC, Chen F. Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta). Process Biochem 2008;43:1288–92. [34] Mulders KJ, Lamers PP, Wijffels RH, Martens DE. Dynamics of biomass composition and growth during recovery of nitrogen-starved Chromochloris zofingiensis. Appl Microbiol Biotechnol 2015;99:1873–84. [35] Zhao WY, Sun H, Ren YY, Wu T, He YJ, Chen F. Chlorella zofingiensis as a promising strain in wastewater treatment. Bioresour Technol 2018;268:286–91. [36] Panis G, Carreon JR. Commercial astaxanthin production derived by green alga Haematococcus pluvialis: a microalgae process model and a techno-economic assessment all through production line. Algal Res 2016;18:175–90. [37] Yu XH, Chen L, Zhang WW. Chemicals to enhance microalgal growth and accumulation of high-value bioproducts. Front Microbiol 2015;6:56. [38] Lee C, Choi YE, Yun YS. A strategy for promoting astaxanthin accumulation in Haematococcus pluvialis by 1-aminocyclopropane-1-carboxylic acid application. J Biotechnol 2016;236:120–7. [39] Perez-Lopez P, Gonzalez-Garcia S, Jeffryes C, Agathos SN, McHugh E, Walsh D, Murray P, Moane S, Feijoo G, Moreira MT. Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae: from lab to pilot scale. J Clean Prod 2014;64:332–44. [40] Huang JC, Zhong YJ, Liu J, Sandmann G, Chen F. Metabolic engineering of tomato for high-yield production of astaxanthin. Metab Eng 2013;17:59–67. [41] Zhang Z, Sun DZ, Mao XM, Liu J, Chen F. The crosstalk between astaxanthin, fatty acids and reactive oxygen species in heterotrophic Chlorella zofingiensis. Algal Res 2016;19:178–83. [42] Zhu S, Huang W, Xu J, Wang Z, Xu J, Yuan Z. Metabolic changes of starch and lipid triggered by nitrogen starvation in the microalga Chlorella zofingiensis. Bioresour Technol 2014;152:292–8. [43] Sudibyo H, Pradana YS, Samudra TT, Budiman A, Indarto, Suyono EA. Study of cultivation under different colors of light and growth kinetic study of Chlorella zofingiensis D€ onz for biofuel production. Energy Procedia 2017;105:270–6. [44] Zhu S, Wang Y, Shang C, Wang Z, Xu J, Yuan Z. Characterization of lipid and fatty acids composition of Chlorella zofingiensis in response to nitrogen starvation. J Biosci Bioeng 2015;120:205–9.

58

Chapter 3

[45] Zemke PE, Sommerfeld MR, Hu Q. Assessment of key biological and engineering design parameters for production of Chlorella zofingiensis (Chlorophyceae) in outdoor photobioreactors. Appl Microbiol Biotechnol 2013;97:5645–55. [46] Zhang J, Liu L, Chen F. Production and characterization of exopolysaccharides from Chlorella zofingiensis and Chlorella vulgaris with anti-colorectal cancer activity. Int J Biol Macromol 2019;134:976–83. [47] Tripathi U, Venkateshwaran G, Sarada R, Ravishankar GA. Studies on Haematococcus pluvialis for improved production of astaxanthin by mutagenesis. World J Microbiol Biotechnol 2001;17:143–8. [48] Becker J, Wittmann C. From systems biology to metabolically engineered cells—an omics perspective on the development of industrial microbes. Curr Opin Microbiol 2018;45:180–8. [49] Huang WP, Ye JR, Zhang JJ, Lin Y, He MX, Huang JC. Transcriptome analysis of Chlorella zofingiensis to identify genes and their expressions involved in astaxanthin and triacylglycerol biosynthesis. Algal Res 2016;17:236–43. [50] Liu J, Sun Z, Mao XM, Gerken H, Wang XF, Yang WQ. Multiomics analysis reveals a distinct mechanism of oleaginousness in the emerging model alga Chromochloris zofingiensis. Plant J 2019;98:1060–77. [51] Wang XF, Wei HH, Mao XM, Liu J. Proteomics analysis of lipid droplets from the oleaginous alga Chromochloris zofingiensis reveals novel proteins for lipid metabolism. Genomics Proteomics Bioinformatics 2019;17:260–72. [52] Cordero BF, Couso I, Leon R, Rodriguez H, Vargas MA. Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis. Appl Microbiol Biotechnol 2011;91:341–51. [53] Cordero BF, Couso I, Leon R, Rodriguez H, Vargas MA. Isolation and characterization of a lycopene ε-cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light. Mar Drugs 2012;10:2069–88. [54] Liu J, Sun Z, Gerken H, Huang JC, Jiang Y, Chen F. Genetic engineering of the green alga Chlorella zofingiensis: a modified norflurazon-resistant phytoene desaturase gene as a dominant selectable marker. Appl Microbiol Biotechnol 2014;98:5069–79. [55] Xu Y, Falarz L, Chen GQ. Characterization of type-2 diacylglycerol acyltransferases in the green microalga Chromochloris zofingiensis. J Agric Food Chem 2019;67:291–8. [56] Fang N, Wang CK, Liu XF, Zhao X, Liu YH, Liu XM, Du YM, Zhang ZF, Zhang HB. De novo synthesis of astaxanthin: from organisms to genes. Trends Food Sci Technol 2019;92:162–71. [57] Saini DK, Chakdar H, Pabbi S, Shukla P. Enhancing production of microalgal biopigments through metabolic and genetic engineering. Crit Rev Food Sci Nutr 2020;60:391–405. [58] Pick U, Zarka A, Boussiba S, Davidi L. A hypothesis about the origin of carotenoid lipid droplets in the green algae Dunaliella and Haematococcus. Planta 2019;249:31–47. [59] Gaj T, Gersbach CA, Barbas 3rd. CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 2013;31:397–405. [60] Ul Ain Q, Chung JY, Kim YH. Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN. J Control Release 2015;205:120–7. [61] Aslanbay Guler B, Deniz I, Demirel Z, Imamoglu E. Computational fluid dynamics simulation in scaling-up of airlift photobioreactor for astaxanthin production. J Biosci Bioeng 2020;129:86–92. [62] Behera B, Acharya A, Gargey IA, Aly N, Balasubramanian P. Bioprocess engineering principles of microalgal cultivation for sustainable biofuel production. Bioresour Technol Rep 2019;5:297–316. [63] Johnson TJ, Katuwal S, Anderson GA, Gu LP, Zhou RB, Gibbons WR. Photobioreactor cultivation strategies for microalgae and cyanobacteria. Biotechnol Prog 2018;34:811–27. [64] Khoo KS, Lee SY, Ooi CW, Fu XT, Miao XL, Ling TC, Show PL. Recent advances in biorefinery of astaxanthin from Haematococcus pluvialis. Bioresour Technol 2019;288:121606. [65] Do TT, Ong BN, Nguyen Tran ML, Nguyen D, Melkonian M, Tran HD. Biomass and astaxanthin productivities of Haematococcus pluvialis in an angled twin-layer porous substrate photobioreactor: effect of inoculum density and storage time. Biology 2019;8:68.

Astaxanthin from Chromochloris zofingiensis: Feasibility analysis 59 [66] Yusoff FM, Nagao N, Imaizumi Y, Toda T. Bioreactor for microalgal cultivation systems: strategy and development. In: Asghar RA, Nath YA, Arti G, editors. Prospects of renewable bioprocessing in future energy systems. Biofuel and biorefinery technologies, vol. 10. Cham: Springer; 2019. p. 117–59. [67] Kiperstok AC, Sebestyen P, Podola B, Melkonian M. Biofilm cultivation of Haematococcus pluvialis enables a highly productive one-phase process for astaxanthin production using high light intensities. Algal Res 2017;21:213–22. [68] Ooms MD, Dinh CT, Sargent EH, Sinton D. Photon management for augmented photosynthesis. Nat Commun 2016;7:12699. [69] Pierobon SC, Cheng X, Graham PJ, Nguyen B, Karakolis EG, Sinton D. Emerging microalgae technology: a review. Sustain Energy Fuels 2018;2:13–38. [70] Wang S-K, Stiles AR, Guo C, Liu C-Z. Microalgae cultivation in photobioreactors: an overview of light characteristics. Eng Life Sci 2014;14:550–9. [71] OzkanA A, Kinney K, Katz L, Berberoglu H. Reduction of water and energy requirement of algae cultivation using an algae biofilm photobioreactor. Bioresour Technol 2012;114:542–8. [72] Khan MI, Shin JH, Kim JD. The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microb Cell Factories 2018;17:36. [73] Moreno-Garcia L, Adjalle K, Barnabe S, Raghavan GSV. Microalgae biomass production for a biorefinery system: recent advances and the way towards sustainability. Renew Sust Energ Rev 2017;76:493–506. [74] Slade R, Bauen A. Micro-algae cultivation for biofuels: cost, energy balance, environmental impacts and future prospects. Biomass Bioenergy 2013;53:29–38. [75] Chew KW, Yap JY, Show PL, Suan NH, Juan JC, Ling TC, Lee DJ, Chang JS. Microalgae biorefinery: high value products perspectives. Bioresour Technol 2017;229:53–62. [76] t’Lam GP, Vermue MH, Eppink MHM, Wijffels RH, van den Berg C. Multi-product microalgae biorefineries: from concept towards reality. Trends Biotechnol 2018;36:216–27. [77] Ruiz J, Olivieri G, de Vree J, Bosma R, Willems P, Reith JH, Eppink MHM, Kleinegris DMM, Wijffels RH, Barbosa MJ. Towards industrial products from microalgae. Energy Environ Sci 2016;9:3036–43. [78] Singh G, Patidar SK. Microalgae harvesting techniques: a review. J Environ Manag 2018;217:499–508. [79] Mayers JJ, Landels AR, Allen MJ, Albers E. An energy and resource efficient alkaline flocculation and sedimentation process for harvesting of Chromochloris zofingiensis biomass. Bioresour Technol Rep 2020;9:100358. [80] Rammuni MN, Ariyadasa TU, Nimarshana PHV, Attalage RA. Comparative assessment on the extraction of carotenoids from microalgal sources: astaxanthin from H. pluvialis and beta-carotene from D. salina. Food Chem 2019;277:128–34. [81] Saini RK, Keum YS. Carotenoid extraction methods: a review of recent developments. Food Chem 2018;240:90–103. [82] Hariskos I, Posten C. Biorefinery of microalgae—opportunities and constraints for different production scenarios. Biotechnol J 2014;9:739–52. [83] Gifuni I, Pollio A, Safi C, Marzocchella A, Olivieri G. Current bottlenecks and challenges of the microalgal biorefinery. Trends Biotechnol 2019;37:242–52. [84] Orr VCA, Rehmann L. Ionic liquids for the fractionation of microalgae biomass. Curr Opin Green Sustain Chem 2016;2:22–7. [85] Ansari FA, Shriwastav A, Gupta SK, Rawat I, Bux F. Exploration of microalgae biorefinery by optimizing sequential extraction of major metabolites from Scenedesmus obliquus. Ind Eng Chem Res 2017;56:3407–12.

This page intentionally left blank

CHAPTER 4

Astaxanthin in microalgae Eustigmatophyceae ˜o, Joana D. Ferreira, and Lı´lia M.A. Santos Clara B. Martins, Mariana F.G. Assunc¸ a Coimbra Collection of Algae (ACOI), Department of Life Sciences, University of Coimbra, Coimbra, Portugal

Abbreviations ACOI DMAPP DNA DW EU GGPP GGPS HDL IPP LDL MAV MEP USFDA

Coimbra Collection of Algae dimethylallyl diphosphate deoxyribonucleic acid dry weight European Union geranylgeranyl pyrophosphate geranylgeranyl pyrophosphate synthase high-density lipoprotein isopentenyl pyrophosphate low-density lipoprotein mevalonate pathway nonmevalonate pathway United States Food and Drug Administration

1 Introduction Currently, there is a growing interest in bioactive compounds from natural sources. Carotenoids of microalgae have been highlighted in the last years due to their structure and bioactivities [1–4]. The main microalgal carotenoids are astaxanthin, β-carotene, lutein, lycopene, zeaxanthin, violaxanthin, and fucoxanthin, the first three being the most studied [4]. The first description of astaxanthin in microalgae was performed by Tischer [5] who called it “haematochrome.” Astaxanthin (3,30 -dihydroxy-β,β-carotene-4,40 -dione) is a red secondary carotenoid synthesized de novo by some microalgae, plants, yeasts, and bacteria and present in seafood such as salmon, trout, red sea bream, shrimp, lobster, and fish eggs [6]. Astaxanthin shows high biological activity, namely, a strong antioxidant capacity, and is already available in the market [7]. Haematococcus pluvialis is considered the microalga with the highest astaxanthin accumulation capacity; however, the pigment has also been reported in other groups of microalgae like the Eustigmatophyceae [8, 9]. The current review provides Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00011-8 Copyright # 2021 Elsevier Inc. All rights reserved.

61

62

Chapter 4

information on the biological activities of astaxanthin, its health benefits, commercial applications, and the potential of other groups of microalgae to produce it, namely, the Eustigmatophyceae class.

2 Astaxanthin structure Astaxanthin is a xanthophyll with a chemical formula of C40H52O4 and a molecular weight of 596.86 gmol1 [1]. Its unique molecular structure with polar end groups, which quenches free radicals and the double bonds in their middle segment with the capacity to remove high-energy electrons, explains its antioxidant activity that is higher than in other carotenoids [10]. It is a molecule sensitive to light, heat, and oxygen due to its structural characteristics and exists in stereoisomers, geometric isomers, and free and esterified forms [11]. The stereoisomers (3S, 30 S) and (3R, 30 R) are the most abundant in nature, while synthetic astaxanthin contains a mixture of isomers (3S, 30 S), (3R, 30 S), and (3R, 30 R). The predominant form found in the microalga H. pluvialis is the stereoisomer form (3S, 30 S) while in the yeast Xanthophyllomyces dendrorhous, it is, (3R, 30 R) [7]. The polyene chain may exist in two different configurations, geometric isomers cis and trans. The cis isomers are thermodynamically less stable than trans isomers. In nature, most carotenoids are predominantly in the form of trans isomers [12]. Astaxanthin is often in free form or esterified with one or both hydroxyl groups linked to several fatty acids (e.g., palmitic, stearic, and oleic acids), while the synthetic astaxanthin is usually in free form [1]. The esterification with fatty acids allows astaxanthin to be concentrated within cytoplasmic globules to maximize its photoprotective efficiency [13]. In algae, astaxanthin is found mainly in the esterified form (about 70% monoester and 10% diester) [1, 7]. It is thought that the fatty acid accumulation may be related to astaxanthin ester accumulation [14]. The fatty acid production enables the oil globules to hold high content of astaxanthin esters. In some microalgal species, the synthesis of fatty acids may stimulate carotenoid accumulation by providing sequestering structures and creating a plastid-localized sink for the end product of the carotenoid biosynthetic pathway [14].

3 Synthesis and accumulation of astaxanthin In microalgae the process of carotenogenesis occurs as a defense mechanism in response to adverse conditions, in particular, the accumulation of the higher amount of astaxanthin in cells enhances the cell resistance to oxidative stress, limiting the photoinhibition and photodamage caused by intense solar radiation [13]. This resistance is triggered by the quenching of oxygen atoms for the synthesis itself as well as from the antioxidant properties of the astaxanthin molecules. Thus the accumulation of secondary carotenoids is considered a safe way to achieve a sustained increase in photoprotective capacity [15, 16]. The accumulation of astaxanthin is a way to store energy and carbon, which is later used for further synthesis under less stressful conditions

Astaxanthin in microalgae Eustigmatophyceae 63 [16, 17]. An important key intermediate of carotenoid synthesis is isopentenyl pyrophosphate (IPP) and can be originated by two pathways: mevalonate pathway (MAV) located in the cytosol and nonmevalonate pathway (MEP) located in the chloroplast. In H. pluvialis, IPP is produced inside the chloroplast through the MEP pathway, since this microalga does not have the key enzymes for the catalysis of IPP through the MVA pathway [6]. Then, IPP is isomerized in dimethylallyl diphosphate (DMAPP), and the elongation of the isoprenoid chain is initiated with the molecule of DMAPP and subsequent linear addition of three IPP molecules catalyzed by an enzyme geranylgeranyl pyrophosphate synthase (GGPS) (Fig. 4.1). At the end of this step, a C20 compound is obtained, geranylgeranyl pyrophosphate (GGPP). The condensation of two GGPP

Fig. 4.1 Synthesis pathway of astaxanthin in H. pluvialis. Adapted from Shah M, Mahfuzur R, Liang Y, Cheng JJ, Daroch M, Astaxanthin-producing green microalga Haematococcus pluvialis: from single cell to high value commercial products. Front Plant Sci. 2016;7:531.

64

Chapter 4

molecules results in a phytoene, which is a precursor for astaxanthin and other carotenoids [6, 16]. Desaturation reactions convert the colorless phytoene to pink-colored lycopene, and the cyclization of lycopene originates β-carotene (precursor of other carotenoids including astaxanthin) and α-carotene (precursor of lutein). Posteriorly, the synthesis of xanthophylls by the addition of oxygen groups to carotenes occurs (Fig. 4.1) [1, 6].

4 Bioactivity of astaxanthin Essentially due to its strong antioxidant activity, astaxanthin has been showing nutraceutical and pharmaceutical effects against cardiovascular diseases, degenerative eye diseases, diabetes, and inflammation and activity against some types of cancer like oral, colon, and liver cancers [11, 17–21]. Its antioxidant power is 500 times stronger than vitamin E and 65 times more active than vitamin C and is much more effective than other carotenoids as lutein and lycopene [22]. It is more effective in scavenging-free radicals allowing reduction of DNA damage and protection of eye and skin cells from photooxidation and improving heart health by modifying blood levels of LDL and HDL cholesterol [1]. Furthermore, astaxanthin has also been applied in other areas such as aquaculture, being used as a common coloring agent of animal bodies like salmon [10, 17]. In addition to pigmentation, astaxanthin has already been shown to benefit the growth and survival of larval fish and shrimp [1]. The search of astaxanthin for cosmetic products and supplementation in foods increased the demand for natural astaxanthin [11].

5 Current global market of astaxanthin Astaxanthin is already on the current market in the dosage forms as tablets, capsules, syrups, oils, soft gels, creams, and biomass and granulated powders [7]. It having been approved for the first time in 1987, by the United States Food and Drug Administration (USFDA), as a feed additive for use in the aquaculture industry, and in 1999 it was approved for use as a dietary supplement [23]. The astaxanthin market reached $288.7 million in 2017 and is expected to increase to $426.9 million in 2022. Currently the value of astaxanthin is between $2500 and $7000/kg, and several production companies are now well established, as Cyanotech (Hawaii, USA), Alga Technologies (Israel), Astareal (Japan), and Algacan (Canada) [4]. Astaxanthin is considered by the European Union (EU) as a food dye with the E number E161j; however, in the United States, it has been approved only for specific uses in animal and fish foods [9]. More than 95% of the astaxanthin world market is produced synthetically, but this is expected to decline to around 55% by the end of 2024 [22]. Since natural astaxanthin is three or four times more valuable than the synthetic in nutraceuticals and pharmaceuticals market, there is increasing demand for natural astaxanthin for specific commercial applications. Thus the cultivation of H. pluvialis on an industrial scale has generated great interest and is considered an attractive business opportunity. However, due to the high price of natural

Astaxanthin in microalgae Eustigmatophyceae 65 astaxanthin related to the expensive and time-consuming cultivation of H. pluvialis, the commercialized microalgal-derived astaxanthins 90 and >130 mgg-1 FW, respectively), without formation of ketolutein. Thus overexpression of a single ketolase results in efficient redirection of the vast majority of the carotenoid pool of carrot roots into the formation of β-ketocarotenoids. Very recently, engineering of carotenoid biosynthesis for the production of ketocarotenoids has been achieved in several species containing carotenogenic flower petals potentially representing new sources of ketocarotenoids. Petals of many plants contain considerably higher concentrations of carotenoids. For both Lotus japonicus [189] and N. glauca [187], a crtW-type ketolase was used for transformation. Although L. japonicus exhibited the formation of astaxanthin and other keto intermediates, 4-ketozeaxanthin was the only ketolated product in N. glauca. Also the ketocarotenoid yields were lower than those reported for L. japonicus. In a paper, which compared the interaction of

170 Chapter 9 ketolase and hydroxylase genes, the use of the bkt was suggested as an alternative for heterologous ketocarotenoid formation [190]. To increase the antioxidant capacity, β-carotene ketolase (bkt) and β-carotene hydroxylase (crtR-B) were introduced into apple trees [161]. Transgenic lines were reported to exhibit high antioxidant activity due to the production of ketocarotenoids, namely, astaxanthin and canthaxanthin. Metabolic engineering of carotenoid biosynthesis in plants has given recently several successful results. Depending on the plant species, seeds, fruit, flower, and tuber were the preferred organs for modified carotenoid production [121]. In general, transformation with a β-carotene ketolase gene may result in the formation of different 3-hydroxy-4-keto β-carotene derivatives since a 3-hydroxylase already exists in plants. The high expression of only the ketolase gene may seem enough for producing large amounts of astaxanthin, judging from the successful expression of bkt1in carrot [130]. It was also reported that the simultaneous high expression of the carotenoid 3,3-hydroxylase gene crtZ (SD212) in addition to the ketolase gene is useful for increasing astaxanthin levels [131]. Strong constitutive promoters such as CaMV 35S promoter and tissue-specific promoters are used in plants for the high expression of the ketolase and hydroxylase genes. Table 9.2 represents organisms bioengineered for astaxanthin production. 4.2.2 Role of promoters in metabolic pathway engineering The selection of promoter for the gene expression depends on the gene function and the type of host species for the successful of pathway engineering. When observing the suitability of the promoter sequences for the transgenic production in microalgae, the endogenous promoter like Rubisco small subunit (RbcS2) or the ubiquitin (Ubi1) promoter or the heterologous promoters CaMV35S and SV40 has been successfully used for microalgal genetic transformation. The CaMV35S, being a constitutive promoter, has been studied universally for higher plants, which also works well in several algal stains and showed strong expression [112–114]. Also the SV40, a polyomavirus promoter, has been shown to work in H. pluvialis and in C. reinhardtii [191]. The efficiency of heterologous promoters in microalgae and the use of different transformation methods were described in a study on C. reinhardtii [192]. The efficiency was evaluated using heterologous promoter, cauliflower mosaic virus 35S (CaMV 35S), and Agrobacterium nopaline synthase (NOS). These promoters were fused to the paromomycin conferring resistance aminoglycoside 30 -phosphotransferase encoding gene (APHVIII), and C. reinhardtii was transformed by the glass bead agitation method. Higher transformation efficiencies and higher level of APHVIII expression were identified in those transformants harboring the NOS promoter than CaMV 35S promoter. And hence the NOS promoter is widely used for genetic manipulation of higher plants that has been very rarely used for the transformation of microalgae. For astaxanthin overexpression bkt gene in the same homologous host H. pluvialis, CaMV 35S promoter was successfully used [173]. At the same time, for D. salina Rubisco smaller subunit promoter along with its transit peptide sequence was used for the astaxanthin production [36].

Metabolic engineering of astaxanthin pathway 171

5 Conclusion The increased demand of astaxanthin in industries has raised the market demand for natural astaxanthin. However, there is limitation for natural astaxanthin production as the production is restricted to very few microbes, algae, and plants among which microalgae are ranked as a better choice due to its feasibility for industrial production. Although mutagenesis and culture conditions have improved the production efficiency of algal strains, they are far from meeting market demand. Genetic engineering of microalgae is necessary to produce economically feasible strains for metabolite production. Metabolic engineering provides an alternate strategy to create highly efficient transgenic algal systems through which natural astaxanthin may be produced in industrial scale. Although the results of metabolic engineering are predictable to some extent, many a times quantitative prediction fails due to the lack of knowledge in comprehensive understanding of different metabolic pathways. A better understanding of astaxanthin biosynthetic pathway and its intricate relation with other metabolic pathway that has been recently understood in the past decade may considerably improve the production further. Current efforts are focused on the manipulation of individual metabolic genes, but the outcomes are not stable and/or efficient for large-scale production. Genetic modifications that enhance the physiological properties of algal strains and optimization of metabolite production are further needed to improve the potential of this promising technology in the future.

References [1] Lim KC, Yusoff FM, Shari M, Kamarudin MS. Astaxanthin as feed supplement in aquatic animals. Rev Aquac 2018;10(3):738–73. [2] Barredo JL, Garcı´a-Estrada C, Kosalkova K, Barreiro C. Biosynthesis of astaxanthin as a main carotenoid in the Heterobasidiomycetous yeast Xanthophyllomyces dendrorhous. J Fungi 2017;3(3):44. [3] Panis G, Carreon JR. Commercial astaxanthin production derived by green alga Haematococcus pluvialis: a microalgae process model and a techno-economic assessment all through production line. Algal Res 2016;18:175–90. [4] Ambati RR, Phang SM, Ravi S, Aswathanarayana RG. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications—a review. Mar Drugs 2014;12(1):128–52. [5] Sarada R, Tripathi U, Ravishankar GA. Influence of stress on astaxanthin production in Haematococcus pluvialis grown under different culture conditions. Process Biochem 2002;37(6):623–7. [6] Choi YE, Yun YS, Park JM. Evaluation of factors promoting astaxanthin production by a unicellular green alga, Haematococcus pluvialis, with fractional factorial design. Biotechnol Prog 2002;18(6):1170–5. [7] Ho YH, Leung HM, Yuen SY, Ng KS, Li TS, Yuen LM, Wong YK. Maximization of astaxanthin production from green microalga Haematococcus pluvialis using internally-illuminated photobioreactor. Adv Biosci Bioeng 2018;6:10–22. [8] Butler TO, McDougall GJ, Campbell R, Stanley MS, Day JG. Media screening for obtaining Haematococcus pluvialis red motile macrozooids rich in astaxanthin and fatty acids. Biology 2017;7(1):2. [9] Liu J, van der Meer JP, Zhang L, Zhang Y. Cultivation of Haematococcus pluvialis for astaxanthin production. In: Stephen PS, John RB, editors. Microalgal production for biomass and high-value products. Boca Raton: CRC Press; 2016. p. 267. [10] Zhang WW, Zhou XF, Zhang YL, Cheng PF, Ma R, Cheng WL, Chu HQ. Enhancing astaxanthin accumulation in Haematococcus pluvialis by coupled light intensity and nitrogen starvation in column photobioreactors. J Microbiol Biotechnol 2018;28(12):2019–28.

172 Chapter 9 [11] Kang CD, Lee JS, Park TH, Sim SJ. Complementary limiting factors of astaxanthin synthesis during photoautotrophic induction of Haematococcus pluvialis: C/N ratio and light intensity. Appl Microbiol Biotechnol 2007;74(5):987–94. [12] Johnson EA, An GH. Astaxanthin from microbial sources. Crit Rev Biotechnol 1991;11:297–326. [13] Schmidt I, Schewe H, Gassel S, Jin C, Buckingham J, H€ umbelin M, et al. Biotechnological production of astaxanthin with Phaffia rhodozyma/Xanthophyllomyces dendrorhous. Appl Microbiol Biotechnol 2011; 89(3):555–71. [14] Fontana JD, Guimaraes MF, Martins NT, Fontana CA, Baron M. Culture of the astaxanthinogenic yeast Phaffia rhodozyma in low-cost media. Appl Biochem Biotechnol 1996;57–58:413–22. [15] Hayman TG, Mannarelli BN, Leathers TD. Production of carotenoids by Phaffia rhodozyma grown on media composed of corn wet-milling co-products. J Ind Microbiol 1995;115:173–83. [16] Leathers DT. Bioconversions of maize residues to value-added coproducts using yeast-like fungi. FEMS Yeast Res 2003;3:133–40. [17] Jiang G, Yang Z, Wang Y, Yao M, Chen Y, Xiao W, Yuan Y. Enhanced astaxanthin production in yeast via combined mutagenesis and evolution. Biochem Eng J 2020;156:107519. [18] Kim JH, Kang SW, Kim SW, Chang HI. High-level production of astaxanthin by Xanthophyllomyces dendrorhous mutant JH1 using statistical experimental designs. Biosci Biotechnol Biochem 2005;69(9):1743–8. [19] Zhuang Y, Jiang GL, Zhu MJ. Atmospheric and room temperature plasma mutagenesis and astaxanthin production from sugarcane bagasse hydrolysate by Phaffia rhodozyma mutant Y1. Process Biochem 2020;91:330–8. [20] Tripathi U, Venkateshwaran G, Sarada R, Ravishankar G. Studies on Haematococcus pluvialis for improved production of astaxanthin by mutagenesis. World J Microbiol Biotechnol 2001;17(2):143–8. [21] Kamath BS, Vidhyavathi R, Sarada R, Ravishankar G. Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Haematococcus pluvialis mutants. Bioresour Technol 2008;99(18):8667–73. [22] Chen Y, Li D, Lu W, Xing J, Hui B, Han Y. Screening and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis. Biotechnol Lett 2003;25(7):527–9. [23] Wang N, Guan B, Kong Q, Sun H, Geng Z, Duan L. Enhancement of astaxanthin production from Haematococcus pluvialis mutants by three-stage mutagenesis breeding. J Biotechnol 2016;236:71–7. [24] Dominguez-Bocanegra AR, Ponce-Noyola T, Torres-Mun˜oz JA. Astaxanthin production by Phaffia rhodozyma and Haematococcus pluvialis: a comparative study. Appl Microbiol Biotechnol 2007;75:783–91. [25] Henry LK, Thomas ST, Widhalm JR, Lynch JH, Davis TC, Kessler SA, Dudareva N. Contribution of isopentenyl phosphate to plant terpenoid metabolism. Nat Plants 2018;4(9):721–9. [26] Huang J-C, Zhong Y-J, Sandmann G, Liu J, Chen F. Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants. Planta 2012;236(2):691–9. [27] Saini RK, Keum Y-S. Microbial platforms to produce commercially vital carotenoids at industrial scale: an updated review of critical issues. J Ind Microbiol Biotechnol 2019;46(5):657–74. [28] Jagadevan S, Banerjee A, Banerjee C, et al. Recent developments in synthetic biology and metabolic engineering in microalgae towards biofuel production. Biotechnol Biofuels 2018;11:185. [29] Lior D, Na’ama S, Michal S. Transgene expression in microalgae—from tools to applications. Front Plant Sci 2016;7:505. [30] Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25:294–306. [31] Croft T, Moulin M, Webb E, Smith G. Thiamine biosynthesis in algae is regulated by riboswitches. Proc Natl Acad Sci 2007;104:20770–5. [32] Shao N, Bock R. A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii. Curr Genet 2008;53:381–8. [33] Surzycki R, Cournac L, Peltier G, Rochaix D. Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas. Proc Natl Acad Sci U S A 2007;104:17548–53. [34] Bogen C, Al-Dilaimi A, Albersmeier A, Wichmann J, Grundmann M, Rupp O, et al. Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production. BMC Genomics 2013;14:926.

Metabolic engineering of astaxanthin pathway 173 [35] Fajardo C, De Donato M, Carrasco R, Martı´nez-Rodrı´guez G, Mancera JM, Ferna´ndez Acero FJ. Advances and challenges in genetic engineering of microalgae. Rev Aquac 2020;12(1):365–81. [36] Anila N, Daris PS, Chandrashekar A, Ravishankar GA, Sarada R. Metabolic engineering of Dunaliella salina for production of ketocarotenoids. Photosynth Res 2016;127:321–33. [37] Ahn M-J, Noh SA, Ha S-H, Back K, Lee SW, Bae JM. Production of ketocarotenoids in transgenic carrot plants with an enhanced level of ß-carotene. Plant Biotechnol Rep 2012;6(2):133–40. [38] Pierce EC, LaFayette PR, Ortega MA, Joyce BL, Kopsell DA, Parrott WA. Ketocarotenoid production in soybean seeds through metabolic engineering. PLoS One 2015;10(9)e0138196. [39] Rodriguez-Concepcion M, Boronat A. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 2002;130(3):1079–89. https://doi.org/10.1104/pp.007138. [40] Lichtenthaler HK. The 1-Deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 1999;50:47–65. [41] Schwender J, Gem€unden C, Lichtenthaler H. Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/ 2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids. Planta 2001;212:416–23. [42] Fang N, Wang C, Liu X, Zhao X, Liu Y, Liu X, Du Y, Zhang Z, Zhang H. De novo synthesis of astaxanthin: from organisms to genes. Trends Food Sci Technol 2019;92:162–71. [43] Sandmann G. Carotenoid biosynthesis in microorganisms and plants. In: EJB reviews 1994. vol. 1994. Berlin, Heidelberg: Springer; 1994. [44] Cunningham Jr. FX, Gantt E. Identification of multi-gene families encoding isopentenyl diphosphate isomerase in plants by heterologous complementation in Escherichia coli. Plant Cell Physiol 2000;41 (1):119–23. [45] Margalith P. Production of ketocarotenoids by microalgae. Appl Microbiol Biotechnol 1999;51:431–8. [46] Lotan T, Hirschberg J. Cloning and expression in Escherichia coli of the gene encoding β-C-4-oxygenase, that converts β-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis. FEBS Lett 1995;364 (2):125–8. [47] Misawa N, Satomi Y, Kondo K, Yokoyama A, Kajiwara S, Saito T, Ohtani T, Miki W. Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J Bacteriol 1995;177:6575–84. [48] Breitenbach J, Misawa N, Kajiwara S, Sandmann G. Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus pluvialis. FEMS Microbiol Lett 1996;140(2–3):241–6. [49] Boussiba S. Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Physiol Plant 2000;108(2):111–7. [50] Gr€unewald K, Hagen C. β-Carotene is the intermediate exported from the chloroplast during accumulation of secondary carotenoids in Haematococcus pluvialis. J Appl Phycol 2001;13(1):89–93. [51] Hirschberg J. Carotenoid biosynthesis in flowering plants. Curr Opin Plant Biol 2001;4(3):210–8. [52] Demain AL. Mutation and the production of secondary metabolites 11 contribution no. 2050 of the Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. In: Perlman D, editor. Advances in applied microbiology. Academic Press; 1973. p. 177–202. [53] Schiano di Visconte G, Spicer A, Chuck CJ, Allen MJ. The microalgae biorefinery: a perspective on the current status and future opportunities using genetic modification. Appl Sci 2019;9:4793. [54] Cheng J, Li K, Yang Z, Zhou J, Cen K. Enhancing the growth rate and astaxanthin yield of Haematococcus pluvialis by nuclear irradiation and high concentration of carbon dioxide stress. Bioresour Technol 2016;204:49–54. [55] Hu Z, Li Y, Sommerfeld M, Chen F, Hu Q. Enhanced protection against oxidative stress in an astaxanthin-overproduction Haematococcus mutant (Chlorophyceae). Eur J Phycol 2008;43:365–76. [56] Hong M-E, Choi SP, Park Y-I, Kim Y-K, Chang WS, Kim BW, Sim SJ. Astaxanthin production by a highly photosensitive Haematococcus mutant. Process Biochem 2012;47:1972–9.

174 Chapter 9 [57] Peng J, Xiang W, Tang Q, Sun N, Chen F, Yuan J. Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column. Sci China C Life Sci 2008;51:1108–15. [58] Go´mez PI, Inostroza I, Pizarro M, Perez J. From genetic improvement to commercial-scale mass culture of a Chilean strain of the green microalga Haematococcus pluvialis with enhanced productivity of the red ketocarotenoid astaxanthin. AoB Plants 2013;5:plt026. https://doi.org/10.1093/aobpla/plt026. [59] Tjahjono AE, Kakizono T, Hayama Y, Nishio N, Nagai S. Isolation of resistant mutants against carotenoid biosynthesis inhibitors for a green alga Haematococcus pluvialis, and their hybrid formation by protoplast fusion for breeding of higher astaxanthin producers. J Ferment Bioeng 1994;77:352–7. [60] Hong ME, Choi HI, Kwak HS, Hwang S-W, Sung YJ, Chang WS, Sim SJ. Rapid selection of astaxanthin-hyperproducing Haematococcus mutant via azide-based colorimetric assay combined with oil-based astaxanthin extraction. Bioresour Technol 2018;267:175–81. [61] Rosa FF. Towards improvement of Haematococcus pluvialis cultures by cell sorting and auv mutagenesis. [Doctoral dissertation]. [62] Liu J, Huang Q. Screening of astaxanthin-hyperproducing Haematococcus pluvialis using Fourier transform infrared (FT-IR) and Raman microspectroscopy. Appl Spectrosc 2016;70:1639–48. [63] Liu J, Zhong Y, Sun Z, Huang J, Sandmann G, Chen F. One amino acid substitution in phytoene desaturase makes Chlorella zofingiensis resistant to norflurazon and enhances the biosynthesis of astaxanthin. Planta 2010;232:61–7. [64] An GH, Schuman DB, Johnson EA. Isolation of Phaffia rhodozyma mutants with increased astaxanthin content. Appl Environ Microbiol 1989;55:116–24. [65] Calo P, Velazquez JB, Sieiro C, Blanco P, Longo E, Villa TG. Analysis of astaxanthin and other carotenoids from several Phaffia rhodozyma mutants. J Agric Food Chem 1995;43:1396–9. [66] Bon JA, Leathers TD, Jayaswal RK. Isolation of astaxanthin-overproducing mutants of Phaffia rhodozyma. Biotechnol Lett 1997;19:109–12. [67] Pala´gyi ZS, Linka B, Papp T, Va´gv€olgyi CS. Isolation and characterization of Xanthophyllomyces dendrorhous mutants with altered carotenoid content. Acta Aliment 2006;35:223–8. [68] Meyer PS, du Preez JC, Kilian SG. Selection and evaluation of astaxanthin-overproducing mutants of Phaffia rhodozyma. World J Microbiol Biotechnol 1993;9:514–20. [69] Ang FS, Khaw SY, Few LL, See Too WC, Chew AL. Isolation of a stable astaxanthin-hyperproducing mutant of Xanthophyllomyces dendrorhous through random mutagenesis. Appl Biochem Microbiol 2019;55:255–63. [70] Kong M, Xu W, Wang H, Tang H, Zhang L. A rapid and simple strategy for the formation and isolation of astaxanthin high-yielding Xanthophyllomyces dendrorhous mutant variants. 7:6. [71] Kim YT, Ji K, Jeong TH. Improved production of astaxanthin of the Paracoccus haeundaensis by mutagenesis. FASEB J 2010;24:682.3. [72] Chatdumrong W, Yongmanitchai W, Limtong S, Worwattanamateekul W. Optimization of docosahexaenoic acid (DHA) production and improvement of astaxanthin content in a mutant Schizochytrium limacinum isolated from mangrove forest in Thailand. Kasetsart J 2007;41:324–34. [73] Chen Y, Li D, Lu W, Xing J, Hui B, Han Y. Screening and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis. Biotechnol Lett 2003;25(7):527–9. [74] Stachowiak B. Efficiency of selected mutagens in generating Xanthophyllomyces dendrorhous strains hyperproducing astaxanthin. Pol J Microbiol 2013;62:67–72. [75] Ukibe K, Katsuragi T, Tani Y, Takagi H. Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry. FEMS Microbiol Lett 2008;286:241–8. [76] Brehm-Stecher BF, Johnson EA. Isolation of carotenoid hyperproducing mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by flow cytometry and cell sorting. Methods Mol Biol 2012;898:207–17. [77] Emmerstorfer-Augustin A, Moser S, Pichler H. Screening for improved isoprenoid biosynthesis in microorganisms. J Biotechnol 2016;235:112–20. [78] Visser H, van Ooyen AJJ, Verdoes JC. Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous. FEMS Yeast Res 2003;4:221–31.

Metabolic engineering of astaxanthin pathway 175 [79] Park BJ, Kim BM, Shim SH, Kim JD, Lee CG. Enhancement of astaxanthin production of Haematococcus pluvialis by mutation. Microbiol Biotechnol Lett 2006;34(2):136–42. [80] Chun SB, Chin JE, Bai S, An G-H. Strain improvement of Phaffia rhodozyma by protoplast fusion. FEMS Microbiol Lett 1992;93:221–6. [81] Kajiwara S, Fraser PD, Kondo K, Misawa N. Expression of an exogenousisopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli. Biochem J 1997;324:421–6. [82] Farmer WR, Liao JC. Improving lycopene production in Escherichia coli by engineering metabolic control. Nat Biotechnol 2000;18(5):533–7. [83] Misawa N, Shimada H. Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts. J Biotechnol 1998;59(3):169–81. [84] Wang CW, Oh MK, Liao JC. Engineered isoprenoid pathway enhances astaxathin production in Escherichia coli. Biotechnol Bioeng 1999;62:235–41. [85] Albrecht M, Misawa N, Sandmann G. Metabolic engineering of the terpenoidbiosynthetic pathway of Escherichia coli for the production of the carotenoids ß-carotene and zeaxanthin. Biotechnol Lett 1999;21:791–5. [86] Mann V, Harker M, Pecker I, Hirschberg J. Metabolic engineering of astaxanthin production in tobacco flowers. Nat Biotechnol 2000;18:888–92. [87] Sandmann G, Albrecht M, Schnurr G, Knorzer O, Boger P. The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli. Trends Biotechnol 1999;17:233–7. [88] Schmidt-Dannert C. Engineering novel carotenoid in microorganisms. Curr Opin Biotechnol 2000;11:255–61. [89] Potrykus I. Gene transfer to cereals: an assessment. Nat Biotechnol 1990;8:535–42. [90] Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M. Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta 2009;229(4):873–83. [91] Kindle KL. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 1990;87:1228–32. [92] Hawkins RL, Nakamura M. Expression of human growth hormone by the eukaryotic alga, Chlorella. Curr Microbiol 1999;38:335–41. [93] Eric JE, Brown LM. Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea. Curr Genet 1991;19:317–21. [94] Jin ES, Polle JE, Melis A. Involvement of zeaxanthin and of the Cbrprotein in the repair of photosystem II from photoinhibition in the green alga Dunaliella salina. Biochem Biophys Acta 2001;1506:244–59. [95] Dunahay TG. Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. Biotechniques 1993;15:452–60. [96] Ten Lohuis MR, Miller DJ. Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. Plant J 1998;13:427–35. [97] Lumbreras V, Stevens DR, Purton S. Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J 1998;14:441–7. [98] Boynton JE, Gillham NW, Harris EH, Hoslerj P, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein T, Shark KB, Sanford JC. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 1988;240:1534–8. [99] Schiedlmeier B, Schmitt R, Muller W, Kirk MM, Gruber H, Mages W, Kirk DL. Nuclear transformation of Volvox carteri. Proc Natl Acad Sci U S A 1994;91:5080–4. [100] Hallmann A, Rappel A, Sumper M. Gene replacement by homologous recombination in the multicellular green alga Volvox carteri. Proc Natl Acad Sci U S A 1997;94:7469–74. [101] Apt KE, Kroth-Pancic PG, Grossman AR. Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol Gen Genet 1996;252:572–9. [102] Zaslavskaia LA, Lippmeier JC, Kroth PG, Grossman AR, Apt KE. Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. J Phycol 2000;36:379–86.

176 Chapter 9 [103] Brown LE, Sprecher SL, Keller LR. Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation. Mol Cell Biol 1991;11:2328–32. [104] Minoda A, Sakagami R, Yagisawa F, Kuroiwa T, Tanaka K. Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, Cyanidioschyzon merolae. Plant Cell Physiol 2004;45:667–71. [105] Geng D, Wang Y, Wang P, Li W, Sun Y. Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta). J Appl Phycol 2003;15:451–6. [106] Chow KC, Tung WL. Electrotransformation of Chlorella vulgaris. Plant Cell Rep 1999;18:778–80. [107] Sun Y, Yang ZY, Gao XS, Li QY, Zhang QQ, Xu ZK. Expression of foreign genes in Dunaliella by electroporation. Mol Biotechnol 2005;30:185–92. [108] Degui G, Yiqin W, Wenbin L, Yongru S. Transient expression of gus gene in Dunaliella salina. High Technol Lett 2002;2:35–9. [109] Henry EC, Meints RH. Recombinant viruses as transformation vectors of marine macroalgae. J Appl Phycol 1994;6:247–53. [110] Etten V, Meints RH. Giant viruses infecting algae. Annu Rev Microbiol 1999;53:447–94. [111] Cheney D, Metz B, Stiller J. Agrobacterium mediated genetic transformation in the macroscopic marine red alga Porphyra yezoensis. J Phycol 2001;37:11. [112] Kumar SV, Misquitta RW, Reddy VS, Rao BJ, Rajam MV. Genetic transformation of the green alga Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci 2004;166(3):731–8. [113] Kathiresan S, Chandrashekar A, Ravishankar GA, Sarada R. Agrobacterium mediated transformation in the green alga Haematococcus pluvialis (Chlorophyceae, Volvocales). J Phycol 2009;45:642–9. [114] Anila N, Chandrashekar A, Ravishankar GA, Sarada R. Establishment of Agrobacterium mediated genetic transformation in Dunaliella bardawil. Eur J Phycol 2011;46(1):36–44. [115] Cha TS, Yee W, Aziz A. Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris. World J Microbiol Biotechnol 2012;28:1771–9. [116] Rathod JP, Prakash G, Pandit R, et al. Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri. Photosynth Res 2013;118:141–6. [117] Hoshino T, Fujii R, Nakahara T. Overproduction of carotenoids in Thermus thermophilus. J Ferment Bioeng 1994;77:423–4. [118] Papp T, Velayos A, Barto´k T, Eslava AP, Va´gv€olgyi C, Iturriaga EA. Heterologous expression of astaxanthin biosynthesis genes in Mucor circinelloides. Appl Microbiol Biotechnol 2006;69(5):526–31. [119] Beyer P, Potrykus I. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 2000;287:303–5. [120] Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY, Hinchuliffe E, Adams JL, Silverstone AL, et al. Improving the nutritional value of golden rice through increased pro-vitamin A content. Nat Biotechnol 2005;23:482–7. [121] Sandmann G. Genetic manipulation of carotenoid biosynthesis: strategies, problems and achievements. Trends Plant Sci 2001;6:14–7. [122] Kajiwara S, Kakizono T, Saito T, Kondo K, Ohtani T, Nishio N, Nagai S, Misawa N. Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli. Plant Mol Biol 1995;29:343–52. [123] Ojima K, Breitenbach J, Visser H, Setoguchi Y, Tabata K, Hoshino T, van den Berg J, Sandmann G. Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a beta carotene 3-hydroxylase/4-ketolase. Mol Genet Genomics 2006;275:148–58. [124] Tramontin LRR, Kildegaard KR, Sudarsan S, Borodina I. Enhancement of Astaxanthin biosynthesis in oleaginous yeast Yarrowia lipolytica via microalgal pathway. Microorganisms 2019;7(10):472. [125] Park SY, Binkley RM, Kim WJ, Lee MH, Lee SY. Metabolic engineering of Escherichia coli for high-level astaxanthin production with high productivity. Metab Eng Sep 2018;49:105–15. [126] Harker M, Hirschberg J. Biosynthesis of keto-carotenoids in transgenic cyanobacteria expressing the algal gene for ß-C-4-oxygenase, crtO. FEBS Lett 1997;404:129–34. [127] Kildegaard KR, Adiego-Perez B, Domenech Belda D, Khangura JK, Holkenbrink C, Borodina I. Engineering of Yarrowia lipolytica for production of astaxanthin. Synth Syst Biotechnol 2017;2(4):287–94.

Metabolic engineering of astaxanthin pathway 177 [128] Miura Y, Kondo K, Saito T, Shimada H, Fraser P, Misawa N. Production of the carotenoid lycopene, beta-carotene, and astaxanthin in the food yeast Candida utilis. Appl Environ Microbiol 1998;64:1226–9. [129] Stalberg K, Lindgren O, Ek B, Hoglund AS. Synthesis of keto-carotenoids in the seed of Arabidopsis thaliana. Plant J 2003;36:771–9. [130] Jayaraj J, Devlin R, Punja Z. Metabolic engineering of novel keto-carotenoid production in carrot plants. Transgenic Res 2008;17:489–501. [131] Hasunuma T, Miyazawa S, Yoshimura S, Shinzaki Y, Tomizawa K, Shindo K, Choi SK, Misawa N, Miyake C. Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering. Plant J 2008;55:857–68. [132] Zhong YJ, Huang JC, Liu J, Li Y, Jiang Y, Xu ZF, Sandmann G, Chen F. Functional characterization of various algalcarotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis. J Exp Bot 2011;62(10):3659–69. https://doi.org/10.1093/jxb/err070. [133] Huang JC, Zhong YJ, Liu J, Sandmann G, Chen F. Metabolic engineering of tomato for high-yield production of astaxanthin. Metab Eng 2013;17:59–67. [134] Harada H, Maoka T, Osawa A, Hattan J, Kanamoto H, Shindo K, et al. Construction of transplastomic lettuce (Lactuca sativa) dominantly producingastaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids. Transgenic Res 2014;23(2):303–15. [135] Li S, Huang JC. Assessment of expression cassettes and culture media for different Escherichia coli strains to produce astaxanthin. Nat Prod Bioprospect 2018;8(5):397–403. [136] Lu Q, Bu YF, Liu JZ. Metabolic engineering of Escherichia coli for producing astaxanthin as the predominant carotenoid. Mar Drugs 2017;15(10):296. [137] Chou YL, Ko CY, Yen CC, Chen LO, Shaw JF. Multiple promoters driving the expression of astaxanthin biosynthesis genes can enhance free-form as taxanthin production. J Microbiol Methods 2019;160:20–8. [138] Fraser PD, Miura Y, Misawa N. In vitro characterization of astaxanthin biosynthetic enzymes. J Biol Chem 1997;272(10):6128–35. [139] Zhao M, Huang D, Zhang X, Koffas MAG, Zhou J, Deng Y. Metabolic engineering of Escherichia coli for producing adipic acid through the reverse adipate-degradation pathway. Metab Eng 2018;47:254–62. [140] Henke NA, Heider SAE, Peters-Wendisch P, Wendisch VF. Production of the marine carotenoid astaxanthin by metabolically engineered Corynebacterium glutamicum. Mar Drugs 2016;14(7):124. [141] Jin J, Wang Y, Yao M, Gu X, Li B, Liu H, et al. Astaxanthin over- production in yeast by strain engineering and new gene target uncovering. Biotechnol Biofuels 2018;11:230. [142] Zhou P, Ye L, Xie W, Lv X, Yu H. Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt. Appl Microbiol Biotechnol 2015;99(20):8419–28. [143] Zhou P, Xie W, Li A, Wang F, Yao Z, Bian Q, et al. Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in saccharomyces cerevisiae. Enzyme Microb Technol 2017;100:28–36. [144] Lin YJ, Chang JJ, Lin HY, Thia C, Kao YY, Huang CC, et al. Metabolic engineering a yeast to produce astaxanthin. Bioresour Technol 2017;245:899–905. [145] Galarza JI, Gimpel JA, Bertha VR, Vega OA, Henrı´quez V. Over-accumulation of astaxanthin in Haematococcus pluvialis through chloroplast genetic engineering. Algal Res 2018;31:291–7. [146] Leo´n R, Couso I, Ferna´ndez E. Metabolic engineering of ketocarotenoids biosynthesis in the unicelullar microalga Chlamydomonas reinhardtii. J Biotechnol 2007;130(2):143–52. [147] Perozeni F, Cazzaniga S, Baier T, Zanoni F, Zoccatelli G, Lauersen KJ, et al. Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii. Plant Biotechnol J 2020; https://doi.org/10.1111/pbi.13364. [148] Zhong YJ, Huang JC, Liu J, Li Y, Jiang Y, Xu ZF, Sandmann G, Chen F. Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis. J Exp Bot 2011;62(10):3659–69. [149] Mortimer CL, Misawa N, Ducreux L, Campbell R, Bramley PM, Taylor M, et al. Product stability and sequestration mechanisms in Solanum tuberosum engineered to biosynthesize high value ketocarotenoids. Plant Biotechnol J 2016;14(1):140–52.

178 Chapter 9 [150] Ralley L, Enfissi EM, Misawa N, Schuch W, Bramley PM, Fraser PD. Metabolic engineering of ketocarotenoid formation in higher plants. Plant J 2004;39(4):477–86. [151] Lu Y, Stegemann S, Agrawal S, Karcher D, Ruf S, Bock R. Horizontal transfer of a synthetic metabolic pathway between plant species. Curr Biol 2017;27(19):3034–41. [152] Fang N, Wang C, Liu X, Zhao X, Liu Y, Liu X, et al. Identification of eukaryotic enzymes compatible with tobacco bioreactor for astaxanthin synthesis. Chin Tob Sci 2019;40(1):9–16. [153] Enfissi EMA, Nogueira M, D’Ambrosio C, Stigliani AL, Giorio G, Misawa N, et al. The road to astaxanthin production in tomato fruit reveals plastid and metabolic adaptation resulting in an unintended high lycopene genotype with delayed over-ripening properties. Plant Biotechnol J 2019;17(8):1501–13. [154] Nogueira M, Enfissi EMA, Martinez Valenzuela ME, Menard GN, Driller RL, Eastmond PJ, Fraser PD. Engineering of tomato for the sustainable production of ketocarotenoids and its evaluation in aquaculture feed. Proc Natl Acad Sci U S A 2017;114(41):10876–81. [155] Bai C, Berman J, Farre G, Capell T, Sandmann G, Christou P, et al. Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity. Transgenic Res 2017;26(1):13–23. [156] Ha SH, Kim JK, Jeong YS, You MK, Lim SH, Kim JK. Stepwise pathway engineering to the biosynthesis of zeaxanthin, astaxanthin and capsanthin in rice endosperm. Metab Eng 2019;52:178–89. [157] Zhu Q, Zeng D, Yu S, Cui C, Li J, Li H, et al. From golden rice to a STARice: bioengineering astaxanthin biosynthesis in rice endosperm. Mol Plant 2018;11(12):1440–8. [158] Farre G, Perez-Fons L, Decourcelle M, Breitenbach J, Hem S, Zhu C, Capell T, Christou P, Fraser PD, Sandmann G. Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid. Transgenic Res 2016;25(4):477–89. [159] Campbell R, Morris WL, Mortimer CL, Misawa N, Ducreux LJ, Morris JA, Hedley PE, Fraser PD, Taylor MA. Optimising ketocarotenoid production in potato tubers: effect of genetic background, transgene combinations and environment. Plant Sci 2015;234:27–37. [160] Fujisawa M, Takita E, Harada H, Sakurai N, Suzuki H, Ohyama K, Shibata D, Misawa N. Pathway engineering of Brassica napus seeds using multiple key enzyme genes involved in ketocarotenoid formation. J Exp Bot 2009;60(4):1319–32. [161] Jia D, Fan L, Shen J, Qin S, Li F, Yuan Y. Genetic transformation of the astaxanthin biosynthetic genes bkt and crtR-B into apple tree to increase photooxidation resistance. Sci Hortic 2019;243:428–33. [162] Zhang C, Seow VY, Chen X, Too H-P. Multidimensional heuristic process for high-yield production of astaxanthin and fragrance molecules in Escherichia coli. Nat Commun 2018;9(1):1858. [163] Lee PC, Schmidt-Dannert C. Metabolic engineering towards biotechnological production of carotenoids in microorganisms. Appl Microbiol Biotechnol 2002;60:1–11. [164] Miura Y, Kondo K, Shimada H, Saito T, Nakamura K, Misawa N. Production of lycopene by the food yeast, Candida utilis that does not naturally synthesize carotenoid. Biotechnol Bioeng 1998;58:306–8. [165] Shimada H, Kondo K, Fraser PD, Miura Y, Saito T, Misawa N. Increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway. Appl Environ Microbiol 1998;64:2676–80. [166] Yamano S, Ishii T, Nakagawa M, Ikenaga H, Misawa N. Metabolic engineering for production of beta-carotene and lycopene in Saccharomyces cerevisiae. Biosci Biotech Bioch 1994;58:1112–4. [167] Lagarde D, Beuf L, Vermaas M. Increased production of zeaxanthin and other pigments by application of genetic engineering techniques to Synechocystis sp. strain PCC 6803. Appl Environ Microbiol 2000;66:64–72. [168] Chi S, He Y, Ren J, Su Q, Liu X, Chen Z, et al. Overexpression of a bifunctional enzyme, CrtS, enhances astaxanthin synthesis through two pathways in Phaffia rhodozyma. Microb Cell Fact 2015;14:90. [169] Sarnaik A, Nambissa V, Pandit R, Lali A. Recombinant Synechococcus elongatus PCC 7942 for improved zeaxanthin production under natural light conditions. Algal Res 2018;36:139–51. [170] Cunningham FX, Gantt E. Genes and enzymes of carotenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 1998;49:557–83.

Metabolic engineering of astaxanthin pathway 179 [171] Steinbrenner J, Linden H. Regulation of two carotenoid biosynthesis genes coding for phytoene synthase and carotenoid hydroxylase during stress-induced astaxanthin formation in the green alga Haematococcus pluvialis. Plant Physiol 2001;125(2):810–7. [172] Yan Y, Zhu YH, Jiang JG, Song DL. Cloning and sequence analysis of the phytoene synthase gene from a unicellular chlorophyte, Dunaliella salina. J Agric Food Chem 2005;53(5):1466–9. [173] Kathiresan S, Chandrashekar A, Ravishankar GA, Sarada R. Regulation of astaxanthin and its intermediates through cloning and genetic transformation of β-carotene ketolase in Haematococcu spluvialis. J Biotechnol 2015;196:33–41. [174] Vidhyavathi R, Venkatachalam L, Sarada R, Ravishankar GA. Regulation of carotenoid biosynthetic genes expression and carotenoid accumulation in the green alga Haematococcus pluvialisunder nutrient stress conditions. J Exp Bot 2008;59(6):1409–18. [175] Kim DK, Hong SJ, Bae JH, Yim N, Jin ES, Lee CG. Transcriptomic analysis of Haematococcus lacustris during astaxanthin accumulation under highirradiance and nutrient starvation. Biotechnol Bioprocess Eng 2011;16:698–705. [176] Scaife MA, Ma CA, Ninlayarn T, Wright PC, Armenta RE. Comparative analysis of β-carotene hydroxylase genes for astaxanthin biosynthesis. J Nat Prod 2012;75:1117–24. [177] Gao Z, Meng C, Gao H, Zhang X, Xu D, Su Y, Wang Y, Zhao Y, Ye N. Analysis of mRNA expression profiles of carotenogenesis and astaxanthin pro-duction of Haematococcus pluvialis under exogenous 2,4epibrassinolide (EBR). Biol Res 2013;46:201–6. [178] Meng CX, Teng CY, Jiang P, Qin S, Tseng CK. Cloning and characterization of β-carotene ketolase gene promoter in Haematococcus pluvialis. Acta Biochim Biophys Sin 2005;37(4):270–5. [179] Huang JC, Chen F, Sandmann G. Stress-related differential expression of multiple β-carotene ketolase genes in the unicellular green alga Haematococcus pluvialis. J Biotechnol 2006;122(2):176–85. [180] Tao L, Wilczek J, Odom JM, Cheng Q. Engineering a β-carotene ketolase for astaxanthin production. Metab Eng 2006;8(6):523–31. ´ ngeles VM. Enhancement of carotenoids biosynthesis in [181] Baldo FC, Inmaculada C, Leo´n R, Rodrı´guez H, A Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis. Appl Microbiol Biotechnol 2011;91:341–51. [182] Simon DP, Anila N, Gayathri K, Sarada R. Heterologous expression of β-carotene hydroxylase in Dunaliella salina by agrobacterium-mediated genetic transformation. Algal Res 2016;18:257–65. [183] Davison PA, Hunter CN, Horlon P. Overexpression of ß-carotene hydroxylase enhances stress tolerance in Arabidopsis. Nature 2002;418:203–6. [184] Cho DH, Jung YJ, Choi C-S, Lee HJ, Park JH, Dane F, Kang KK. Astaxantlhin production in transgenic Arabidopsis with chyB gene encoding ß-carotene hydroxylase. J Plant Biol 2008;58:58–63. [185] Romer S, Lubeck J, Kauder F, Steiger S, Adomat C, Sandmann G. Genetic engineering of a zeaxanthin-rich potato by antisense inactivation and cosuppression of carotenoid epoxidation. Metab Eng 2002;4:263–72. [186] Gerjets T, Sandmann G. Keto-carotenoid formation in transgenic potato. J Exp Bot 2006;57:3639–45. [187] Gerjets T, Sandmann M, Zhu C, Sandmann G. Metabolic engineering of ketocarotenoid biosynthesis in leaves and flowers of tobacco species. Biotechnol J 2007;2:1263–9. [188] Morris WL, Ducreux LJ, Fraser PD, Millam S, Taylor MA. Engineering ketocarotenoid biosynthesis in potato tubers. Metab Eng 2006;8:253–63. [189] Suzuki S, Nishihara M, Nakatsuka T, Misawa N, Ogiwara I, Yamamura S. Flower color alteration in Lotus japonicus by modification of the carotenoid biosynthetic pathway. Plant Cell Rep 2007;26:951–9. [190] Choi S, Matsuda S, Hoshino T, Peng X, Misawa N. Characterization of bacterial ß-carotene 3, 3-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adinorubin production by gene combination in Escherichia coli. Appl Microbiol Biotechnol 2006;72:1238–46. [191] Coll JM. Methodologies for transferring DNA into eukaryotic microalgae: a review. Span J Agric Res 2006;4 (4):316–30. [192] Dı´az-Santos E, De La Vega M, Vila M, Vigara J, Leo´n R. Efficiency of different heterologous promoters in the unicellular microalga Chlamydomonas reinhardtii. Biotechnol Prog 2013;29(2):319–28.

This page intentionally left blank

CHAPTER 10

Revealing mechanisms of algal astaxanthin production and bioengineering potential using multiomics Tim L. Jeffers and Melissa S. Roth Department of Plant and Microbial Biology, University of California, Berkeley, CA, United States

Abbreviations ABC Ax BKT CHX CHYB CRTISO CVDE/VDE CYP DGAT ER HXK LCYB LCYE LD MEP PDS PSY ROS TAG Zea βc

ATP-binding cassette astaxanthin β-carotene ketolase cycloheximide β-ring carotenoid hydroxylase prolycopene isomerase chlorophycean/plant-like violaxanthin deepoxidase cytochrome P450 diacylglycerol acyltransferase endoplasmic reticulum hexokinase lycopene β-ring cyclase lycopene ε-ring cyclase cytoplasmic lipid droplet methylerythritol-4-phosphate pathway phytoene desaturase phytoene synthase reactive oxygen species triacylglycerol zeaxanthin β-carotene

1 Introduction: Astaxanthin, a carotenoid for human health and industry Astaxanthin (Ax) is a blood-red, lipid-soluble ketocarotenoid and a pigment responsible for red coloration across nature. The red or pink hues of crustacean shells [1], the scales and muscles of salmon [2], and the feathers of flamingos and scarlet tanagers are due to astaxanthin deposition [3, 4]. However, most Ax in nature is synthesized de novo by bacteria, yeast, or algae. In Chlorophycean algae such as Haematococcus pluvialis and Chromochloris zofingiensis, Ax Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00010-6 Copyright # 2021 Elsevier Inc. All rights reserved.

181

182 Chapter 10 accumulates in cultures transitioning from green to orange-red pigmentation, often in response to environmental stress or excess carbon (Fig. 10.1A) [5, 6]. Like most carotenoids, Ax contains 40 carbon atoms. In addition, Ax has two terminal β-ionone rings, each containing a hydroxyl group and a ketone, giving each molecule four oxygen atoms (Fig. 10.1B). The two double bonds from the ketone groups extend the length of conjugation (13 double bonds) allowing Ax to be red, compared with its precursors, orange β-carotene (βc) and zeaxanthin (Zea) (11 double bonds) [7]. The extended conjugation and its terminal rings likely make Ax a potent antioxidant [8, 9]. Ax quenches several reactive oxygen species (ROS), including superoxide, singlet oxygen, and hydroxyl radical [9]. The ROS scavenging ability is more potent than other carotenoids and has been measured to be 100 times more potent than α-tocopherol, another lipophilic antioxidant [10, 11]. Because of its coloring and antioxidant properties, Ax has a wide variety of commercial applications from feed to cosmetics to nutraceuticals to pharmaceuticals. Historically, Ax has been primarily used commercially as a coloring agent in the aquaculture industry. Crustaceans and salmonids are fed Ax for enhanced pigmentation. In addition, Ax also enhances their reproduction, growth, survival, and stress tolerance [2]. In 2019, the global Ax market was estimated between USD 0.1 and 1 billion and is projected to increase over the next decade as the marketplace continues to expand into high-value nutraceuticals and pharmaceuticals [12, 13]. Traditionally, almost all Ax (>95%) in the market was synthetically derived from petrochemicals, raising issues of safety, sustainability, and consumer preference [2, 14, 15]. Synthetic Ax, which is used in animal feed, lacks esterification, has duller pigmentation, and is less stable compared with Ax produced by algae [16]. Additionally, the stereochemistry is different for 75% of molecules and may explain why synthetic Ax has substantially lower (>20 times) antioxidant potency than biologically produced Ax [17]. Only natural Ax has been approved for human consumption [17]. Numerous studies in animals and humans now show that Ax is broadly beneficial as an anticarcinogenic, cardioprotective, and neuroprotective molecule that modulates the immune system and enhances skin and eye health, in addition to several other positive outcomes reported [14, 18–21]. Unfortunately the high costs of naturally derived Ax are not competitive with its synthetic form [15]. Understanding natural accumulation of Ax in biological organisms could provide bioengineering strategies to meet the growing demand from biologically produced Ax. The biological sources of Ax span several taxa, but green algae are the most promising source for commercial naturally derived Ax [22]. While certain bacteria and yeast synthesize free Ax [23], algae also produce esterified Ax, which has enhanced stability and potency [24, 25]. Most organisms share homologous enzymes for the majority of the carotenoid biosynthesis pathway, but there are some unique features including enzyme fusion and nonhomologous ketolases whose function arose by convergent evolution for Ax production [26, 27]. In this review, we will focus on green algae, close relatives of vascular plants, where decades of research have unveiled the enzymatic pathways and mechanistic function of Ax and its relevance to photosynthesis [5, 6].

Fig. 10.1 Astaxanthin biosynthesis and accumulation strategies in green algae. (A) Astaxanthin accumulation in C. zofingiensis showing green cultures transitioning to orange-red after glucose application (Continued)

184 Chapter 10 Ax is considered a secondary carotenoid, as it is distinct from the primary carotenoids (lutein, βc, Zea, etc.) that physically associate with the photosynthetic apparatus and are essential for photosynthetic light harvesting and energy dissipation [5, 6]. All enzymes of the carotenoid biosynthesis pathway responsible for the synthesis of precursors to the keto-modified secondary carotenoids like Ax can be identified in most green algal genomes. In vascular plants and green algae, the carotenoid pathway is fed by isoprenoid precursors synthesized through the plastid-localized methylerythritol-4-phosphate pathway (MEP). The enzyme phytoene synthase (PSY) converts two geranylgeranyl diphosphates (C20) to the first carotenoid, phytoene. The rest of the pathway involves desaturations, cyclizations, and further modifications (Fig 10.1C) to make the several carotenoid products with various functions. The green algae H. pluvialis (also referred to as H. lacustris) and C. zofingiensis can accumulate large amounts of Ax outside of the chloroplast in cytoplasmic lipid droplets (LDs). In both organisms the two ketolations occur via β-carotene ketolase (BKT), a critical enzyme for Ax biosynthesis [28, 29] and the two hydroxylations via a β-ring hydroxylase, which is proposed to be either the canonical enzyme for Zea synthesis (CHYB) [30] or a distinct cytochrome P450 (CYP) specialized for secondary carotenogenesis [6, 28, 31]. In H. pluvialis, localization and chemical inhibitor studies show that these two reactions likely occur outside the chloroplast [32–34]. Furthermore, Ax is esterified at the hydroxyl groups by fatty acids, by an unidentified enzymatic esterase [28, 35]. Several reviews have summarized the physiological advantage secondary carotenoids provide algae in stressful conditions including the roles of Ax in ROS scavenging, as a sink for the excess oxygen and electrons produced in photoinhibitory conditions and as “molecular sunscreen” [5, 6, 16, 36, 37]. These two algae have clear differences in molecular strategies to accumulate Ax [35]. Most relevant, H. pluvialis accumulates up to 4% of its biomass as Ax compared with just 0.6% in C. zofingiensis [35]. However, C. zofingiensis grows faster and can reach higher biomass than H. pluvialis [16, 30, 38]. Therefore total C. zofingiensis Ax productivity (mg L
1 day
1) can be comparable with H. pluvialis [39, 40]. With these differences noted, engineering C. zofingiensis to accumulate more Ax, or enhancing biomass accumulation in H. pluvialis, could be effective strategies for dramatically increasing Ax productivity.

Fig.10.1, cont’d (60mM, 3 days). (B) Astaxanthin chemical formula. Fatty acids can be esterified to the two –OH groups on terminal rings. (C) Astaxanthin biosynthesis with C. zofingiensis annotated genes. Three strategy cells or bioengineering can use to increase astaxanthin production: Strategy (1) precursor enhancement by increasing methylerythritol-4-phosphate pathway (MEP) products and rate of lycopene production. Strategy (2) branch prioritization by enhancing β-cyclization, ketolation, and β-hydroxylases while deprioritizing ε-cyclization. Strategy (3) stabilization of astaxanthin through esterification and packaging into lipid droplets. The acyltransferase that esterifies astaxanthin has not been identified.

Multiomics and mechanisms of algal astaxanthin production 185 Recent comprehensive genomic resources for H. pluvialis, C. zofingiensis, and now over 100 algae are published, expanding the evolutionary context, and molecular repertoire biologists can assay to understand Ax accumulation [28, 41, 42]. From the perspective of multiomics experiments these genomes can facilitate, we provide an overview of the distribution of natural Ax accumulation in algae and describe and present a model of the systemic changes that occur in a microalgal cell accumulating Ax, including Ax regulation, biosynthesis, modification, and packaging. We describe three strategies for regulation of Ax accumulation, explore recent multiomics datasets to provide insights into H. pluvialis and C. zofingiensis biology, and frame outstanding questions in the field. We utilize multiomics datasets as platforms for potential gene discovery involved in Ax biosynthesis to expand the bioengineering prospects for making economically feasible, biologically derived Ax.

2 Evolution and distribution of astaxanthin accumulation in algae While Ax biosynthesis is persistent across many algal lineages, Ax accumulation occurs primarily within green algae (division Chlorophyta). The two most studied Ax accumulators are both within the class Chlorophyceae, H. pluvialis (order Chlamydomonadales formerly known as Volvocales) [43], and C. zofingiensis (order Sphaeropleales) [28, 44]. Other Chlamydomonadales Ax accumulators include the snow-dwelling algae Chlamydomonas nivalis, Chloromonas nivalis, and Sanguina spp. where Ax is photoprotective in cold, high light environments [24, 45, 46]. From the order Sphaeropleales, several species of Scenedesmus and Coelastrella produce Ax with varying degrees of accumulation [47]. Other Chlorophyte Ax accumulators include various species of Chlorella (Trebouxiophyceae) [47] and marine Prasinophytes [48]. The only known examples of vascular plants producing Ax are in the flowers of the genus Adonis (Tracheophyte); however, the mechanism of carotenoid ketolation is distinct and unrelated to that of green algae [49]. Considering more distant lineages, there are many species of the Eustigmatophyte Nannochloropsis (Heterokonta) that produce Ax [50]. Additionally, Ax production is noted in species of Euglena (Euglenozoa) [51] and dinoflagellates (Alveolata) [52]. All these organisms have close relatives lacking Ax accumulation, suggesting that Ax has been gained and/or lost several times. The list of Ax-producing organisms continues to grow due to the recent interest in carotenoid nutraceuticals and pharmaceuticals. In addition, the price of DNA sequencing has dropped rapidly, allowing for hundreds of full and draft algal genomes to be released in the past few years [42]. Many organisms have an ortholog of BKT in their genome making it possible that several additional algal species are able to accumulate Ax under unknown circumstances. For example, the genome of Dunaliella salina (Chlamydomonadales) also contains BKT, but there is no known condition where it produces or accumulates Ax [36]. Additionally, the genome of the reference model green alga Chlamydomonas reinhardtii (Chlamydomonadales) contains a BKT gene and is thought to produce Ax it its zygospore stage, but it is not known to

186 Chapter 10 accumulate in vegetative cells [53, 54]. Altogether, it is likely that Ax can occur in several more algae than currently reported. However, it is clear that the well-studied models of H. pluvialis and C. zofingiensis accumulate Ax more readily and at higher concentrations than most other organisms.

3 Comparative genomics of H. pluvialis and C. zofingiensis The first versions of the H. pluvialis and C. zofingiensis genomes and transcriptomes were both recently published [28, 41]. These resources will no doubt expand the technical capacity and number of hypotheses for algal researchers. However, it is important to note that genomes are rarely “finished,” and improvements in the genome assemblies may require iterative reprocessing [42]. For instance, the C. reinhardtii genome assembly has been updated several times since its original 2007 release [55]. The nucleotide assembly was improved by targeted resequencing of gaps and alignment with a genetic map [55]. Furthermore, gene models were improved (e.g., correcting the starting methionine) using updated prediction software that was trained on an expanded range of RNA-seq experiments [55]. The current draft nuclear genome of H. pluvialis (SAG 192.80) is scaffold based, meaning it is in large segments of grouped contigs separated by gaps [41]. Its genome size is 660Mbp in the current assembly, 32% of which is identified as repetitive elements, but the actual genome size is predicted to be 950 Mbp [41]. Strikingly, Haematococcus has the largest chloroplast genome on record (1.35 Mbp, strain UTEX 2505) [56, 57] and one of largest mitochondrial genomes (0.125Mbp) among photosynthetic eukaryotes [58]. Both organellar genomes contain long regions of repetitive elements [58]. Integrating homology- and transcriptome-based gene modeling was used to identify 18,545 protein-coding genes, which covered 59% of universally conserved genes by BUSCO analysis [41]. Further research and sequencing of the complicated H. pluvialis genome will improve the coverage of its assembled sequence and gene models and may potentially provide insight into how its genome size impacts physiology and Ax accumulation. In contrast to H. pluvialis, C. zofingiensis (SAG 211–14) has high-quality, near-complete assembled nucleus, chloroplast, and mitochondrial genomes [28]. Its genome size is 58Mbp, with only 6% repetitive sequence content, and 99% of sequences were assembled across 19 chromosomes [28]. Gene models generated by the software Augustus [59], based on the transcriptomes of 14 RNA-seq experiments, predicted C. zofingiensis has 15,274 protein coding genes with estimates of 92% of gene models represented from BUSCO analyses [28]. The published genomes and transcriptomes of H. pluvialis and C. zofingiensis enable comparative and functional analyses on astaxanthin accumulators.

Multiomics and mechanisms of algal astaxanthin production 187

3.1 Gene family expansions in astaxanthin biosynthesis The addition of annotated genomes from two Ax-accumulating algae allows for early comparative genomic strategies to gain insight into the genomic features that contributed to the emergence of Ax accumulation. For example, there is an expansion of putative extracellular matrix proteins in the multicellular green alga Volvox carteri as compared with unicellular C. reinhardtii, which likely contributes to the developmental complexity of V. carteri [60]. We examined gene family expansions in the microalgal Ax biosynthesis pathway to provide insight into strategies for Ax accumulation. We identified three proteins of phytoene synthase (HpPSY) using blastp in the proteome of H. pluvialis (red_GLEAN_10008751, red_GLEAN_10008753, and red_GLEAN_10019503) ([41], CDS sequences provided by the authors). Additionally, we also confirmed the two reported copies of DsPSY in the proteome of the βc accumulator D. salina (Dusal.0943s00001 and Dusal.0031s00032) [36]. In contrast, C. zofingiensis and C. reinhardtii have one copy [36]. The evolution of this gene duplication for the rate-limiting enzyme of carotenoid biosynthesis is hypothesized to be followed by subfunctionalization; one copy of PSY may be involved in primary carotenoid biosynthesis, while the other is activated for the production of secondary carotenoids to allow for high secondary carotenoid accumulation [36]. Therefore the duplication in PSY in H. pluvialis and D. salina may account for how these organisms can drastically increase carotenoid levels to 4% and 10% of total dry weight, respectively [36]. In contrast, C. zofingiensis (one CzPSY1) accumulates Ax by partitioning more secondary carotenoids from primary carotenoids but exhibits little difference in total carotenoid levels [35]. The BKT protein family also has expansions consistent with enhancing the capacity to accumulate Ax. Originally, three HpBKT proteins were identified in H. pluvialis [61]; however, , six HpBKTs were found using blast against the draft H. pluvialis annotations [41]. The genome of C. zofingiensis reveals two copies of CzBKT [28]. In contrast, there is only a single copy of BKT in low and non-Ax accumulators C. reinhardtii and D. salina. In C. zofingiensis, CzBKT1 is likely to be the primary enzyme involved in Ax accumulation; 17 independent mutants that were unable to accumulate Ax after high light or glucose treatment all had mutations in their CzBKT1 gene [28]. CzBKT2 may synthesize Ax in untested conditions or may serve another function in C. zofingiensis. In H. pluvialis the transcripts of the different HpBKTs are expressed at different levels and vary in response to distinct stressors [61]. Moreover, some HpBKT genes (e.g., red_GLEAN_10016356) are highly expressed in multiple treatments, while other homologs (e.g., red_GLEAN_10009496) have almost no detected expression under specific treatments [41]. While little is known of the benefit of having multiple BKTs or if they serve redundant or specialized conditions, the expansion of the gene family appears linked to the ability to accumulate ketocarotenoids.

188 Chapter 10 Several other carotenoid enzymes seem to have duplicated in H. pluvialis, whereas C. zofingiensis has been predicted to have single copies. Through homology to plant and other algal enzymes, we predict two copies of phytoene desaturase (HpPDS), ζ-carotene desaturase (HpZDS), prolycopene isomerase (HpCRTISO), and Zea epoxidase (HpZEP), but single members of ζ-carotene isomerase (HpZISO) and lycopene ε-cyclase (HpLCYE). Unlike C. zofingiensis, which has both a plant-like and Chlorophycean-like violaxanthin deepoxidase (CzVDE1 and CzCVDE1, respectively) [28, 62], we predict H. pluvialis has two HpCVDEs. Finally, we note expansion of lycopene β-cyclase (HpLCYB, three) and HpCHYB (possibly five), the latter of which we found was unlikely to have multiple paralogs in other algae. HpLCYB and HpCHYB expansions could contribute to increased production of β-carotenoids. These gene family expansions could be a generalized phenomenon associated with the large H. pluvialis genome. However, since the current number of predicted H. pluvialis protein-coding genes (18,545) is not substantially greater than algae with reduced genomes with low gene family expansions (e.g., C. zofingiensis, with 15,274 genes), it may suggest that evolutionary duplications enhance massive Ax accumulation in H. pluvialis.

4 Regulation of carotenoid biosynthesis for astaxanthin accumulation In H. pluvialis and C. zofingiensis, Ax accumulates in high abundance after various stresses or excess carbon treatment, conditions that also inhibit photosynthesis [6, 37, 63]. Ax does not normally participate in photosynthetic light harvesting (yet was recently discovered to associate with photosystems in red H. pluvialis cells [64]). However, Ax biosynthesis shares most of the same enzymes as the primary carotenoid biosynthesis pathway. Therefore the emergence of secondary carotenoid accumulation in algae requires a fundamentally different mRNA regulation of the carotenoid biosynthesis pathway from plants and algae that do not accumulate secondary carotenoids [36]. Here, we describe transcriptional regulation of secondary carotenoids, which unlike that of primary carotenoids, is frequently induced, while photosynthesis is downregulated [35, 36, 63]. Regulation of primary carotenoids has been previously reviewed [65, 66]. In plants and algae, transcript levels of particular carotenoid biosynthesis genes, with few exceptions, are often predictive of increases in carotenoid levels [67]. The transcriptional inhibitor, actinomycin D, substantially reduces total carotenoids by 75% in H. pluvialis, during inducing conditions, providing compelling evidence for their transcriptional regulation [68]. Furthermore, overexpressing a single gene of the carotenoid pathway, such as PSY and BKT, is frequently sufficient to increase total concentrations or alter ratios of types of carotenoids in a transgenic system, respectively [69, 70]. Therefore it is reasonable to interpret carotenoid gene expression patterns as predictive of actual secondary carotenoid abundance and composition. Here, we take advantage of this predictive relationship and analyze RNA-seq data to understand

Multiomics and mechanisms of algal astaxanthin production 189 the gene regulatory networks that lead to secondary carotenoid accumulation. We also discuss cases where posttranslational modifications might enhance Ax production and present a cellular model outlining known components of regulation of Ax accumulation.

4.1 Strategies to accumulate astaxanthin There are three ways in which a cell might accumulate large amounts of secondary carotenoids: (1) increasing total carotenoids and therefore also generating a larger pool of secondary carotenoids, (2) changing the prioritization of the carotenoid biosynthesis branches to increase the amount of secondary carotenoids and decrease the amount of primary carotenoids, and (3) increasing and stabilizing astaxanthin storage (Fig. 10.1C). These nonexclusive approaches can be achieved by altering expression and activities of different carotenoid pathway genes. The enhancement of Ax levels could be achieved, naturally or through genetic engineering, by (1.1) an increase in the pool of isoprenoid precursors, such as via the MEP pathway, which synthesizes geranylgeranyl diphosphate, (1.2) increased expression activity of phytoene biosynthesis and its four consecutive desaturations, (2.1) prioritization of the β-cyclization (via LCYB) branch over the ε-cyclization (via LCYE) branch that leads to lutein, (2.2) upregulation of direct Ax biosynthesis from βc, by BKT and CHYB, and (3) increasing accumulation of esterified Ax and minimizing turnover. Strategies (1.1) and (1.2) would cause an increase in total carotenoids resulting in a higher abundance of secondary carotenoids. Strategies (2.1) and (2.2) would divert primary carotenoids to secondary carotenoids leaving the total amount of carotenoids the same. Strategy (3) would stabilize Ax through esterification and packaging into LDs (see Sections 5.3 and 5.4) and prevent metabolic feedback inhibition of ketolation by free Ax. Either naturally or through bioengineering, employing all three strategies synergistically will provide the greatest accumulation of Ax. Analytical chemistry experiments and gene expression patterns of H. pluvialis and C. zofingiensis indicate that H. pluvialis may employ all three strategies to accumulate Ax, whereas C. zofingiensis most likely uses strategies (2) and (3) [35, 63]. This difference might result in significantly higher Ax content per biomass in H. pluvialis than in C. zofingiensis [16, 35]. A detailed time series carotenoid profiling after nitrogen deprivation of C. zofingiensis reveals a slight increase in total carotenoids but a dramatic change in proportion of primary and secondary carotenoids resulting in a loss of primary carotenoids and an increase in secondary carotenoids, 70% of which are Ax [35]. Consistent with these data, most genes in the MEP pathway and the following enzymes that synthesize and desaturate phytoene have little expression difference in Ax-inducing conditions, whereas enzymes that act on βc and Ax biosynthesis are upregulated, and CzLCYE1 is downregulated [28, 35, 63]. In contrast, H. pluvialis increases total carotenoid content 10-fold under the Ax-inducing condition, usually concurrent with an increase in expression of at least one of its HpPSYs and HpPDS [32, 41, 71, 72]. High temporal resolution analyses show that H. pluvialis first converts βc to Ax (phase I, 3h

190 Chapter 10 after induction), followed by new biosynthesis of βc to enhance total carotenoid levels (phase II, >6h) during high light stress [6, 31]. This example shows both primary carotenoid conversion (strategy 2.2) followed by new carotenoid biosynthesis (strategy 1 and 2.1) being involved in H. pluvialis accumulation. To improve understanding of regulation of the carotenoid pathway at the mRNA level, we generated a transcriptomic database of several published C. zofingiensis transcriptomes that were aligned to the published genome, including experiments from Ax-inducing conditions such as high light, glucose treatment, and nitrogen deprivation [28, 63, 73]. Specifically, we looked at the variation in carotenoid gene expression across these transcriptomes of C. zofingiensis (Fig. 10.2) [28, 63, 73]. Notably, CzBKT1 and CzCHYB1, enzymes that directly precede Ax biosynthesis, have some of the widest ranges of expressions (900 and 600 FPKM range, respectively) of the carotenoid pathway. The gene with the highest mRNA abundance was CzBKT1 after H2O2 treatment, followed by treatment with the singlet oxygen-generator Rose Bengal and followed by a nitrogen-deprivation time course (Fig. 10.2A) [28, 73]. CzBKT2, the duplicated member of the BKT family with unknown function, has much lower mRNA abundance than most of the carotenoid genes (and 6.5 mean lower expression than CzBKT1) but does appear to be induced by the Ax-inducing condition of H2O2. Because CzBKT1 expression is the highest in Ax-inducing conditions, and CzBKT1 is the only carotenoid biosynthesis pathway enzyme that exclusively produces secondary carotenoids, we used it as a reference to compare other carotenoid biosynthesis enzymes (Fig. 10.3B and D). We found that CzCHYB1 and CzLCYB1 reach similar FPKM levels and are positively correlated (comparing FPKMs) with CzBKT1 in Ax-inducing conditions (r¼0.46 and 0.72, respectively) (Fig. 10.2C), while CzLCYE1 is downregulated in Ax-inducing conditions and is negatively correlated with CzBKT1 (r ¼
0.36) (Fig. 10.2D). These results are consistent with C. zofingiensis differentially regulating the β- and ε-cyclization branches of the pathway. Notably, CzPSY1 expression does not continue to elevate as CzBKT1 expression continues to increase in most transcriptomes (Fig. 10.2B), which supports the analytical chemistry work that increasing phytoene production is not a prominent mechanism of Ax accumulation for C. zofingiensis [35]. Genes that are not part of the carotenoid biosynthesis that are highly correlated with CzBKT1 across these published transcriptomes are discussed in the succeeding text as potentially novel genes that may be involved in the regulation of Ax accumulation.

4.2 Integrating varied signals to lead to astaxanthin accumulation To change transcript levels within an organism, an environmental signal has to be perceived via a sensor that starts a relay of modifications often ending with a transcription factor (TF) that modulates expression. A potential complexity to untangling the regulation of Ax accumulation

500 250

CzCYP97A

CzCYP97C

CzNSY1

CzLCYE1

CzVDE1

CzZEP1

CzCVDE1

CzBKT1

CzBKT2

CzLCYB1

CzCHYB1

CzZDS1

CzCRTISO1

(A)

CzZISO1

CzPSY1

0 CzPDS1

FPKM (all transcriptomes)

750

Gene Name

400

CzBKT1

300 FPKM

CzPSY1 CzPDS1

200

CzZISO1

100

CzZDS1

0

CzCRTISO1

0

(B)

100

200 300 CzBKT1 FPKM

400

600

Gene Name CzBKT1

FPKM

400

CzLYB1 CzCHYB1

200

CzBKT2

0 0

(C)

100

200 300 CzBKT1 FPKM

400

400 Gene Name 300 FPKM

CzBKT1

200

CzLCYE1 CzCYP97A

100

CzCYP97C

0

(D)

0

100

200 300 CzBKT1 FPKM

400

Fig. 10.2 Transcriptional patterns of the carotenoid biosynthesis pathway in C. zofingiensis. (A) FPKMs of carotenoid pathway genes from several published C. zofingiensis transcriptomes (Continued)

192 Chapter 10 is that diverse signals, such as high light, nutrient deprivation, and glucose addition, modulate the carotenoid pathway [28, 35, 63, 74]. Therefore regulation of secondary carotenogenic gene expression could be the result of (1) several diverse signal transduction pathways intersecting with the same result of Ax upregulation (e.g., by modifications to a master regulator of carotenogenesis) and/or (2) various stressors causing similar stress responses in the cell that are directly perceived for Ax accumulation. There have been recent advances in understanding the signal transduction pathways that lead to Ax accumulation. C. zofingiensis is known to accumulate Ax in response to glucose [63], but recent work has shown that hexokinase1 mutants in C. zofingiensis are insensitive to Ax accumulation with glucose [75]. Because HXK is considered to be both an enzyme and glucose sensor in yeast and plants [75–77], these results suggest that Ax accumulation is HXK dependent [75]. Another study using hexokinase inhibitors and glucose analogs showed that hexokinase signaling is necessary for the upregulation of CzCHYB1 in a response that was distinct from glucose metabolism [78]. The application of plant hormones to H. pluvialis and C. zofingiensis cultures can modulate Ax abundance [40, 72], suggesting that conservation of hormone receptor pathways, across plants and green algae, may mediate Ax accumulation. Exploring other homologs of canonical environmental sensors discovered in plants, such as the nitrate transceptor for nitrogen deficiency or the blue light–responsive cryptochromes, may show evidence of environmental sensors for triggering Ax accumulation [79, 80]. Altogether, these studies support that Ax accumulation in algae could result from the intersection of diverse signal transduction pathways, which could provide new hypotheses on regulatory circuits that lead to Ax accumulation. In addition to diverse signal transduction pathways intersecting to cause Ax accumulation, various environmental stressors could cause similar cellular stress responses. Both ROS and overreduction of the plastoquinone (PQ) pool are influenced by high light, nutrient deprivation, salt, and low temperature and have been known to relay gene expression signals in other photosynthetic organisms [81]. They are similarly implicated in Ax accumulation

Fig.10.2, cont’d including a diverse array of treatments [28, 63, 73]. CzBKT1 and CzCHYB1 have the highest expression ranges in the pathway and reach the highest mRNA abundances. Highest transcript level of CzBKT1 and CzBKT2 is after H2O2 treatment. Color key from Fig. 10.1C. (B–D) Specific sections of the pathway plotted against CzBKT1 (red) expression. CzBKT1 is plotted against itself as a reference. Estimated linear model (line) and 95% confidence interval (transparent error bars) of each transcript FPKM as a function CzBKT1. H2O2 datapoints are removed to avoid skewing linear models. Color key from Fig. 10.1C. (B) Under most conditions, phytoene and lycopene synthesis enzymes (CzPSY1 to CzCRTISO1) are not induced with CzBKT1 nor at the same levels. (C) In β-cyclization branch, CzLCYB1 and CzCHYB1 are induced with near equal abundance as BKT1 expression in most cases. (D) ε-Cyclization transcripts are not induced or repressed (CzLCYE1) as CzBKT1 increases.

Multiomics and mechanisms of algal astaxanthin production 193

Fig. 10.3 Algal astaxanthin accumulation model. (A) Environmental signals are perceived by largely unknown receptors to alter transcription factor (TF) activity and carotenogenic gene expression. Translation may also be regulated, and certain enzymes are targeted to either chloroplast or endoplasmic reticulum (ER). In the magnified section (dashed box), we show β-carotene as a dominant plastidderived precursor of astaxanthin (solid line), although zeaxanthin may also be a major precursor in C. zofingiensis (dotted line). β-Carotene and zeaxanthin are synthesized in the chloroplast envelope (uncertain if either or both membranes) and exported to the ER, possibly by an ABC transporter. Ketolation, hydroxylation, and esterification most likely take place in ER membrane. Esterified astaxanthin is then deposited into budding lipid droplets (LD), which grow, pinch off, and travel to the cell periphery to serve as protectant against damaging light. The protein scaffold major lipid droplet protein (MLDP) may play an important role in LD size, number, and stabilization. The LD contains not only astaxanthin but also triacylglycerols.

of green algae [6]. For instance, blocking PQ reduction via the inhibitor DCMU prevents mRNA increases in PSY in high light in H. pluvialis [82]. Future research on additional carotenogenic enzymes and in multiple organisms including H. pluvialis, C. zofingiensis, and D. salina will help validate and distinguish Ax accumulation from other signaling pathways and the PQ redox state’s effect on carotenoid gene expression. The benefit of signal perception leading to Ax accumulation is that in many cases, particularly nutrient deprivation, it could allow carotenoid gene expression to be activated before ROS is generated. In other cases—high light, external ROS application—PQ reduction would be very rapid. Thus it would be possible for multiple signals and gene regulatory networks to work synergistically to quickly tune Ax accumulation.

194 Chapter 10

4.3 Strategies to find candidate transcription factors that initiate astaxanthin accumulation Transcriptional regulation of the carotenoid biosynthesis pathway is fundamental for Ax accumulation. However, there are few or no validations of specific TFs that bind directly to carotenogenic gene cis elements to activate or repress parts of the pathways for the biosynthesis of both primary and secondary carotenoids in plants and algae [83]. Genetic engineering of TFs can increase production of useful molecules, because they modulate several downstream genes [84]. For example, attenuating the expression of a TF that is downregulated upon nitrogen deprivation increased triacylglycerol (TAG) storage lipid productivity twofold in Nannochloropsis gaditana [85]. Using statistical correlation with Ax accumulation or carotenogenic transcripts, recent publications have proposed candidate TFs that could initiate Ax gene expression. A recent study in C. zofingiensis identifies 60 TFs including MYB (Cz10g24240) and Nin-like (Cz01g40030) genes, which are significantly induced by nitrogen deprivation and had high coexpression with several carotenogenic genes [35]. In H. pluvialis a de novo transcriptome assembly following Ax-inducing phytohormone treatments identifies 26 TF gene families important for salicylic and jasmonic acid and also includes MYB TFs [72]. Because a given environmental stress has a variety of metabolic responses, it is difficult to initially know whether the TF is regulating Ax accumulation, another nitrogen-deprivation response, or is a master regulator of several responses. Due to this inherent complexity of biological networks, it is important to employ more advanced computational strategies beyond correlation in a single condition to differentiate between master regulators of a process and a specific regulation of a metabolic pathway [86]. Applying this strategy to carotenogenic algae could yield new insights into regulation of carotenoid biosynthesis. One method to distinguish a generalized stress response from Ax accumulation per se is to conduct correlation analyses of the carotenogenic pathway across several transcriptomes. Similarly, comparative transcriptome studies in C. reinhardtii successfully determine the extent to which certain stresses overlap or are distinct in their mRNA response [87, 88]. We applied this approach by looking across several published C. zofingiensis transcriptomes to isolate the genes involved in Ax accumulation rather than specific nutrient stress responses. The correlation between CzBKT1 and a list of TFs generated by PlantTFDB v4.0 [89] revealed putative genes that could be involved in Ax accumulation in microalgae including bZIP and C3H family members (Table 10.1). One caveat of using correlation to identify candidate TFs is that correlation may not reveal all TFs that are necessary for a metabolic process. For example, constitutively expressed TFs may be modified very early after stress induction to initiate expression of several response genes, but would not regulate their own expression. Therefore, it is important to also consider the role of classical genetics strategies in unveiling novel TFs. However, upregulation of HpBKTs and

Multiomics and mechanisms of algal astaxanthin production 195 Table 10.1: C. zofingiensis transcription factors with high Pearson correlation to CzBKT1 (r≥0.85) across several published transcriptomes including a diverse array of treatments. Gene ID Cz10g24190 Cz06g16250 Cz04g06060 Cz10g15210 Cz06g16030 Cz17g02030 Cz05g16110 Cz02g34190 Cz10g12010 Cz04g21220

TF family

Correlation with CzBKT1(r)

bZIP C3H CO-like C3H bZIP B3 C3H ERF G2-like bZIP

0.92 0.91 0.89 0.88 0.87 0.86 0.86 0.86 0.86 0.85

subsequent Ax accumulation were found to be dependent on de novo protein translation in H. pluvialis, suggesting that a TF synthesized in response to stress might further enhance Ax production [68]. While correlation is a good approach to unveil putative TFs, genetic manipulation and further analyses are necessary to confirm roles and test whether TFs regulate carotenogenesis specifically or a range of metabolic pathways.

4.4 Posttranslational regulation of the carotenoid biosynthesis pathway Some environmental signals lead to de novo translation of carotenogenic enzymes at the induction of stress signals to accumulate Ax. For example, treating H. pluvialis with the transcriptional inhibitor actinomycin D or cytoplasmic translational inhibitor cycloheximide (CHX) prevents Ax accumulation in the Ax-inducing conditions of high light or the carbon source acetate [90, 91]. Intriguingly, when high concentrations of Fe2+, which creates ROS by Fenton chemistry, are added with acetate, Ax still accumulates despite treatment with CHX [91]. These results may suggest that specific conditions, perhaps involving rapid induction by ROS, can cause Ax accumulation by posttranslational modifications of already abundant proteins. Because H. pluvialis is predicted to have six copies of HpBKT [41], it is possible that some may be specifically activated posttranslationally to produce BKT in response to ROS, while others may be regulated by de novo protein translation in response to other nutrient stresses. Future experiments with CHX across several environmental stresses could reveal which induction treatments are posttranslationally regulated. Moreover, expanding this research to C. zofingiensis will test if these mechanisms of Ax accumulation are conserved. It is also worth noting that there are some conflicting data regarding whether chloramphenicol, a chloroplast translational inhibitor, also prevents Ax accumulation in H. pluvialis [90, 91]. Overall, using inhibitors could be a valuable area to improve understanding of posttranscriptional and posttranslational regulation of Ax accumulation in algae.

196 Chapter 10

5 Biosynthesis, modification, and packaging of astaxanthin in algae After translation in the cytoplasm, carotenogenic enzymes have to be exported to the appropriate organelles to allow for the biosynthesis, transport, esterification, and packaging of Ax to different compartments in the cell. This task is considerable because C40 carotenoids such as Ax are large, hydrophobic molecules, and they likely involve transport systems similar to those needed for other large lipids. Therefore, besides factors that modulate gene expression, identifying genes involved in Ax processing may equally inform bioengineering strategies to increase Ax productivity. In H. pluvialis, export of carotenoids from the chloroplast and esterification were shown to be necessary to drive the total accumulation of Ax [33]. In plants, several feedback mechanisms dependent on protein sublocalization, developmental requirements, metabolic flux, and surrounding environments regulate carotenoid biosynthesis (see review [83]). We propose a hypothetical model of the subcellular localization of Ax biosynthesis, based primarily on biochemical assays, organellar fractionation, and demonstrated or predicted protein localization (Fig. 10.3). This model shows Ax accumulation from its chloroplast precursors to esterification to deposition into the LD and can be summarized by the following steps: (1) The MEP and carotenoid pathways synthesize βc in the chloroplast; (2) βc is transported to the endoplasmic reticulum (ER), where two consecutive ketolations (by BKT) and two hydroxylations (by CHYB or an unknown CYP) form free Ax; (3) hydroxyl groups of Ax are esterified, potentially by a diacylglycerol acyltransferase (DGAT) [33]; and (4) esterified Ax is deposited into a LD forming in the ER membrane (Fig. 10.3) [92]. ER-localized steps of Ax biosynthesis from βc to canthaxanthin to Ax is considered the dominant order of reactions in H. pluvialis (Fig. 10.3) [93]. This has been demonstrated by in vitro assays that HpBKT preference for βc [94], transgenic experiments in carotenoid producing E. coli that show at least three HpBKT isoforms have been shown to ketolate βc at much higher efficiencies than Zea [61, 93, 95–97] and by inhibitors of Ax biosynthesis that show increases in βc or canthaxanthin but not Zea [31, 32]. The dominant pathway to Ax is still under debate in C. zofingiensis because an CzBKT1 can ketolate both βc and Zea as precursors in vitro [95], CzBKT1 mutants accumulate both βc and Zea [28, 29], and C. zofingiensis accumulates a larger fraction of intermediates to Ax biosynthesis [16, 35].

5.1 Synthesizing astaxanthin precursors in the chloroplast The MEP pathway precedes phytoene biosynthesis, and most of the carotenoid biosynthesis pathway is thought to occur in the chloroplast with membrane-bound enzymes. Proteomic studies in plants identify the majority of pathway enzymes in envelope membranes as opposed to thylakoid membranes [98, 99]. However, PSY is detected in the plastoglobuli in Arabidopsis thaliana [99], but not in the βc-rich plastoglobuli of D. bardawil [100]. Whether Ax is made

Multiomics and mechanisms of algal astaxanthin production 197 during accumulation from newly synthesized or recycled carotenoids may differ between organisms and could affect how it is formed. During Ax induction, H. pluvialis appears to synthesize Ax de novo, whereas C. zofingiensis could mainly recycle its primary carotenoids [35]. If Ax accumulates during photoinhibitory conditions in both algae, then it is possible that primary carotenoids released from degraded photosystems may be recycled into Ax. In all photosynthetic organisms a structural and biophysical understanding of the suborganellar localization and protein arrangement of the carotenoid biosynthetic metabolon is lacking, which partially inhibits the ability to make amino acid level edits to the pathway that would make carotenoid biosynthesis more efficient [99].

5.2 Exporting astaxanthin precursors from the chloroplast The last enzymatic steps that produce Ax (likely by CHYBs or CYPs and BKTs), postcyclization, likely occur outside the chloroplast (Fig. 10.3) [32]. Therefore βc (and/or potentially Zea in C. zofingiensis) must be exported from the chloroplast to the ER membrane, where ketolation, hydroxylation, and esterification likely take place. The mechanism of this transfer is unknown, but diffusion across the membrane seems unlikely given the large and hydrophobic nature of carotenoids. More likely a transporter protein is necessary to traffic the Ax precursor out of the chloroplast for the final steps of its biosynthesis (Fig. 10.3). It is possible that ATP-binding cassette (ABC) transporters export the hydrophobic βc out of the chloroplast. ABC transporters, an ancient protein family found throughout all kingdoms of life, evolved to transport a wide variety of substrates [101]. In plants, trigalactosyldiacylglycerol (TGD) proteins 1–3 form an ABC transporter complex to aid the trafficking of fatty acids from the ER to the plastid [101–104]. Recently, four ABC transporter components in C. zofingiensis were identified that were highly upregulated shortly after high light treatment, an Ax-inducing condition [28]. We confirmed that three ABC transporters transcripts (Cz05g17060, Cz09g27180, and Cz04g21110) maintain high correlation with CzBKT1 across several published transcriptomes (r¼0.86, 0.86, 0.78, respectively). Using the bioinformatic software DeepLoc [105], which can distinguish plastid versus ER localization, two (Cz09g27180 and Cz04g21110) are predicted to be plastid localized and one (Cz05g17060) is predicted to be lysosome and vacuole localized. In addition to a protein transport model, there could be contact or even fusion of the chloroplast and the ER, which could make carotenoid transfer feasible even in the absence of a transporter. Electron microscopy of green algae and plants shows continuity of chloroplast and ER membranes in some species [102, 106]. In plants the ER structure is dynamic, branches throughout the cell in a lace-like network, and has specific attachment domains with other organelle membranes like the chloroplast, allowing for short-distance transport of hydrophobic molecules [102, 107]. Recent work with trans-organellar complementation in A. thaliana suggests that these membranes are biochemically continuous, because nonpolar chemicals,

198 Chapter 10 including carotenoids, can transfer from the chloroplast to the ER without a transport protein [103, 108]. Future research in H. pluvialis and C. zofingiensis investigating how contact and spatial orientation of the ER and the chloroplast may change from green cells to Ax-accumulating cells will elucidate the potential roles of organelle structure in Ax biosynthesis. Understanding known mechanisms of carotenoid and lipid transport in other kingdoms of life may give insight into transport of carotenoids in algae. Because animals cannot make carotenoids de novo, carotenoids must be ingested and transported from their gut and deposited to other body parts. In animals cells, carotenoids including Ax combine with lipoproteins such as the surface membrane protein CD36 [109]. The scavenger receptor B (SCARB1) recognizes lipoproteins and transports them across cell membranes [110]. SCARB1 mutants in flies and canaries have lost the ability to transport carotenoids and thus are lacking coloration in, respectively, their eyes and feathers [111, 112]. In mammalian systems, it has also been hypothesized that the other scavenger receptors (including CD36), sterol transporters (NPC1L), and ABC transporter family proteins (ABCG5 and ABCB1) can transport carotenoids [110]. Intriguingly, most of these putative transporters have homologs in H. pluvialis and C. zofingiensis, but further research is necessary to confirm a role in algal carotenoid transport.

5.3 Final steps of astaxanthin biosynthesis and esterification likely occur at the ER The leading theory is that Ax is synthesized and esterified in the ER membrane of Ax accumulators. Early electron microscopy images of H. pluvialis show the first deposits of Ax are visible just outside the ribosome-rich ER near the nucleus, and these deposits accumulate with age [92]. Algal LDs are formed via enzymes associated with the ER in the same way that Ax deposits are formed, which offers the opportunity for them to develop simultaneously (Fig. 10.3) [113]. Fractionation of H. pluvialis into different suborganellar compartments shows that only ER-enriched fractions were able to synthesize Ax from βc in vitro, which indicates that HpBKTs and hydroxylases are localized in the ER [33]. Another study detects HpBKT both in the chloroplast and LD (they do not isolate ER), but only the extraplastidic LD fraction can synthesize Ax [34]. When we analyzed protein sequences with the software DeepLoc [105], we found three HpBKTs (red_GLEAN_10016156, red_GLEAN_10009496, and red_GLEAN_10016356) were predicted to be localized to the ER membrane and one (red_GLEAN_10003056) predicted to the plastid. In the absence of ER fractionation studies in C. zofingiensis, we ran DeepLoc to find that CzBKT1 and CzBKT2 were predicted to have a 97% likelihood of localizing to the ER membrane, while precursor enzymes CzPSY1 and CzLCYB1 were predicted likely to be membrane-bound plastid proteins. Because CHYB is upregulated in Ax-accumulating conditions and contributes to Ax biosynthesis in transgenic systems, it has been suggested to play a β-hydroxylation role in Ax biosynthesis in algae [30, 95], but CYPs have also been implicated [6, 28, 31]. The protein sequence of CzCHYB1 (manually updated from the public sequence based on transcriptome data [28]) and three putative HpCHYBs are predicted to the plastid, where they are thought to be

Multiomics and mechanisms of algal astaxanthin production 199 essential for Zea biosynthesis, a primary carotenoid involved in many types of photoprotection of photosystem II [114]. As CHYB is proposed to be the protein involved in secondary carotenoid hydroxylation as well, this localization may suggest that zeaxanthin is an important astaxanthin precursor or that there may be a mechanism where its localization changes upon induction of Ax accumulation. Another possibility is that an enzyme besides CHYB can hydroxylate canthaxanthin or βc in the ER. For instance, in land plants, CHYBs may not be the sole enzymes that hydroxylate β-ring carotenoids, since double knockout mutations of both AtCHYB isoforms in A. thaliana reduce, but do not eliminate, β-ring hydroxylated carotenoids [115]. A CYP enzyme is implicated as a hydroxylase in high-light treated H. pluvialis, since cotreatment with a CYP inhibitor prevents Ax accumulation, while canthaxanthin and echinenone levels increase [31]. Furthermore, one of the CzCYPs (Cz10g28330) induced by high light in C. zofingiensis [28] has high Pearson correlation with CzBKT1 (r¼0.85) and a 92% likelihood of ER-localization by DeepLoc. In both organisms, knockout genetics and localization studies of CHYB and candidate CYPs are necessary to validate the identity of the hydroxylase in Ax accumulation, its position in the cell, and how C. zofingiensis and H. pluvialis might differ in their accumulation strategies. In microalgae, large amounts of accumulated Ax are esterified to fatty acids in either mono- or diesterified forms (Fig. 10.3) [5, 6, 35]. This increase in hydrophobicity likely allows for deposition in TAG-filled LDs [6]. Ax esterification has only been observed in ER- or LD-associated fractions, suggesting that esterification occurs in these membranes [33, 34]. Furthermore, in vitro and in vivo experiments on H. pluvialis show that total Ax formation is enhanced by supplying either oleic (C16) or palmitic (C18) fatty acids to induce esterification [33]. Because esterification has a positive role inducing Ax formation, it could also be enhanced genetically to engineer higher Ax production. Moreover the esterified forms of Ax and other carotenoids could be more valuable commercially because of their potential improvements in stability, potency, and nutritional benefits as compared with free forms [25], although this needs further testing. The enzyme that esterifies Ax in microalgae remains unknown. In plants and animals, membrane-binding diacylglycerol acyltransferases (DGATs) esterify carotenoids and apocarotenoids [116], making them possible candidates of Ax esterification in algae. In H. pluvialis a chemical inhibitor of DGAT1 and DGAT2 decreases total Ax formation, providing evidence that these DGATs have roles in esterification in algae [33]. In C. zofingiensis, DGATs may also perform this role, and two DGAT2s (Cz11g21100 and Cz06g22030) have high gene expression correlation (r¼0.84, 0.77, respectively) with CzBKT1 across several published transcriptomes. Another acyltransferase (Cz02g29020) is also proposed to be an esterase based on upregulation after high light treatment [28], has very high correlation (r¼0.95) with CzBKT1, and is given a 70% likelihood of localization to the ER by DeepLoc. A recent study expressed this enzyme and 10 CzDGATs heterologously in a free astaxanthin-producing yeast strain, and could not detect esterification of astaxanthin by any of the enzymes, leaving the identity of the astaxanthin esterase still unknown [117].

200 Chapter 10

5.4 Formation of the astaxanthin-rich lipid droplet Esterified Ax is found in TAG-filled cytoplasmic LDs [118]. Generally, in eukaryotic organisms, LDs form in the membrane of an ER tubule from a localized site of TAG biosynthesis [119]. TAG lipids accumulate and expand in the membrane, causing a LD to eventually bud off the ER and travel through the cytoplasm, where it may be involved in interactions with other organelles or move through the cell via membrane trafficking (Fig. 10.3) [119,120]. The major lipid droplet protein (MLDP1) stabilizes mature TAG LDs in green algae [121–123], its expression is coordinated with Ax accumulation in C. zofingiensis [63], and it is the most abundant protein in the C. zofingiensis LD proteome [123]. While this evidence suggests MLDP1 may affect the size and number of Ax LDs, it needs to be experimentally confirmed in H. pluvialis and C. zofingiensis. There are several general questions of what factors, purely biophysical or initiated by proteins, drive LD formation and initiation in eukaryotes [119]. In Ax-accumulating algae the relationship between Ax biosynthesis and deposition of the carotenoid in the forming LD is also unknown. In both H. pluvialis and C. zofingiensis, the LDs accumulate near the cell periphery in Ax-inducing conditions [28,63,124]. However, how the LD movement may be activated and what membrane trafficking players could be involved remain unresolved questions in the field. Omics technologies can help provide insight into the connection between LD formation, Ax biosynthesis, and how Ax is oriented within the LD. A recent study on the proteome of isolated Ax-rich LDs of C. zofingiensis reveals 163 proteins, including 94 that are not shared by C. reinhardtii LDs [123]. Importantly, this proteome was devoid of carotenoid biosynthesis proteins, suggesting that Ax biosynthesis and esterification occur in the ER preceding LD formation in C. zofingiensis. Curiously the authors identify two abundant proteins that are homologs of mammalian retinol dehydrogenases, which are important for apocarotenoid processing [123]. These enzymes could play a role in Ax activity or stability in the LD and would be an interesting subject for future investigations. By contrast, HpBKT has been detected in the LD of H. pluvialis, and isolated LD fractions could produce Ax from βc, suggesting Ax biosynthesis continues after LD are secreted from ER [34]. A proteomics analysis of the H. pluvialis LD to compare with C. zofingiensis may reveal further divergent strategies of Ax biosynthesis between the two species.

5.5 Carotenoid and fatty acid biosynthesis: Coordination or competition? The similarities between secondary carotenoid biosynthesis and fatty acid biosynthesis are notable. Both processes are thought to have early precursors synthesized in chloroplast envelope membranes [83, 113]. When environmental stress represses photosynthesis, thylakoid lipids and primary carotenoids associated with photosystems are remobilized [35,63,125].

Multiomics and mechanisms of algal astaxanthin production 201 During this stress response, both fatty acids and carotenoid products are exported to the ER, undergo additional modifications, and ultimately end up in Ax-rich LDs. Because of these similarities, researchers have investigated whether these two pathways have regulatory or metabolic feedback with each other. In H. pluvialis, adding either a carotenoid or a fatty acid biosynthesis inhibitor decreases both Ax and fatty acids suggesting that these two pathways are coupled and coordinated by metabolic feedback [31, 33]. Supplementing with fatty acids during fatty acid inhibition reinstates Ax accumulation, providing further evidence for a metabolic feedback model [33]. Moreover, gene transcripts from each pathway in H. pluvialis were insensitive to the inhibitor from the other pathway, showing these two processes are not transcriptionally coordinated [33]. Interestingly the metabolic coordination was not found to be true in heterotrophically grown C. zofingiensis: chemically inhibiting fatty acid biosynthesis actually leads to an increase in total Ax compared with control treatments [126]. Recently, the increase in astaxanthin with fatty acid inhibition was also confirmed in photoautotrophic nitrogen starved cells [117]. These results suggest that Ax and fatty acids compete for carbon resources in heterotrophic C. zofingiensis, though more research needs to be conducted to see if the effect is the same in photoautotrophic conditions.

5.6 What happens to astaxanthin when red cells regreen? Both H. pluvialis and C. zofingiensis can revert to green pigmentation, and H. pluvialis can restore its vegetative growth stage after removal from the Ax-inducing treatment [63,127]. In H. pluvialis, total carotenoid and Ax levels per biomass decrease during recovery, which is attributed to dilution as the growth is recovered [127]. Precise time-resolved analytical chemistry would elucidate whether carotenoid composition, esterification, and localization change during recovery. However, it is interesting to note that during removal of the glucose Ax-inducing condition in C. zofingiensis, TAG levels decrease and transcripts of the ß-oxidation pathway for fatty acid degradation are upregulated, suggesting that the Ax-containing LD is targeted [63]. Mechanistically, degradation of the LD could be the result of lipolysis or lipophagy [120]. If the LD is actively degraded to support growth after stress recovery, it would be interesting to follow the fate of esterified Ax.

6 Conclusion: From mechanistic models to engineering strategies There are several missing pieces in our mechanistic knowledge of the regulation and life cycle of the Ax in an algal cell. Improving understanding of algal Ax accumulation will help increase economic viability of biologically derived Ax, and likewise the path to commercial production of natural Ax may also advance the knowledge of Ax regulation, biosynthesis, and accumulation. An integrated metabolic bioengineering approach synergistically targeting the three strategies (1) increasing flux through the pathway though additional precursors, (2)

202 Chapter 10 prioritizing the branch for Ax biosynthesis, and (3) stabilizing accumulated Ax through esterification and packaging into LDs and minimizing turnover will be most effective for improving Ax production. Multiomics insight of the system-wide transformation that allows H. pluvialis and C. zofingiensis to produce and store Ax expands the list of possible genes involved and offers novel gene targets for enhancing Ax production. Distinguishing Ax regulation per se from a generalized stress response will also be an essential contribution of multiomics analysis to engineering. While many of the conditions that cause Ax accumulation (e.g., nitrogen deprivation) also negatively impact growth and biomass [16,128], some treatments (e.g., glucose and high light) enhance both Ax accumulation and biomass [63,129]. Therefore it is possible to precisely engineer the simultaneous upregulation of Ax productivity and biomass. Aside from bioengineering Ax accumulators themselves, another bioengineering strategy is to produce Ax in organisms that do not naturally produce and/or accumulate it. This could be achieved, for example, by introducing the carotenogenic genes of H. pluvialis and C. zofingiensis into C. reinhardtii, cyanobacteria, and vascular plants [69,130,131]. Because these organisms have not evolved the machinery to export and store Ax, understanding the mechanistic model of Ax accumulation in the native systems will also enhance the engineering ability in nonnative organisms. The end goal of bioengineering Ax accumulation in microalgae is to meet the growing commercial demands for biologically derived Ax and increase the sustainability of carotenoid production. However, increasing productivity of natural Ax producers not only is a significant economic interest but also would lower the price of Ax for applications in human health and diseases as well as potentially replacing synthetic Ax and improving the feasibility of green technologies in aquaculture and other markets.

Acknowledgments We would like to thank Chao Bian of the Shenzhen Key Lab of Marine Genomics in China for providing us with the H. pluvialis genome annotations and FASTA format of all CDS [41]. We also thank Oliver Dautermann, Olga Gaidarenko, Krishna Niyogi, and Daniel Westcott for helpful discussion and comments on the manuscript. This material is based upon work supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, under Award Number DE-SC0018301.

References [1] Budd AM, Hinton TM, Tonks M, Cheers S, Wade NM. Rapid expansion of pigmentation genes in penaeid shrimp with absolute preservation of function. J Exp Biol 2017;220(22):4109–18. [2] Lim KC, Yusoff FM, Shariff M, Kamarudin MS. Astaxanthin as feed supplement in aquatic animals. Rev Aquac 2018;10(3):738–73. [3] Fox DL. Astaxanthin in the American flamingo. Nature 1955;175(4465):942–3. [4] Lopes RJ, Johnson JD, Toomey MB, Ferreira MS, Araujo PM, Melo-Ferreira J, et al. Genetic basis for red coloration in birds. Curr Biol 2016;26(11):1427–34.

Multiomics and mechanisms of algal astaxanthin production 203 [5] Solovchenko A, Neverov K. Carotenogenic response in photosynthetic organisms: a colorful story. Photosynth Res 2017;133(1–3):31–47. [6] Lemoine Y, Schoefs B. Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress. Photosynth Res 2010;106(1–2):155–77. [7] Mel
endez-Martı´nez AJ, Britton G, Vicario IM, Heredia FJ. Relationship between the colour and the chemical structure of carotenoid pigments. Food Chem 2007;101(3):1145–50. [8] Higuera-Ciapara I, F
elix-Valenzuela L, Goycoolea FM. Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr 2006;46(2):185–96. [9] Kogure K. Novel antioxidative activity of astaxanthin and its synergistic effect with vitamin E. J Nutr Sci Vitaminol 2019;65:S109–12. [10] Miki W. Biological functions and activities of animal carotenoids. Pure Appl Chem 1991;63(1):141–6. [11] Palozza P, Krinskyl NI. Astaxanthin and canthaxanthin are potent antioxidants in a membrane model. Arch Biochem Biophys 1992;297(2):291–5. [12] Natural astaxanthin market 2020: top countries data, growth factors, defination, business opportunity, trend, segmentation and forecast to 2026, The Express Wire; 2020. [Internet]. [cited 14 April 2020]. Available from: https://www.theexpresswire.com/pressrelease/Natural-Astaxanthin-Market-2020-Top-CountriesData-Growth-Factors-Defination-Business-Opportunity-Trend-Segmentation-and-Forecast-to-2026_ 10806761. [13] Astaxanthin market size, share global industry report, 2027, 1–76. [Internet]. [cited 16 April 2020]. Available from: https://www.grandviewresearch.com/industry-analysis/global-astaxanthin-market. [14] Fakhri S, Abbaszadeh F, Dargahi L, Jorjani M. Astaxanthin: a mechanistic review on its biological activities and health benefits. Pharmacol Res 2018;136:1–20. [15] Panis G, Carreon JR. Commercial astaxanthin production derived by green alga Haematococcus pluvialis: a microalgae process model and a techno-economic assessment all through production line. Algal Res 2016;18:175–90. [16] Liu J, Sun Z, Gerken H, Liu Z, Jiang Y, Chen F. Chlorella zofingiensis as an alternative microalgal producer of astaxanthin: biology and industrial potential. Mar Drugs 2014;12(6):3487–515. [17] Capelli B, Bagchi D, Cysewski GR. Synthetic astaxanthin is significantly inferior to algal-based astaxanthin as an antioxidant and may not be suitable as a human nutraceutical supplement. Forum Nutr 2013; 12(4):145–52. [18] Fakhri S, Aneva IY, Farzaei MH, Sobarzo-Sa´nchez E. The neuroprotective effects of astaxanthin: therapeutic targets and clinical perspective. Molecules 2019;24(14):2460. [19] Davinelli S, Melvang HM, Andersen LP, Scapagnini G, Nielsen ME. Astaxanthin from shrimp cephalothorax stimulates the immune response by enhancing IFN-γ, IL-10, and IL-2 secretion in splenocytes of Helicobacter pylori-infected mice. Mar Drugs 2019;17(7):382. [20] Kim SH, Kim H. Inhibitory effect of astaxanthin on oxidative stress-induced mitochondrial dysfunction–a mini-review. Nutrients 2018;10(9):1137. [21] Zhang L, Wang H. Multiple mechanisms of anti-cancer effects exerted by astaxanthin. Mar Drugs 2015; 13(7):4310–30. [22] Novoveska´ L, Ross ME, Stanley MS, Pradelles R, Wasiolek V, Sassi JF. Microalgal carotenoids: a review of production, current markets, regulations, and future direction. Mar Drugs 2019;17(11):640. [23] Katsumata T, Ishibashi T, Kyle D. A sub-chronic toxicity evaluation of a natural astaxanthin-rich carotenoid extract of Paracoccus carotinifaciens in rats. Toxicol Rep 2014;1:582–8. [24] Ambati RR, Moi PS, Ravi S, Aswathanarayana RG. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications. Mar Drugs 2014;12(1):128–52. [25] Bunea A, Socaciu C, Pintea A. Xanthophyll esters in fruits and vegetables. Not Bot Horti Agrobot ClujNapoca 2014;42(2):310–24. [26] Schmidt I, Schewe H, Gassel S, Jin C, Buckingham J, H€ umbelin M, et al. Biotechnological production of astaxanthin with Phaffia rhodozyma/Xanthophyllomyces dendrorhous. Appl Microbiol Biotechnol 2011; 89(3):555–71.

204 Chapter 10 [27] Ojima K, Breitenbach J, Visser H, Setoguchi Y, Tabata K, Hoshino T, et al. Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a β-carotene 3-hydroxylase/4-ketolase. Mol Genet Genomics 2006;275(2):148–58. [28] Roth MS, Cokus SJ, Gallaher SD, Walter A, Lopez D, Erickson E, et al. Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production. Proc Natl Acad Sci U S A 2017;114(21):E4296–305. [29] Ye Y, Huang JC. Defining the biosynthesis of ketocarotenoids in Chromochloris zofingiensis. Plant Divers 2020;42(1):61–6. [30] Han D, Li Y, Hu Q. Astaxanthin in microalgae: pathways, functions and biotechnological implications. Algae 2013;28(2):131–47. [31] Schoefs B, Rmiki NE, Rachadi J, Lemoine Y. Astaxanthin accumulation in Haematococcus requires a cytochrome P450 hydroxylase and an active synthesis of fatty acids. FEBS Lett 2001;500(3):125–8. [32] Gr€unewald K, Hagen C. β-Carotene is the intermediate exported from the chloroplast during accumulation of secondary carotenoids in Haematococcus pluvialis. J Appl Phycol 2001;13(1):89–93. [33] Chen G, Wang B, Han D, Sommerfeld M, Lu Y, Chen F, et al. Molecular mechanisms of the coordination between astaxanthin and fatty acid biosynthesis in Haematococcus pluvialis (Chlorophyceae). Plant J 2015;81(1):95–107. [34] Gr€unewald K, Hirschberg J, Hagen C. Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis. J Biol Chem 2001;276(8):6023–9. [35] Zhang Y, Shi M, Mao X, Kou Y, Liu J. Time-resolved carotenoid profiling and transcriptomic analysis reveal mechanism of carotenogenesis for astaxanthin synthesis in the oleaginous green alga Chromochloris zofingiensis. Biotechnol Biofuels 2019;12(1):287. [36] Pick U, Zarka A, Boussiba S, Davidi L. A hypothesis about the origin of carotenoid lipid droplets in the green algae Dunaliella and Haematococcus. Planta 2019;249(1):31–47. [37] Solovchenko AE. Recent breakthroughs in the biology of astaxanthin accumulation by microalgal cell. Photosynth Res 2015;125(3):437–49. [38] Chen JH, Liu L, Wei D. Enhanced production of astaxanthin by Chromochloris zofingiensis in a microplate-based culture system under high light irradiation. Bioresour Technol 2017;245(A):518–29. [39] Liu J, Mao X, Zhou W, Guarnieri MT. Simultaneous production of triacylglycerol and high-value carotenoids by the astaxanthin-producing oleaginous green microalga Chlorella zofingiensis. Bioresour Technol 2016;214:319–27. [40] Chen JH, Wei D, Lim PE. Enhanced coproduction of astaxanthin and lipids by the green microalga Chromochloris zofingiensis: selected phytohormones as positive stimulators. Bioresour Technol 2020;295122242. [41] Luo Q, Bian C, Tao M, Huang Y, Zheng Y, Lv Y, et al. Genome and transcriptome sequencing of the astaxanthin-producing green microalga, Haematococcus pluvialis. Genome Biol Evol 2019;11(1):166–73. [42] Blaby-Haas CE, Merchant SS. Comparative and functional algal genomics. Annu Rev Plant Biol 2019; 70(1):605–38. [43] Allewaert CC, Vanormelingen P, Proschold T, Gomez PI, Gonzalez MA, Bilcke G, et al. Species diversity in European Haematococcus pluvialis (Chlorophyceae, Volvocales). Phycologia 2015;54(6):583–98. [44] Fuc´ıkova´ K, Lewis LA. Intersection of Chlorella, Muriella and Bracteacoccus: resurrecting the genus Chromochloris Kol et Chodat (Chlorophyceae, Chlorophyta). Fottea 2012;12(1):83–93. [45] Remias D, L€utz-Meindl U, L€utz C. Photosynthesis, pigments and ultrastructure of the alpine snow alga Chlamydomonas nivalis. Eur J Phycol 2005;40(3):259–68. [46] Procha´zkova´ L, Leya T, Kr´ızˇkova´ H, Nedbalova´ L. Sanguina nivaloides and Sanguina aurantia gen. et spp. nov. (Chlorophyta): the taxonomy, phylogeny, biogeography and ecology of two newly recognised algae causing red and orange snow. FEMS Microbiol Ecol 2019;95(6) fiz064. [47] Minhas AK, Hodgson P, Barrow CJ, Sashidhar B, Adholeya A. The isolation and identification of new microalgal strains producing oil and carotenoid simultaneously with biofuel potential. Bioresour Technol 2016;211:556–65.

Multiomics and mechanisms of algal astaxanthin production 205 [48] Lopes Dos Santos A, Gourvil P, Rodrı´guez F, Garrido JL, Vaulot D. Photosynthetic pigments of oceanic Chlorophyta belonging to prasinophytes clade VII. J Phycol 2016;52(1):148–55. [49] Cunningham FX, Gantt E. Elucidation of the pathway to astaxanthin in the flowers of Adonis aestivalis. Plant Cell 2011;23(8):3055–69. [50] Lubia´n LM, Montero O, Moreno-Garrido I, Huertas IE, Sobrino C, Gonza´lez-Del Valle M, et al. Nannochloropsis(Eustigmatophyceae) as source of commercially valuable pigments. J Appl Phycol 2000; 12(3–5):249–55. [51] Grung M, Liaaen-Jensen S. Algal carotenoids; secondary carotenoids of algae; carotenoids in a natural bloom of Euglena sanguinea. Biochem Syst Ecol 1993;21(8):757–63. [52] Pandeirada MS, Craveiro SC, Daugbjerg N, Moestrup Ø, Domingues P, Calado AJ. Studies on Woloszynskioid dinoflagellates X: ultrastructure, phylogeny and colour variation in Tovellia rubescens n. sp. (Dinophyceae). J Eukaryot Microbiol 2019;66(6):937–53. [53] Lohr M, Im CS, Grossman AR. Genome-based examination of chlorophyll and carotenoid biosynthesis in Chlamydomonas reinhardtii. Plant Physiol 2005;138(1):490–515. [54] Lohr M. Carotenoids. In: Stern D, editor. The Chlamydomonas sourcebook: organellar and metabolic processes. 2nd ed. vol. 2. Oxford, UK: Elsevier; 2009. p. 807. [55] Blaby IK, Blaby-Haas CE, Tourasse N, Hom EFY, Lopez D, Aksoy M, et al. The Chlamydomonas genome project: a decade on. Trends Plant Sci 2014;19(10):672–80. [56] Bauman N, Akella S, Hann E, Morey R, Schwartz AS, Brown R, et al. Next-generation sequencing of Haematococcus lacustris reveals an extremely large 1.35-megabase chloroplast genome. Genome Announc 2018;6(12):e00181–18. [57] Smith DR. Haematococcus lacustris: the makings of a giant-sized chloroplast genome. AoB Plants 2018;10 (5):ply058. [58] Zhang X, Bauman N, Brown R, Richardson TH, Akella S, Hann E, et al. The mitochondrial and chloroplast genomes of the green alga Haematococcus are made up of nearly identical repetitive sequences. Curr Biol 2019;29(15):R736–7. [59] Stanke M, Sch€offmann O, Morgenstern B, Waack S. Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources. BMC Bioinformatics 2006;7:1–11. [60] Prochnik SE, Umen J, Nedelcu AM, Hallmann A, Miller SM, Nishii I, et al. Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri. Science 2010;329(5988):223–6. [61] Huang JC, Chen F, Sandmann G. Stress-related differential expression of multiple β-carotene ketolase genes in the unicellular green alga Haematococcus pluvialis. J Biotechnol 2006;122(2):176–85. [62] Li Z, Peers G, Dent RM, Bai Y, Yang SY, Apel W, et al. Evolution of an atypical de-epoxidase for photoprotection in the green lineage. Nat Plants 2016;2(10):16140. [63] Roth MS, Gallaher SD, Westcott DJ, Iwai M, Louie KB, Mueller M, et al. Regulation of oxygenic photosynthesis during trophic transitions in the green alga Chromochloris zofingiensis. Plant Cell 2019; 31(3):579–601. [64] Mascia F, Girolomoni L, Alcocer MJP, Bargigia I, Perozeni F, Cazzaniga S, et al. Functional analysis of photosynthetic pigment binding complexes in the green alga Haematococcus pluvialis reveals distribution of astaxanthin in photosystems. Sci Rep 2017;7(1):16319. [65] Llorente B, Martinez-Garcia JF, Stange C, Rodriguez-Concepcion M. Illuminating colors: regulation of carotenoid biosynthesis and accumulation by light. Curr Opin Plant Biol 2017;37:49–55. [66] Stanley L, Yuan YW. Transcriptional regulation of carotenoid biosynthesis in plants: so many regulators, so little consensus. Front Plant Sci 2019;10:1017. [67] Cazzonelli CI, Pogson BJ. Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci 2010;15(5):266–74. [68] Vidhyavathi R, Venkatachalam L, Kamath BS, Sarada R, Ravishankar GA. Differential expression of carotenogenic genes and associated changes in pigment profile during regeneration of Haematococcus pluvialis cysts. Appl Microbiol Biotechnol 2007;75(4):879–87.

206 Chapter 10 [69] Perozeni F, Cazzaniga S, Baier T, Zanoni F, Zoccatelli G, Lauersen KJ, et al. Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii. Plant Biotechnol J 2020;https://doi.org/10.1111/pbi.13364. ´ , Rodrı´guez H. Efficient heterologous transformation of Chlamydomonas [70] Couso I, Cordero BF, Vargas MA reinhardtii npq2 mutant with the zeaxanthin epoxidase gene isolated and characterized from Chlorella zofingiensis. Mar Drugs 2012;10(9):1955–76. [71] Wang B, Zarka A, Trebst A, Boussiba S. Astaxanthin accumulation in Haematococcus pluvialis (Chlorophyceae) as an active photoprotective process under high irradiance. J Phycol 2003;39(6):1116–24. [72] Gao Z, Li Y, Wu G, Li G, Sun H, Deng S, et al. Transcriptome analysis in Haematococcus pluvialis: astaxanthin induction by salicylic acid (SA) and jasmonic acid (JA). PLoS One 2015;10(10):e0140609. [73] Liu J, Sun Z, Mao X, Gerken H, Wang X, Yang W. Multiomics analysis reveals a distinct mechanism of oleaginousness in the emerging model alga Chromochloris zofingiensis. Plant J 2019;98(6):1060–77. [74] Mao X, Wu T, Sun D, Zhang Z, Chen F. Differential responses of the green microalga Chlorella zofingiensis to the starvation of various nutrients for oil and astaxanthin production. Bioresour Technol 2018;249:791–8. [75] Roth MS, Westcott DJ, Iwai M, Niyogi KK. Hexokinase is necessary for glucose-mediated photosynthesis repression and lipid accumulation in a green alga. Commun Biol 2019;2(1):347. [76] Li L, Sheen J. Dynamic and diverse sugar signaling. Curr Opin Plant Biol 2016;33:116–25. [77] Entian KD, Kopetzki E, Fr€ohlich KU, Mecke D. Cloning of hexokinase isoenzyme PI from Saccharomyces cerevisiae: PI transformants confirm the unique role of hexokinase isoenzyme PII for glucose repression in yeasts. Mol Gen Genet 1984;198(1):50–4. [78] Li Y, Huang J, Sandmann G, Chen F. Glucose sensing and the mitochondrial alternative pathway are involved in the regulation of astaxanthin biosynthesis in the dark-grown Chlorella zofingiensis (Chlorophyceae). Planta 2008;228(5):735–43. [79] Ho CH, Lin SH, Hu HC, Tsay YF. CHL1 functions as a nitrate sensor in plants. Cell 2009;138(6):1184–94. [80] Kottke T, Oldemeyer S, Wenzel S, Zou Y, Mittag M. Cryptochrome photoreceptors in green algae: unexpected versatility of mechanisms and functions. J Plant Physiol 2017;217:4–14. [81] Rosso D, Bode R, Li W, Krol M, Saccon D, Wang S, et al. Photosynthetic redox imbalance governs leaf sectoring in the Arabidopsis thaliana variegation mutants immutans, spotty, var1, and var2. Plant Cell 2009;21(11):3473–92. [82] Steinbrenner J, Linden H. Regulation of two carotenoid biosynthesis genes coding for phytoene synthase and carotenoid hydroxylase during stress-induced astaxanthin formation in the green alga Haematococcus pluvialis. Plant Physiol 2001;125(2):810–7. [83] Wurtzel ET. Changing form and function through carotenoids and synthetic biology. Plant Physiol 2019;179 (3):830–43. [84] Wang N, Guan B, Kong Q, Sun H, Geng Z, Duan L. Enhancement of astaxanthin production from Haematococcus pluvialis mutants by three-stage mutagenesis breeding. J Biotechnol 2016;236:71–7. [85] Ajjawi I, Verruto J, Aqui M, Soriaga LB, Coppersmith J, Kwok K, et al. Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator. Nat Biotechnol 2017;35 (7):647–52. [86] Marshall-Colo´n A, Kliebenstein DJ. Plant networks as traits and hypotheses: moving beyond description. Trends Plant Sci 2019;24(9):840–52. [87] Schmollinger S, M€uhlhaus T, Boyle NR, Blaby IK, Casero D, Mettler T, et al. Nitrogen-sparing mechanisms in Chlamydomonas affect the transcriptome, the proteome, and photosynthetic metabolism. Plant Cell 2014;26(4):1410–35. [88] Blaby IK, Blaby-Haas CE, P
erez-P
erez ME, Schmollinger S, Fitz-Gibbon S, Lemaire SD, et al. Genome-wide analysis on Chlamydomonas reinhardtii reveals the impact of hydrogen peroxide on protein stress responses and overlap with other stress transcriptomes. Plant J 2015;84(5):974–88. [89] Jin J, Tian F, Yang DC, Meng YQ, Kong L, Luo J, et al. Plant TFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Res 2017;45(D1):D1040–5. [90] Brinda BR, Sarada R, Kamath BS, Ravishankar GA. Accumulation of astaxanthin in flagellated cells of Haematococcus pluvialis – cultural and regulatory aspects. Curr Sci 2004;87:1290–5.

Multiomics and mechanisms of algal astaxanthin production 207 [91] Kobayashi M, Kakizono T, Nagai S. Enhanced carotenoid biosynthesis by oxidative stress in acetate-induced cyst cells of a green unicellular alga, Haematococcus pluvialis. Appl Environ Microbiol 1993;59(3):867–73. [92] Lang NJ. Electron microscopic studies of extraplastidic astaxanthin in Haematococcus. J Phycol 1968; 4(1):12–9. [93] Lohr M. Carotenoid metabolism in phytoplankton. In: Roy S, Llewellyn C, Skarstad Egeland E, Johnsen G, editors. Phytoplankton pigments: characterization, chemotaxonomy and applications in oceanography. Cambridge, UK: Cambridge University Press; 2011. p. 113–65. [94] Fraser PD, Shimada H, Misawa N. Enzymic confirmation of reactions involved in routes to astaxanthin formation, elucidated using a direct substrate in vitro assay. Eur J Biochem 1998;252(2):229–36. [95] Huang J, Zhong Y, Sandmann G, Liu J, Chen F. Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants. Planta 2012;236(2):691–9. [96] Breitenbach J, Misawa N, Kajiwara S, Sandmann G. Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus pluvialis. FEMS Microbiol Lett 1996;140(2–3):241–6. [97] Zhong Y-J, Huang J-C, Liu J, Li Y, Jian Y, Xu Z-F, et al. Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis. J Exp Bot 2011;62(10):3659–69. [98] Shumskaya M, Quinlan RF, Wurtzel ET. Elucidating carotenoid biosynthetic enzyme localization and interactions using fluorescent microscopy. Methods Mol Biol 2020;2083:223–34. [99] Shumskaya M, Wurtzel ET. The carotenoid biosynthetic pathway: thinking in all dimensions. Plant Sci 2013;208:58–63. [100] Davidi L, Levin Y, Ben-Dor S, Pick U. Proteome analysis of cytoplasmatic and plastidic β-carotene lipid droplets in Dunaliella bardawil. Plant Physiol 2015;167(1):60–79. [101] Roston RL, Gao J, Murcha MW, Whelan J, Benning C. TGD1, -2, and -3 proteins involved in lipid trafficking form ATP-binding cassette (ABC) transporter with multiple substrate-binding proteins. J Biol Chem 2012;287(25):21406–15. [102] Wang Z, Benning C. Chloroplast lipid synthesis and lipid trafficking through ER-plastid membrane contact sites. Biochem Soc Trans 2012;40(2):457–63. [103] Mehrshahi P, Johnny C, Della PD. Redefining the metabolic continuity of chloroplasts and ER. Trends Plant Sci 2014;19(8):501–7. [104] Fan J, Zhai Z, Yan C, Xu C. Arabidopsis trigalactosyldiacylglycerol5 interacts with TGD1, TGD2, and TGD4 to facilitate lipid transfer from the endoplasmic reticulum to plastids. Plant Cell 2015;27(10):2941–55. [105] Almagro Armenteros JJ, Sønderby CK, Sønderby SK, Nielsen H, Winther O. DeepLoc: prediction of protein subcellular localization using deep learning. Bioinformatics 2017;33(21):3387–95. [106] McLean B, Whatley JM, Juniper BE. Continuity of chloroplast and endoplasmic reticulum membranes in Chara and Equisetum. New Phytol 1988;109(1):59–65. [107] Sparkes IA, Frigerio L, Tolley N, Hawes C. The plant endoplasmic reticulum: a cell-wide web. Biochem J 2009;423(2):145–55. [108] Mehrshahi P, Stefano G, Andaloro JM, Brandizzi F, Froehlich JE, Della PD. Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts. Proc Natl Acad Sci U S A 2013;110(29):12126–31. [109] Toews DPL, Hofmeister NR, Taylor SA. The evolution and genetics of carotenoid processing in animals. Trends Genet 2017;33(3):171–82. [110] Reboul E. Mechanisms of carotenoid intestinal absorption: where do we stand? Nutrients 2019;11(4):838. [111] Toomey MB, Lopes RJ, Arau´jo PM, Johnson JD, Gazda MA, Afonso S, et al. High-density lipoprotein receptor SCARB1 is required for carotenoid coloration in birds. Proc Natl Acad Sci U S A 2017;114 (20):5219–24. [112] Kiefer C, Sumser E, Wernet MF, Von Lintig J. A class B scavenger receptor mediates the cellular uptake of carotenoids in Drosophila. Proc Natl Acad Sci U S A 2002;99(16):10581–6. [113] Zienkiewicz K, Du Z-Y, Ma W, Vollheyde K, Benning C. Stress-induced neutral lipid biosynthesis in microalgae – molecular, cellular and physiological insights. Biochim Biophys Acta 2016;1861(9 Pt B):1269–81.

208 Chapter 10 [114] Davison PA, Hunter CN, Horton P. Overexpression of β-carotene hydroxylase enhances stress tolerance in Arabidopsis. Nature 2002;418(6894):203–6. [115] Tian L, Magallanes-Lundback M, Musetti V, Della PD. Functional analysis of β- and ε-ring carotenoid hydroxylases in Arabidopsis. Plant Cell 2003;15(6):1320–32. [116] Ariizumi T, Kishimoto S, Kakami R, Maoka T, Hirakawa H, Suzuki Y, et al. Identification of the carotenoid modifying gene PALE YELLOW PETAL 1 as an essential factor in xanthophyll esterification and yellow flower pigmentation in tomato (Solanum lycopersicum). Plant J 2014;79(3):453–65. [117] Zhang Y, Ye Y, Ding W, Mao X, Li Y, Gerken H, et al. Astaxanthin is ketolated from zeaxanthin independent of fatty acid synthesis in Chromochloris zofingiensis. Plant Physiol 2020;183(3):883–97. https://doi.org/ 10.1104/pp.20.00325. [118] Peled E, Leu S, Zarka A, Weiss M, Pick U, Khozin-Goldberg I, et al. Isolation of a novel oil globule protein from the green alga Haematococcus pluvialis (chlorophyceae). Lipids 2011;46(9):851–61. [119] Walther TC, Chung J, Farese RV. Lipid droplet biogenesis. Annu Rev Cell Dev Biol 2017;33(1):491–510. [120] Huang S, Jiang L, Zhuang X. Possible roles of membrane trafficking components for lipid droplet dynamics in higher plants and green algae. Front Plant Sci 2019;10:207. [121] Moellering ER, Benning C. RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii. Eukaryot Cell 2010;9(1):97–106. [122] Tsai CH, Zienkiewicz K, Amstutz CL, Brink BG, Warakanont J, Roston R, et al. Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii. Plant J 2015;83 (4):650–60. [123] Wang X, Wei H, Mao X, Liu J. Proteomics analysis of lipid droplets from the oleaginous alga Chromochloris zofingiensisreveals novel proteins for lipid metabolism. Genomics Proteomics Bioinforma 2019;17 (3):260–72. [124] Ota S, Morita A, Ohnuki S, Hirata A, Sekida S, Okuda K, et al. Carotenoid dynamics and lipid droplet containing astaxanthin in response to light in the green alga Haematococcus pluvialis. Sci Rep 2018;8 (1):5617. [125] Bar E, Rise M, Vishkautsan M, Arad S. (Malis). Pigment and structural changes in Chlorella zofingiensis upon light and nitrogen stress. J Plant Physiol 1995;146(4):527–34. [126] Zhang Z, Sun D, Mao X, Liu J, Chen F. The crosstalk between astaxanthin, fatty acids and reactive oxygen species in heterotrophic Chlorella zofingiensis. Algal Res 2016;19:178–83. [127] Chekanov K, Lukyanov A, Boussiba S, Aflalo C, Solovchenko A. Modulation of photosynthetic activity and photoprotection in Haematococcus pluvialis cells during their conversion into haematocysts and back. Photosynth Res 2016;128(3):313–23. [128] Zhang Z, Huang JJ, Sun D, Lee Y, Chen F. Two-step cultivation for production of astaxanthin in Chlorella zofingiensis using a patented energy-free rotating floating photobioreactor (RFP). Bioresour Technol 2017;224:515–22. [129] Imaizumi Y, Nagao N, Yusoff FM, Taguchi S, Toda T. Estimation of optimum specific light intensity per cell on a high-cell-density continuous culture of Chlorella zofingiensis not limited by nutrients or CO2. Bioresour Technol 2014;162:53–9. [130] Hasunuma T, Takaki A, Matsuda M, Kato Y, Vavricka CJ, Kondo A. Single-stage astaxanthin production enhances the nonmevalonate pathway and photosynthetic central metabolism in Synechococcus sp. PCC 7002. ACS Synth Biol 2019;8(12):2701–9. [131] Hasunuma T, Miyazawa SI, Yoshimura S, Shinzaki Y, Tomizawa KI, Shindo K, et al. Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering. Plant J 2008;55(5):857–68.

CHAPTER 11

Astaxanthin production from Haematococcus pluvialis by using illuminated photobioreactor Yiu-Hang Hoa, Yee-Keung Wongb, and Ambati Ranga Raoc a

School of Engineering, The Hong Kong University of Science and Technology, Hong Kong, China Episteme Company Limited, Hong Kong, China cCentre of Excellence, Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research (Deemed to be University), Vadlamudi, Guntur, Andhra Pradesh, India

b

Abbreviations BBM BG-11 CO2 H. pluvialis HCO32 IPBRs LEDs PBRs UV UV-VIS

bold basal medium blue-green 11 medium carbon dioxide Haematococcus pluvialis bicarbonate illuminated photobioreactors light-emitting diodes photobioreactors ultraviolet ultraviolet-visible spectroscopy

1 Introduction Haematococcus pluvialis (H. pluvialis) is the most abundant microalgal source of natural astaxanthin [1–4]. When H. pluvialis is exposed to an oxidative stress environment, astaxanthin accumulation occurs in the algal cells. Astaxanthin production is a cell-protective mechanism against long-term oxidative stress in the encysted stage of H. pluvialis [3]. Astaxanthin provides the neutralizing effect against singlet oxygen and exhibits a powerful scavenging ability for free radicals [5]. Due to its powerful antioxidative ability, astaxanthin is applied as a nutraceutical and pharmaceutical agents in commercial applications [6,7]. Photobioreactors (PBRs) currently are a promising approach to achieve high productivity of the H. pluvialis biomass and astaxanthin pigment. PBRs provide a highly controlled environmental condition, including lighting, carbon dioxide, and temperature control. Among this the illuminated photobioreactors (PBRs) are effective for employing the two-stage process for biomass and astaxanthin Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00030-1 Copyright # 2021 Elsevier Inc. All rights reserved.

209

210 Chapter 11 production. PBRs are crucial in the scaling-up process from laboratory to commercial production. An efficient design of the PBR system and production plant, together with the investigation for production procedures, could help to overcome the current difficulties such as low production rate, complexity in regulating physical and chemical factors, complicated working processes, and contamination issues. Use of PBRs for biomass and astaxanthin production can help to overcome the hurdles often confronted in raceway pond mediated production of biomass. The characteristics, design, scaling-up process for the PBRs, and commercial production overview have been discussed in this chapter.

2 Comparison between the closed and open system The microalgae cultivation system generally separated into an open or closed system. Each system provides different environmental conditions and culture volume that greatly affects the biomass productivities of H. pluvialis [6,8,9]. The open pond is a typical open system to be conducted in a natural lake, artificial ponds, or trench under the exposed condition with large culture volume. Raceway ponds can be set up or purchased by the production companies [10,11]. The raceway pond is a recirculation system with the closed-loop channel. The microalgal cells and nutrients are circulated inside the channel by pumping and by paddle wheels. Mixing with rotating arms helps to minimize the optically dark zone inside the raceway pond [12,13]. Although raceway ponds are useful to cultivate a large volume of the algal culture, huge space required, weather, water evaporation, and contamination create the difficulties to produce astaxanthin. Based on the aforementioned restriction, it leads to the development of the closed system for microalgal cultivation. Various types of close system PBRs have been developed, which including closed system, bubble column, airlift, and tubular. Closed system photobioreactors (PBRs) promote microalgae productivity. PBRs provide highly controlled environmental conditions, reduce bacterial contamination, and can carry out either batch or continuous cultivation (Table 11.1). In the current development of PBR technology, photosynthetic efficiency always affected the biomass production of H. pluvialis. The balance between cell density, light quality, light penetration through the reactor’s materials, and mixing properties are a popular research area. Inefficient lighting is the primary cause for increasing cultivation cost and lowering the microalgae productivity. When compared with open ponds, PBRs have a lower risk of contamination, however with a higher investment, operation, and maintenance cost [9,10].

3 Physical and chemical configuration of the illuminated photobioreactors The growth of H. pluvialis requires light, carbon source, water, macro- and micronutrient, and trace elements. Artificial culturing of H. pluvialis inside the IPBRs generally includes several physical and chemical parameters that need to be controlled, including light supply, gaseous input or output, temperature, and culture medium nutrients. Physical and chemical parameters decide the final growth rate, biomass, composition, and astaxanthin productivity. The

Astaxanthin production from Haematococcus pluvialis

211

Table 11.1: Comparison between the closed and open system. Characteristics

Open system (open pond)

Closed system (photobioreactors)

Scale Area required Algal-specific growth rate CO2 fixation rate Areal productivities Control on the operational conditions (chemical and physical) Cost (capital and operational costs) Auxiliary energy demand

Large Large Lower Lower Flexible volume No

Small Smaller higher higher Difficult to scale up Yes

Low

expensive

Low

Risk of contamination Water loss

High High

High (when operated indoors with artificial lighting and power input for aeration) Low Low

advantages of the PBRs make them as a promising method to have high H. pluvialis biomass and astaxanthin productivity.

3.1 Light conditions in the biomass and astaxanthin production period Light is an important environmental factor for algal growth by regulating the rate of photosynthesis. Under the photoautotrophic photosynthetic process, H. pluvialis converts carbon dioxide into organic compounds such as carbohydrates, sugar, and biomass [14,15]. Light condition is varied in indoor, laboratory, or outdoor. To maximize the growth of H. pluvialis, cultivation needs to consider the light intensity, wavelength (light color), and photoperiod (light and dark) cycles. Generally, light intensities of 50–150 μmol m 2 s 1 are required for artificial H. pluvialis biomass production in PBRs. Increasing light intensity may further enhance the H. pluvialis growth rate [16]. With a further increase of light intensity, photoadaption or photoacclimation happens. The photosynthetic efficiency increases with the availability of light result in changes in cell properties and mechanisms, which affect the final types and quantities of the astaxanthin pigments and phospholipids [17,18]. However, once the maximal growth rate is achieved at the saturation intensity, a decreasing growth rate will be observed if it is out of the saturation range, also called photoinhibition [19]. While the light intensity is low, which is insufficient for algal growth, photoinhibition associated with a slow biomass growth rate will occur [20]. H. pluvialis under photoinhibition will lead to attenuation of growth or even cell death. This will create irreversible damage to the photosynthetic organs inside the cell body [21]. Also the high light intensity may disrupt the chloroplast lamellae and inactive the enzyme of CO2 fixation [22]. Therefore it is necessary to avoid photoinhibition to achieve better biomass productivity [23]. With a further increase of light intensity,

212 Chapter 11 photoadaption or photoacclimation happens. The photosynthetic efficiency increases with the availability of light result in changes in cell properties and mechanisms related to the types and quantities of pigments and lipids [17,18]. Giannelli et al. [24] reported that the requirement of light for optimal astaxanthin synthesis must be differentiated between the cell growth stage and the astaxanthin accumulation stage. To accumulate the highest amounts of astaxanthin, the two-stage cultivation process is generally adopted. In the first stage the low light intensity is used to promote H. pluvialis growth until the optimized cell density is reached. In the later stages, high radiance together with nitrogen limitation or starvation is used as a stress condition to induce the accumulation of astaxanthin [2,25]. Carotenoid and chlorophyll in H. pluvialis can absorb visible light range, in which carotenoids are around 400–550nm [26]. A higher amount of the total carotenoids for H. pluvialis can be obtained with a combination of blue and ultraviolet light, while a combination of red and ultraviolet light may use for enhancing biomass productivity [26]. Apart from the light intensity and wavelength, the illumination duration is also a concern. Light/dark cycles can be adjusted to optimize cell growth and astaxanthin accumulation. Examples for triggering the astaxanthin accumulation includes the light condition of blue/UV and blue/red light under 3hday 1 with 500 lux and 21h day 1 with 1500 lux, respectively [27]. The highest amount of carotenoids was observed with the highest amount under 150-μmolm 2 s 1 light intensity [28]. Another investigation by Kobayashi et al. [29] showed that a 24:0 photoperiod was more effective than a 12:12 photoperiod for carotenoid formation under the tested light intensity of 68, 139, 210, and 281 μmol m 2 s 1. Concerning the physiological effects of spectral quality of light, Katsuda et al. [30] reported that the higher cell density and astaxanthin concentration were obtained under blue flashing light of 2–12 μmol m 2 s 1. Moreover the illumination requirements vary against the culture depth, cell density, and condition during the cultivation period. Prolonging the cultivation periods for achieving a high cell concentration inside the PBRs or other container, sufficient light is needed to penetrate the cell culture depth [31]. Inevitably, photooxidation and photoinhibition effects usually happen during the microalgal cultivation [32]. An internally or externally illuminated PBRs with light-emitting diodes (LEDs) provide an adjustable lighting condition (light-dark cycle, light wavelength, and light intensity) during the two-stage cultivation process of H. pluvialis [33]. Optimized light conditions in PBRs are crucial to enhance the biomass productivity and astaxanthin synthetic rate for cost balancing.

3.2 Design of the illuminance system for the photobioreactors Light intensity is one of the essential limiting factors for the growth of H. pluvialis under the photosynthesis process, if the light exposure is only present in the outer surface, which is called the light or photic zone [34]. The interior part of the PBRs, which the light is not able to penetrate, is called the dark zone. The dark zone is an unavoidable area in the PBRs. The algal

Astaxanthin production from Haematococcus pluvialis

213

growth suffered from the fluctuation of the light regime inside the PBRs. Different geometries and designs of the PBRs are specialized in hydrodynamic circulation and light utilization, which affect the light distribution and liquid mixing. Fu et al. [35] reported that the dark zone is created by poor mixing and lighting design. The light/dark zone is affecting the performance of PBRs on algal biomass production. Apart from the geometry of the PBRs, the distribution of light intensity and light path length are also influenced by the algal cells. Posten [36] reported that the light attenuation is due to the absorption of light by the surface’s cells or cell shading effect. Ranjbar et al. [37] reported that in high cell density cultures, mutual shading caused by the cells is occurring, and the light intensity gradient inside the PBR is low. Dependent on the cell biomass concentration in the bioreactor, the dark zone area relates to the increase of the cell concentration [38]. Light limitation (photoinhibition) could occur when the light intensity is not enough. When the intensity surpasses a critical level, light saturation and photooxidation might occur. To solve the light distribution problem in the PBR, strategies including improving mixing, limiting the length of light path (e.g., thinner or small diameter materials), adjustable lighting system, and internally illuminated are commonly adopted.

3.3 Air mixing in the photobioreactors Agitation is one of the factors that affect the growth of H. pluvialis. H. pluvialis is a relatively dense algal cell that always sinks at the bottom of the container. The advantage of keeping the algal suspension in movement can sediment and ensure all the cells are equally exposed to the light and nutrients. Air mixing can improve the growth of H. pluvialis by preventing the nutrients in active contact with the algal cell surface and promote the stimulation of the nutrient uptake and utilization of the incident light. Air mixing of the PBRs system can be applied in both the industry and laboratory. In industry, modifying the cultivation tanks (large-scale cultivation system, which PBRs and the algal stock tank are separated) with air pumping, water lifting, or plastic sheet movement can promote algae mixing [39]. The pumping of carbon dioxide (usually 0.03% CO2) to the culture can promote algae growth when the medium is limited in carbon content. Pumping CO2 can also be the buffers to adjust the CO2/HCO3 balance in culture medium [40].

3.4 Temperature control in the photobioreactors Temperature does play an important role in the growth and carotenoid accumulation in H. pluvialis. For low temperature, three mechanisms may be triggered: (1) reduction of electron transport at a given photon flux rate due to the slow rate of CO2 fixation, (2) inhibition of the active oxygen species to protect PSII, and (3) inhibition of the D1 protein degradation during photoinhibition to induce PSII repair cycle [41]. Using high temperature is a technique for triggering the astaxanthin synthesis in H. pluvialis. Due to the increased oxidative photodamaging effects under high temperature, a threefold astaxanthin formation is observed

214 Chapter 11 when the temperature increased from 25°C to 35°C [42,43]. Moreover the optimum temperature of daytime and nighttime is between 22°C and 28°C for better cell growth and astaxanthin accumulation [44]. During the cultivation period the temperature can be controlled in the indoor environment, while heater can be installed inside the PBRs system.

3.5 Hydrodynamic and mass transfer in the photobioreactors H. pluvialis can achieve an effective growth rate inside the PBRs together with optimum culture condition, for example light input, nutrients, pH, temperature, CO2 supply, etc. Moreover the nutrients available in PBRs are significantly affected by the aeration rate, gas holdup, and mixing for the algal culture. Besides, dissolve oxygen will be accumulated in alga cultures due to the oxygen production in photosynthetic reactions. If excessive oxygen is present inside culture, the photosynthesis rate may be suppressed [45]. Generally the productivity of the PBRs depends on the hydrodynamic and mass transfer [46]. Efficient mixing helps to reduce the dissolved oxygen inside the cultures and provide good mass transfer of oxygen and CO2 in the culture system, simultaneously improve the heat transfer [47]. Hence the design of the PBR needs to evaluate several parameters, such as gas holdup, liquid volume, mass transfer coefficient (kLa), and mixing time.

3.6 Chemicals factors of the illuminated photobioreactors Culture media is the artificial supply of chemical nutrients that is required by the algae during the cultivation period. The choice of medium is depending on the nutrient needs of H. pluvialis during the two-phase cultivation strategies. Depending on the research approach, nutrient content shows a direct effect on the cell content, such as biomass, carbohydrate, protein, chlorophyll, lipids, and astaxanthin pigments in different production phases. Defined media (e.g., blue-green BG-11 or bold basal medium BBM) with sterilized and pH-controlled are usually adopted while altering the nutrient’s components to fulfill specific chemical requirements in the two-stage process [48,49].

4 Process for scaling up the production from laboratory to pilot and mass scale 4.1 Selection of photobioreactors at different production stages In the commercial astaxanthin production, PBRs are an important and expensive investment that affects the biomass production and final astaxanthin yield in production. Purchasing an existing design of PBRs or constructing a self-oriented PBRs can lower the cost and adjust the

Astaxanthin production from Haematococcus pluvialis

215

reactor’s component according to the actual condition. Different designs of PBRs, such as airlift, bubble column, and tubular, have their characteristics and reported that H. pluvialis can be cultivated effectively (Table 11.2). Bubble column reactor is a simple tubular reactor design with a random air mixing produced by ascending bubbles. Bubble column reactor provides a relatively homogeneous culture environment with good air mixing in the cell culture. Optimal dimensions of the bubble column are about 4-m height and 0.2m in diameter and are practicable according to the need for light penetration [36,56]. Bubble column reactors are practical for culturing a small amount of biomass with higher photosynthetic efficiency to the airlift and tubular reactors. The airlift and tubular reactors can be operated both in indoor and outdoor conditions. Airlift reactors compose with a draft tube to divide the culture liquid into a riser and down comer. The draft tube is installed at the center of the column. The gas that holds up in riser is usually higher than the downcomer. The difference between the riser and downcomer creates a liquid circulation in the PBR. Airlift reactors produce a regular mixing and evenly distribute light/dark zone inside the culture. The light regime inside the reactor is improved, and the algal cells are more evenly distribute and circulate between the light/dark zones in the PBRs [38]. Hence, airlift reactors are suitable for medium-density (up to 10 L) growth of microalgae. Tubular reactors can be set up from small- or large-scale and operate under for indoor or outdoor conditions. The tubular reactors divided into vertical or horizontal design and vertical design with artificial lighting provide a homogeneous light distribution and high biomass productivity. Moreover, horizontal design is beneficial under outdoor production as more of the surface area is exposed to the sunlight. Outdoor cultivation of H. pluvialis is challenging due to the contamination, slow cell growth, and low biomass productivity. For minimizing the risk of contamination during the cultivation of H. pluvialis, enclosed tubular reactors usually have higher biomass productivity than the open pond or raceway pond. However, in mass-scale production, the lower cost for scaling up with raceway ponds can compromise with low biomass productivity and contamination. PBRs are important to produce H. pluvialis biomass and astaxanthin efficiently and environmentally friendly. However, PBR production efficiency is affected by several factors, like light input, air mixing, mass transfer, nutrient content, pH, temperature, and salinity [57]. Moreover, preparation of the stock medium and cell harvesting from the PBRs is a time-consuming and labor-intensive process once having a large culture volume. The life span, periodic cleaning, parameter control, and maintenance are also important for long-term running of the PBR. Hence, there is no perfect design of the PBRs. The design of the PBRs needs to compromise with the capital cost, space area, and other practical difficulties in the production process.

216 Chapter 11 Table 11.2: The biomass and astaxanthin productivity under different condition and photobioreactor.

Reactor type

Culture volume Indoor/outdoor

Airlift PBR

2L

Indoor

Tubular PBR

50 L

Indoor

Tubular PBR

50 L

Outdoor

Airlift PBR

30 L

Indoor

Bubble PBR

55 L

Outdoor

Tubular PBR

55 L

Outdoor

Enclosed PBR (Aquasearch Growth Module, AGM) Pond system

25,000 L

Outdoor

6 Pond units with 15-cm pond depth

Outdoor

8000 L 1000 m2

Outdoor Outdoor

PBR Pond

Light condition

Biomass/ carotenoid content

References

110  10 cells/ Autotrophic 12/12 light-dark mL for cycle Haematococcus pluvialis 3 days, 16/8 0.9-g L 1 light dark cycle biomass concentration 24-h growth Biomass period production of 15.7 g m 2 Carotenoid production 104 mg m 2 h 1 Carotenoid Four fluorescent content of 2.7% tubes, which algal dry weight supplied with 50 PAR unit Sunlight 1.4-g L 1 biomass concentration, 4.4-mg day 1 astaxanthin productivity Sunlight 7.0-g L 1 biomass concentration after 16 days, 0.12-mg day 1 astaxanthin productivity Sunlight 0.2 g L 1 in January 0.36 g L 1 in September

[50]

3% astaxanthin Cell transfer content from AGM, astaxanthin harvest on day 5 in the pond system Sunlight 5  105 cells/mL Batch mode 6  104 cells/mL under sunlight

[54]

4

[51]

[51]

[52]

[53]

[53]

[54]

[55] [55]

Astaxanthin production from Haematococcus pluvialis

217

4.2 Mass culture of H. pluvialis in open versus closed systems H. pluvialis is cultivated as a source for astaxanthin and is produced in commercial scale. Haematococcus biomass production is being innovated continuously for enhanced growth of algae and production of astaxanthin. Table 11.3 provides comparative yield of biomass yield of Table 11.3: Mass culture of Haematococcus pluvialis for astaxanthin production in raceway and photobioreactors.

Size and type of reactor Outdoor tubular Open Pond (25,000 L) Indoor, tubular (50 L) semicontinuous Indoor bubbling column (1.8 L), batch Air light tubular (55 L); batch

Astaxanthin Biomass yield Content (% or (g L21 or g m22 mg L21 or mg or mgL21 day21 L21 day21 or g m23 day21) or g L21 day21)

Culture conditions

Growth medium

16–34°C, day light cycle

Modified bold’s

50–90

2.8–3.0

[54]

25°C, 7.0, continuous

BG-11 and nitrogen deficient

0.8

4.4 (80% astaxanthin monoestser)

[58]

25°C, 7.0, continuous

Basal inorganic

1.2

0.8

[59]

20°C, 8.0

Inorganic medium free of acetate Inorganic medium free of acetate

7.0 0.41

1.1

[53]

0.06

0.06

[53]

11.5

4

[60]

1.9

20

[61]

0.7

8

[62]

1.8

2.79

[63]

2.62

78.37

[64]

20°C, 8.0 Bubble column reactor (55 L); batch 24–26°C, BG11 Outdoor tubular 6.4–7.0, dark/ (200 L), batch light (14:10 h) mode Indoor bubbling 25°C, 7.0–7.1, Basal inorganic continuous column (1.8 L), light batch mode 20°C, 8.0; dark/ Standard Air lift tubular culture photobioreactor light (12:12 h) (50L), continuous mode 15–25, 7–10, BG11 Open pond light/dark (14:10 h) 23°C, 7.5, dark/ Continuous Airlift column light (12:12 h) light, NIES-C indoor (6 L), batch mode

References

Continued

218 Chapter 11 Table 11.3:

Mass culture of Haematococcus pluvialis for astaxanthin production in raceway and photobioreactors—cont’d Astaxanthin Biomass yield Content (% or (g L21 or g m22 mg L21 or mg or mgL21 day21 L21 day21 or g m23 day21) or g L21 day21)

Size and type of reactor

Culture conditions

Growth medium

Bubbling column (0.6 L) outdoor/ batch Glass column photobioreactors Photobioreactor (5 L)

20°C, 7–8, continuous light

BG-11

0.5–0.8

3.8

[65]

BG-11

0.8

2.7

[66]

MCM

1.87

–

[67]

Blue-green medium 11-H

19.4

2.5

[68]

Bold’s basal

0.16

46

[69]

FI medium

–

1.29

[7]

FI medium

–

1.88

[7]

FI medium

–

1.83

[7]

FI medium

–

1.77

[7]

20–25°C, day light 16–24°C, 7.5, dark/light (12:12 h) 23°C, 7.3–7.4, Twin layer light/dark photobioreactor (14:10 h) 25°C, light/dark Glass cylinder (12:12 h) photobioreactors – Bubble column photobioreactor (1.5 L) – Flat panel photobioreactor (17 L) – Flat panel photobioreactor (50 L) – Flat panel photobioreactor (90 L)

References

H. pluvialis, and astaxanthin production in raceway and photobioreactors in various studies reported in the literature.

4.3 Astaxanthin production process at commercial scale The current commercial production process splits into biomass production, astaxanthin induction, cell harvesting, processing (dewatering), and producing final products. The producers generally adopt the two-stage strategy for astaxanthin production: the first phase to produce biomass at with fast growth rate and follow with a second phase to induce astaxanthin

Astaxanthin production from Haematococcus pluvialis

219

accumulation. The first growth phase usually conducted in closed indoor PBRs or enclosed outdoor PBR systems to minimize contamination. The indoor culturing includes the preparation of stock culture library, follow with the inoculation inside the vertical column or vertical tubular PBRs. The cultivation scale enlarged from the stock solutions to PBRs (approximately 10–50L) and finally to vertical tubular PBRs (1000L or more). Most of the research and testing (e.g., culture methods and physical and chemical conditions) are performed with artificial lighting under indoor conditions. For the outdoor biomass cultivation, depending on the environmental condition (e.g., sunlight, risk of contamination, and evaporation rate), either raceway ponds or tubular PBRs system can be the major component for cell culturing at commercial-scale production. H. pluvialis can be cultivated inside the vertical or horizontal tubular PBRs (starting from 1000L or more) and raceway ponds (1000m2 or more). The outdoor PBRs are located in the sunny and open area to acquire solar radiation for a faster algal growth rate. The tubular PBR system and raceway pond may obtain the photosynthetic efficiencies of 3% and 1%, respectively. BGG company based in Yunnan Province operates the vertical tubular PBRs for both the biomass growth and astaxanthin accumulation phases [70,71]. The 80-acre facility produces an annual output of over 2 tons of astaxanthin. Aquasearch in Hawaii operated the 25,000-L Aquasearch Growth Module, which is the horizontal tubular closed PBR for the growth of H. pluvialis. Then the cells were transferred into an open pond for 5-day astaxanthin induction [54]. Algatech operates the 600-km vertical tubular PBRs in Arava desert, Israel, for taking advantage of its extreme climate condition [72]. Over 600-km vertical tubular PBRs are operated for the production in both phases. Cyanotech started to cultivate H. pluvialis since 1988 with raceway ponds. The BioAstin produced by Cyanotech achieved the generally regarded as safe status with the US-FDA in 2010. Cyanotech adopts the commercial supercritical carbon dioxide extraction plant for extracting astaxanthin since 2015 [73]. Fuji Chemical is a Japanese company that sets up the production plants in Sweden for using fermenters and artificial lighting inside the sterile plant [74]. Table 11.4 shows an overview of the commercial production of natural astaxanthin. Closed tubular PBRs currently are the dominant methods for H. pluvialis cultivation, as it can minimize the risk of contamination; simultaneously, it can be operated indoor or outdoor with large-scale production. Currently the commercial astaxanthin production faces different challenges and problems: slow cell growth and astaxanthin accumulation; cost ineffective in cultivation, extraction, harvesting, and drying process; lack of skilled researchers, environmental engineers, and scientists; and lack of solutions to the parasite and predator contamination when under large-scale culture volume. Furthermore, analyzing the production data and developing research knowledge for the closed system PBRs, cultivation, and astaxanthin induction techniques may help to overcome the current difficulties and transform the production from laboratory to commercial scale, which is important for running a sustainable astaxanthin production business.

Table 11.4: Current status of companies for natural astaxanthin production with Haematococcus pluvialis. Company BGG Aquasearch

Location Shilin, Yunnan Province, China Hawaii

Majority for production

Indoor/outdoor

Cultivation mode

Outdoor

Autotrophic

Outdoor

Autotrophic

Products

Status

References

Astaxanthin AstaZine N.A.

Expanding

[70,71]

Closed

[54]

Vertical tubular PBR Horizontal tubular PBRs Outdoor, over 600-km vertical tubular PBRs Raceway ponds

Outdoor

Autotrophic

AstaPure Astaxanthin

Operating

[72]

Outdoor

Autotrophic

BioAstin

Operating

[73]

Fermenters

Indoor

Mixotrophic

AstaReal

Operating

[74,75]

Enclosed PBRs

Indoor

N.A.

AstaSuper Astaxanthin Astaxanthin powder Astaxanthinrich oily extract Carotenoid astaxanthin

Operating

[76]

Operating

[77]

Operating

[78]

Operating

[79]

Algatech

Arava desert, Israel

Cyanotech

Fenchem

Hawaii, United States Sweden and Moses Lake, United States Nanjing, China

MicroA

Norway

Column PBRs

Indoor

Autotrophic

Neoalgae

´n, Spain Gijo

Column PBRs

Indoor

Autotrophic

Indoor

Autotrophic

Open ponds

Outdoor

Autotrophic

Zanthin

Operating

[80]

Vertical tubular PBRs

Indoor

Autotrophic

Ingredients and softgel capsules

Operating

[81]

Fuji Chemical

Haliae and Sincere Corporation Valensa Algalif

Saga City, Japan Vertical tubular PBRs, Raceway ponds Elqui Valley, Chile Reykjanesbaer, Iceland

Astaxanthin production from Haematococcus pluvialis

221

5 Conclusion The design, operating factors, and scaling-up process of the PBRs for the cultivation of microalgae H. pluvialis to produce biomass and astaxanthin pigments are described in this chapter. IPBRs are introduced as a promising way to enhance biomass and astaxanthin productivity. The astaxanthin processes in laboratory and commercial generally include the strain selection, two-stage cultivation process, harvesting, astaxanthin extraction, and conversion to final products. Among them the design of a highly efficient PBR system is pivotal importance. Large-scale cultivation of H. pluvialis forms the foundation of astaxanthin production industries. Many are resorting to set up an open pond or raceway pond systems due to the land space and contamination problem. Currently the high cost of astaxanthin is involved in installing, maintaining closed-type PBRs, and running in an indoor environment. Thus more efforts are still needed in developing more versatile, economic, and effective integrated indoor and outdoor PBRs for large-scale cultivation of H. pluvialis. The emerging algae-based biofuels, supplements, and chemicals industry are the success evidences of such studies and are expected to grow shortly with the aid of advanced PBR and algal-related technologies.

Acknowledgment This work was supported by the School of Engineering of The Hong Kong University of Science and Technology, and Episteme Company Limited. ARR acknowledge Vignan’s Foundation for Science, Technology and Research University for providing facility for this work.

References [1] Ambati RR, Phang SM, Ravi S, Aswathanarayana RG. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications—a review. Mar Drugs 2014;12(1):128–52. [2] Boussiba S. Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Physiol Plant 2000;108(2):111–7. [3] Lemoine Y, Schoefs B. Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress. Photosynth Res 2010;106(1–2):155–77. [4] Shah M, Mahfuzur R, Liang Y, Cheng JJ, Daroch M. Astaxanthin-producing green microalga Haematococcus pluvialis: from single cell to high value commercial products. Front Plant Sci 2016;7:531. [5] Dose J, Matsugo S, Yokokawa H, Koshida Y, Okazaki S, Seidel U, Eggersdorfer M, Rimbach G, Esatbeyoglu T. Free radical scavenging and cellular antioxidant properties of astaxanthin. Int J Mol Sci 2016;17(1):103. [6] Ranga Rao A, Ravishankar GA. Potential health and nutraceutical applications of astaxanthin and astaxanthin esters from microalga. In: Ravishankar GA, Ranga Rao A, editors. Handbook of Algal Technologies and Phytochemicals: volume-I: food, health and nutraceutical applications. USA: CRC Press; 2019. p. 121–37. [7] Poonkum W, Powtongsook S, Pavasant P. Astaxanthin induction in microalga H. pluvialis with flat panel airlift photobioreactors under indoor and outdoor conditions. Prep Biochem Biotechnol 2015;45(1):1–17. [8] Madhubalaji CK, Shekh A, Sijil PV, Mudliar S, Chauhan VS, Sarada R, Ranga Rao A, Ravishankar GA. Open cultivation system and closed photobioreactors for microalgal cultivation and biomass production. In: Ravishnkar GA, Ranga Rao A, editors. Handbook of algal technologies and phytochemicals: volume-II phycoremediation, biofuels and global biomass production. vol. 2. USA: CRC Press; 2019. p. 179–202.

222 Chapter 11 [9] Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: a review. Renew Sustain Energy Rev 2010;14(1):217–32. [10] Patel A, Gami B, Patel P, Patel B. Microalgae: antiquity to era of integrated technology. Renew Sustain Energy Rev 2017;71:535–47. [11] Singh RN, Sharma S. Development of suitable photobioreactor for algae production—a review. Renew Sustain Energy Rev 2012;16(4):2347–53. [12] Faried M, Samer M, Abdelsalam E, Yousef RS, Attia YA, Ali AS. Biodiesel production from microalgae: processes, technologies and recent advancements. Renew Sustain Energy Rev 2017;79:893–913. [13] Tan XB, Lam MK, Uemura Y, Lim JW, Wong CY, Lee KT. Cultivation of microalgae for biodiesel production: a review on upstream and downstream processing. Chin J Chem Eng 201826(1):17–30. [14] Ankita J, Ruben MC, Ganti SM. Effects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production: a review. Energies 2013;6:4607–38. [15] Stockenreiter M, Haupt F, Graber AK, Sepp€al€a J, Spilling K, Tamminen T, Stibor H. Functional group richness: implications of biodiversity for light use and lipid yield in microalgae. J Phycol 2013;49(5):838–47. [16] Radakovits R, Jinkerson RE, Darzins A, Posewitz MC. Genetic engineering of algae for enhanced biofuel production. Eukaryot Cell 2010;9(4):486–501. [17] Dubinsky Z, Matsukawa R, Karube I. Photobiological aspects of algal mass culture. J Mar Biotechnol 1995;2:61–5. [18] Fabregas J, Maseda A, Domı´nguez A, Otero A. The cell composition of Nannochloropsis sp. changes under different irradiances in semicontinuous culture. World J Microbiol Biotechnol 2004;20:31–5. [19] Sorokin C, Krauss RW. The effects of light intensity on the growth rates of green algae. Plant Physiol 1958;33 (2):109–13. [20] Liu J, Sun Z, Gerken H, Liu Z, Jiang Y, Chen F. Chlorella zofingiensis as an alternative microalgal producer of astaxanthin: biology and industrial potential. Mar Drugs 2014;12:3487–515. [21] Suzuki T, Matsuo T, Ohtaguchi K, Koide K. Gas-sparged bioreactors for CO2 fixation by Dunaliella tertiolecta. J Chem Technol Biotechnol 1995;62(4):351–8. [22] Iqbal M, Zafar S. Effects of photon flux density, CO2, aeration rate, and inoculum density on growth and extracellular polysaccharide production by Porphyridiumcruentum. Folia Microbiol 1993;38:509–14. [23] Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25(3):294–306. [24] Giannelli L, Yamada H, Katsuda T, Yamaji H. Effects of temperature on the astaxanthin productivity and light harvesting characteristics of the green alga Haematococcus pluvialis. J Biosci Bioeng 2015;119(3):345–50. [25] Boussiba S, Vonshak A. Astaxanthin accumulation in the green alga Haematococcus pluvialis. Plant Cell Physiol 1991;32(7):1077–82. [26] Carvalho AP, Silva SO, Baptista JM, Malcata FX. Light requirements in microalgal photobioreactors: an overview of biophotonic aspects. Appl Microbiol Biotechnol 2011;89(5):1275–88. [27] Suyono EA, Aminin, Pradani L. Combination of blue, red, white, and ultraviolet lights for increasing carotenoids and biomass of microalga Haematococcus pluvialis. Procedia Environ Sci 2015;28:309–405. [28] Goksan T, Ak I, Kılıc¸ C. Growth characteristics of the alga Haematococcus pluvialis flotow as affected by nitrogen source, vitamin, light and aeration. Turkish J Fish Aquat Sci 2011;11:377–83. [29] Kobayashi M, Kakizono T, Yamaguchi K, Nishio N, Nagai S. Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic conditions. J Ferment Bioeng 1992;74(1):17–20. [30] Katsuda T, Shimahara K, Shiraishi H, Yamagami K, Ranjbar R, Katoh S. Effect of flashing light from blue light emitting diodes on cell growth and astaxanthin production of Haematococcus pluvialis. J Biosci Bioeng 2006;102(5):442–6. [31] Wahidin S, Idris A, Shaleh SRM. The influence of light intensity and photoperiod on the growth and lipid content of microalgae Nannochloropsis sp. Bioresour Technol 2013;129:7–11. [32] Chen CY, Yeh KL, Aisyah R, Lee DJ, Chang JS. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresour Technol 2011;102(1):71–81. [33] Fabregas J, Otero A, Maseda A, Domı´nguez A. Two-stage cultures for the production of astaxanthin from Haematococcus pluvialis. J Biotechnol 2001;89(1):65–71.

Astaxanthin production from Haematococcus pluvialis

223

[34] Bitog JP, Lee IB, Lee CG, Kim KS, Hwang HS, Hong SW, Seo IH, Kwon KS, Mostafa E. Application of computational fluid dynamics for modeling and designing photobioreactors for microalgae production: a review. Comput Electron Agric 2011;76(2):131–47. [35] Fu CC, Wu WT, Lu SY. Performance of airlift bioreactors with net draft tube. Enzyme Microb Technol 2003;33(4):332–42. [36] Posten C. Design principles of photo-bioreactors for cultivation of microalgae. Eng Life Sci 2009;9(3):165–77. [37] Ranjbar R, Inoue R, Katsuda T, Yamaji, Ranjbar R, Inoue R, Katsuda T, Yamaji H, Katoh S. High efficiency production of astaxanthin in an airlift photobioreactor. J Biosci Bioeng 2008;106(2):204–7. [38] Janssen MGJ. Cultivation of microalgae: effect of light/dark cycles on biomass yield [PhD thesis]. Wageningen, The Netherlands: Wageningen University; 2002183. [39] Persoone G, Morales J, Verlet H, De Pauw N. Air-lift pumps and the effect of mixing on algal growth. Algal Biomass 1980;11:505–22. [40] Barsanti L, Gualtieri P. Algae culturing. In: Algae: anatomy, biochemistry, and biotechnology. 2nd ed. vol. 1. CRC Press; 2014. p. 253–5. [41] Vonshak A, Torzillo G. Environmental stress physiology. In: Handbook of microalgal culture. Blackwell; 2004. p. 57–82. [42] Hata N, Ogbonna JC, Hasegawa Y. Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture. J Appl Phycol 2001;13:395–402. [43] Tripathi U, Sarada R, Ravishankar GA. Effect of culture conditions on growth of green alga—Haematococcus pluvialis and astaxanthin production. Acta Physiol Plant 2002;24(3):323–9. [44] Wan M, Zhang J, Hou D, Fan J, Li Y, Huang J, Wang J. The effect of temperature on cell growth and astaxanthin accumulation of Haematococcus pluvialis during a light–dark cyclic cultivation. Bioresour Technol 2014;167:276–83. [45] Ugwu CU, Aoyagi H, Uchiyama H. Influence of irradiance, dissolved oxygen concentration, and temperature on the growth of Chlorella sorokiniana. Photosynthetica 2007;45(2):309–11. [46] Jacobi DBA, Ivanova DID, Posten IC. Photobioreactors: hydrodynamics and mass transfer. IFAC Proc Vol 2010;43(6):162–7. [47] Chisti MY. Airlift bioreactors. London and New York: Elsevier Applied Science; 19891. [48] Nichols HW, Bold HC. Trichosarcina polymorpha gen. et sp. nov. J Phycol 1965;1(1):34–8. [49] Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 1979;111(1):1–61. [50] Vega-Estrada J, Montes-Horcasitas MC, Domı´nguez-Bocanegra AR, Canizares-Villanueva RO. Haematococcus pluvialis cultivation in split-cylinder internal-loop airlift photobioreactor under aeration conditions avoiding cell damage. Appl Microbiol Biotechnol 2005;68(1):31–5. [51] Chaumont D, Thepenier C. Carotenoid content in growing cells of Haematococcus pluvialis during a sunlight cycle. J Appl Phycol 1995;7(6):529–37. [52] Harker M, Tsavalos AJ, Young AJ. Autotrophic growth and carotenoid production of Haematococcus pluvialis in a 30 liter air-lift photobioreactor. J Ferment Bioeng 1996;82(2):113–8. [53] Lopez MGM, Sa´nchez EDR, Lo´pez JC, Ferna´ndez FA, Sevilla JF, Rivas J, Guerrero MG, Grima EM. Comparative analysis of the outdoor culture of Haematococcus pluvialis in tubular and bubble column photobioreactors. J Biotechnol 2006;123(3):329–42. [54] Olaizola M. Commercial production of astaxanthin from Haematococcus pluvialis using 25,000-liter outdoor photobioreactors. J Appl Phycol 2000;12(3–5):499–506. [55] Li J, Zhu D, Niu J, Shen S, Wang G. An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis. Biotechnol Adv 2011;29(6):568–74. [56] Merchuk JC, Garcia Camacho F. Bioreactors: airlift reactors. In: Encyclopedia of industrial biotechnology: bioprocess, bioseparation, and cell technology. John Wiley & Sons; 2009. p. 887–953. [57] Huang Q, Jiang F, Wang L, Yang C. Design of photobioreactors for mass cultivation of photosynthetic organisms. Engineering 2017;3(3):318–29. [58] Torzillo G, Goksan T, Faraloni C, Kopecky J, Masojı´dek J. Interplay between photochemical activities and pigment composition in an outdoor culture of Haematococcus pluvialis during the shift from the green to red stage. J Appl Phycol 2003;15(2–3):127–36.

224 Chapter 11 [59] Rio ED, Acien FG, Garcia-Malea MC, Rivas J, Molina-Grima E, Guerrero MG. Efficient one-step production of astaxanthin by the microalga Haematococcus pluvialis in continuous culture. Biotechnol Bioeng 2005;91 (7):808–15. [60] Aflalo C, Meshulam Y, Zarka A, Boussiba S. On the relative efficiency of two- vs. one-stage production of astaxanthin by the green alga Haematococcus pluvialis. Biotechnol Bioeng 2007;98(1):300–5. [61] Rio ED, Acien FG, Garcı´a-Malea MC, Rivas J, Molina-Grima E, Guerrero MG. Efficiency assessment of the one-step production of astaxanthin by the microalga Haematococcus pluvialis. Biotechnol Bioeng 2008;100 (2):397–402. [62] Garcia-Malea MC, Acien FG, Del Rı´o E, Fernandez JM, Cero´n MC, Guerrero MG, Molina-Grima E. Production of astaxanthin by Haematococcus pluvialis: taking the one-step system outdoors. Biotechnol Bioeng 2009;102(2):651–7. [63] Zhang BY, Geng YH, Li ZK, Hu HJ, Li YG. Production of astaxanthin from Haematococcus in open pond by two-stage growth one-step process. Aquaculture 2009;295(3–4):275–81. [64] Yoo JJ, Choi SP, Kim BW, Sim SJ. Optimal design of scalable photo-bioreactor for phototropic culturing of Haematococcus pluvialis. Bioprocess Biosyst Eng 2012;35(1–2):309–15. [65] Wang J, Sommerfeld MR, Lu C, Hu Q. Combined effect of initial biomass density and nitrogen concentration on growth and astaxanthin production of Haematococcus pluvialis (Chlorophyta) in outdoor cultivation. Algae 2013;28(2):193–202. [66] Wang J, Han D, Sommerfeld MR, Lu C, Hu Q. Effect of initial biomass density on growth and astaxanthin production of Haematococcus pluvialis in an outdoor photobioreactor. J Appl Phycol 2013;25(1):253–60. [67] Sun H, Kong Q, Geng Z, Duan L, Yang M, Guan B. Enhancement of cell biomass and cell activity of astaxanthin-rich Haematococcus pluvialis. Bioresour Technol 2015;186:67–73. [68] Kiperstok AC, Sebestyen P, Podola B, Melkonian M. Biofilm cultivation of Haematococcus pluvialis enables a highly productive one-phase process for astaxanthin production using high light intensities. Algal Res 2017;21:213–22. [69] Cheng J, Li K, Yang Z, Zhou J, Cen K. Enhancing the growth rate and astaxanthin yield of Haematococcus pluvialis by nuclear irradiation and high concentration of carbon dioxide stress. Bioresour Technol 2016;204:49–54. [70] BGG. Research and technology, Retrieved 1 May 2020, from: https://bggworld.com/about-bgg/research-andtechnology/; 2020. [71] Nutraingredients-usa.com. BGG to double capacity at astaxanthin farm in southwestern China, Retrieved 1 May 2020, from: https://www.nutraingredients-usa.com/Article/2020/04/22/BGG-to-double-capacityat-astaxanthin-farm-in-southwestern-China; 2020. [72] Algatech. Microalgae cultivation technology, leading astaxanthin manufacturer, Retrieved 1 May 2020, from: https://www.algatech.com/our-technology/; 2020. [73] Cyanotech. Our history, Retrieved 1 May 2020, from: https://www.cyanotech.com/our-history/; 2020. [74] Astareal. The first successful commercial production of astaxanthin algae, Retrieved 1 May 2020, from: https:// astareal.com/en/story-2/; 2020. [75] Adams R. AstaReal American facility celebrates one year, Retrieved 1 May 2020, from: https://www. naturalproductsinsider.com/specialty-nutrients/astareal-american-facility-celebrates-one-year; 2020. [76] Fenchem. Nutrition & food – AstaSuper™ Astaxanthin, Retrieved 1 May 2020, from: http://www.fenchem. com/index.php/industry/industrycenter/1.html; 2020. [77] MicroA. Astaxanthin, Retrieved 1 May 2020, from: https://microa.no/product/astaxanthin/; 2020. [78] Neoalgae. The production plant, Retrieved 1 May 2020, from: http://neoalgae.es/en/neoalgae-en/; 2020. [79] Heliae. Sincere corporation working with Heliae in Japan, Retrieved 1 May 2020, from: https://heliaeglobal. com/sincere-corporation-working-with-heliae-in-japan/; 2015. [80] Valensa. Valensa leads astaxanthin industry by partnering man and nature, Retrieved 1 May 2020, from: https://valensa.com/2019/06/11/valensa-leads-astaxanthin-industry/; 2019. [81] Algalif. Indoor, controlled cultivation system, Retrieved 1 May 2020, from: https://algalif.com/ production-process/; 2020.

CHAPTER 12

Recent developments in astaxanthin production from Phaffia rhodozyma and its applications Yuan Zhuanga and Ming-Jun Zhua,b,c a

Guangdong Provincial Engineering and Technology Research Center of Biopharmaceuticals, School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou, China bCollege of Life and Geographic Sciences, Kashi University, Kashi, China cThe Key Laboratory of Biological Resources and Ecology of Pamirs Plateau in Xinjiang Uygur Autonomous Region, Kashi University, Kashi, China

Abbreviations AACT ARTP AX CoA CrtB (PSY) CrtS (Asy) CrtW (BKT) CrtZ (BCH) DCD DCW DMAPP DNA DO EMS FDA FPP GGPP GPP HDCO HMG-CoA HMGR HMGS IPP MEP MK

acetoacetyl-CoA thiolase atmospheric and room temperature plasma astaxanthin coenzyme A phytoene synthase cytochrome P450 monooxygenase β-carotene ketolase β-carotene hydroxylase 3,30 -dihydroxy-β,ψ-carotene-4,40 -dione dry cell weight dimethylallyl pyrophosphate deoxyribonucleic acid dissolved oxygen ethyl methane sulfonate Food and Drug Administration farnesyl pyrophosphate geranylgeranyl pyrophosphate synthase geranyl pyrophosphate 3-hydroxy-30 ,40 -didehydro-β,ψ-carotene-4-one 3-hydroxy-3-methyl-glutaryl-CoA 3-hydroxy-3-methyl-glutaryl-CoA reductase 3-hydroxy-3-methyl-glutaryl-CoA synthase isopentenyl pyrophosphate 2-C-methyl-D-erythritol-4-phosphate mevalonate kinase

Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00006-4 Copyright # 2021 Elsevier Inc. All rights reserved.

225

226 Chapter 12 MPDC MVA Nrf-2 NTG Padh4 PMK ROS TCA UV

mevalonate pyrophosphate decarboxylase mevalonate erythroid 2-related factor 2 nitrosoguanidine alcohol dehydrogenase promoter phosphomevalonate kinase reactive oxygen species tricarboxylic acid ultraviolet

1 Introduction Astaxanthin (AX) (3,30 -dihydroxy-4,40 -diketo-β,β0 -carotene), a liposoluble compound from the carotenoid family, is capable of dissolving in some organic solvents (such as acetone) [1]. AX, which contains 13 conjugated double bonds and α-hydroxyketone, is capable of quenching singlet oxygen and scavenging free radicals [2], so it is endowed with extremely strong antioxidant activity that is 550 times more potent than vitamin E and is more effective in protecting rat liver cell mitochondria from lipid peroxidation [3]. Additionally, it has certain effect on the prevention and treatment of some diseases [4]. These properties determine the potential of AX in the food, health care, cosmetics, and pharmaceutical industries [5]. In addition, AX is popular in the aquaculture as a pigment additive, showing bright red color to the flesh of salmon or trout and enriching their nutritional value [6]. It is the wide application value of AX that responds to the huge demand for AX in the market, especially as a feed additive and colorant. AX in the market is divided into synthetic AX and natural AX, and the former dominates the global AX market due to lower costs [7]. However, with the development of biotechnology and the market demand for safe natural products, natural AX has become more and more popular. For natural AX, Haematococcus pluvialis and Phaffia rhodozyma are two major commercial sources, among which H. pluvialis has the highest AX content in natural organisms producing AX [8]. H. pluvialis has become the best organism for commercial AX, which is also the only AX source for human consumption approved by various countries in the world [9]. Compared with H. pluvialis, P. rhodozyma has some unique characteristics, such as rapid metabolism and high cell density production in bioreactors [10]. However, the content of AX in wild P. rhodozyma is lower than that of H. pluvialis [11]; thus it is of great significance to research the mechanism of AX accumulation in P. rhodozyma and take various technical measures to increase AX production for the further commercial utilization of AX. This article reviews the different characteristics of P. rhodozyma and other AX sources and analyzes the methods used to promote the accumulation of AX. Both methods based on the metabolic pathway and methods from external factors are summarized in detail in the succeeding text. Finally the application research progress of AX in recent years is mentioned.

Recent developments in astaxanthin production from Phaffia rhodozyma 227

2 Characteristics of P. rhodozyma as an astaxanthin source Natural AX is mainly found in fish, crustaceans, Chlorella zofingiensis, H. pluvialis, and P. rhodozyma [12]. The animals themselves cannot synthesize AX, which is mainly transferred by microalgae/phytoplankton through the food chain to some animals, like shrimp, krill, crayfish, or crabs and finally accumulated in fish such as salmon and trout [13]. Compared with synthetic AX and H. pluvialis AX, P. rhodozyma AX has its unique characteristics (Table 12.1) as a natural producer in the AX market, especially in the aquatic feed industry. As far as the structure is concerned, AX from different sources exists in the form of stereoisomers, geometric isomers, esterification, and unesterification. AX itself has three major stereoisomers: 3S-30 S, 3R-30 R, and 3R-30 S [14], among which the first two are the most abundant in nature [5], mainly from H. pluvialis and P. rhodozyma [19]. According to the esterification degree of two hydroxyl groups on the β-ionone ring of AX and fatty acid, it is divided into free (unesterified), monoester and diester [12], which the first one is unstable due to oxidative degradation [20]. In addition, AX is also classified into cis-isomer and transisomer, which the former is thermodynamically unstable [21] and shows a higher antioxidant capacity than the latter, especially 9-cis AX [22]. According to a report on the oxidative stress response induced by feeding a worm named Caenorhabditis elegans with paraquat (a toxic, fast-acting herbicide), H. pluvialis AX had the strongest ability to scavenge reactive oxygen species (ROS), followed by P. rhodozyma AX, and the synthetic AX was the worst [23]. As far as the production of different AX is concerned, synthetic AX has a much larger share (>95%) than the natural AX in the global AX market due to lower cost [7]. However, synthetic AX has a complex process, whose biological function and food safety are still of concern, even related to some undefined diseases [24]; thus it cannot be used for human consumption [25]. At present, only safe natural AX is approved by the Food and Drug Administration (FDA) as an additive for human consumption like food or health care products [25], which is also related to the high bioavailability of natural AX [26]. In addition to accumulating AX under traditional optimized culture conditions, most microalgae currently are induced to accumulate more AX by the combination of abiotic stress (such as nitrogen deficiency, high light, high CO2 concentration, and electrical treatment) and chemical promoters (such as cytokinin, melatonin, and butylated hydroxytoluene) [27–32]. Besides, there are strategies for strain mutagenesis or genetic modification using omics [33, 34]. Large-scale cultivation of microalgae is generally carried out in a two-stage culture mode in a raceway pond and/or a photobioreactor [35, 36]. With these methods, AX content in H. pluvialis can reach 3.8%–5.0% dry cell weight (DCW) [37] or even higher. For example, AstaReal Inc., the famous manufacturer of AX cosmetics, uses a H. pluvialis strain with AX content as high as 60mg/g DCW. However, the long culture period, high cost, and harsh culture

228 Chapter 12 Table 12.1: Comparison of three sources of astaxanthin in the market.

Recent developments in astaxanthin production from Phaffia rhodozyma 229 conditions are the fatal weaknesses of H. pluvialis, and what’s more, the percentage of extracted AX in the total AX content is small [17]. Compared with H. pluvialis, P. rhodozyma is another major natural source of AX in the market. Although the content of AX in P. rhodozyma wild strain is not high [11] and the fermentation technology requirements are relatively high (such as low temperature and high energy consumption) [16], P. rhodozyma has some characteristics in the production of AX that are not found in H. pluvialis. P. rhodozyma does not require light and can quickly carry out heterotrophic metabolism with various sugars as carbon sources [10]. In addition, the yeast cells are rich in protein, minerals, and vitamins, which can be processed to form yeast extract for use in food and pharmaceutical industries [38]. P. rhodozyma AX is superior to H. pluvialis AX and synthetic AX in some aspects of production, but its low AX content leads to high production costs, which severely limits AX commercial development.

3 Biosynthesis of astaxanthin from P. rhodozyma P. rhodozyma is a red yeast belonging to the Basidiomycetes [39]. About 60years ago, Hermazn Phaff et al. discovered this strain in the secretions from the birch wounds in high-altitude forests in Alaska, USA, and Japan [16]. Later, Miller identified the strain and named it Rhodozyma montanae because it was orange-red in color and was found in the mountains [40]. Soon after, to commemorate Phaff’s outstanding contribution to yeast biology, people named this strain “Phaffia rhodozyma.” In 1995, Golubev found that P. rhodozyma has sexual state and asexual state, namely, Xanthophyllomyces dendrorhous and P. rhodozyma, respectively [39]. The characteristic that P. rhodozyma can accumulate AX was discovered by Andrews et al. in 1976 [41]. AX is the most abundant of the more than 10 carotenoids produced by P. rhodozyma, accounting for more than 70% [42]. Carotenoids are classified into xanthophylls (high degree of oxidation and strong polarity) and carotenes (low degree of oxidation and low polarity) [43]. Xanthophylls are kinds of oxygenated molecules (such as AX) containing hydroxyl and methoxy group, which are modified by a series of cyclization, oxidation, and other structural modification of carotenes. On the contrary, carotenes are kinds of strict hydrocarbons (such as lycopene). AX is the main end product of carotenoid synthesis in P. rhodozyma, whose synthetic pathway has been extensively studied (Fig. 12.1). It is mainly divided into three stages. The first stage is the synthesis of the precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which have different synthetic routes in different organisms. In the majority of prokaryotes and plant plastids, these two substances are produced with pyruvate and glyceraldehyde-3-phosphate as precursors via the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway [44] while produced by mevalonate (MVA) pathway in most eukaryotic organisms such as P. rhodozyma [45].

Fig. 12.1 Biosynthesis of AX from P. rhodozyma (X. dendrorhous). AACT, acetoacetyl-CoA thiolase; HMGR, 3-hydroxy-3-methyl-glutaryl-CoA reductase; HMGS, 3-hydroxy-3-methyl-glutaryl-CoA synthase; MK, mevalonate kinase; MPDC, mevalonate pyrophosphate decarboxylase; PMK, phosphomevalonate kinase; H (discontinuous line) the hydroxylase activity of cytochrome P450 monooxygenase; K (discontinuous line) the ketolase activity of cytochrome P450 monooxygenase [7].

Recent developments in astaxanthin production from Phaffia rhodozyma 231 In the MVA pathway, two acetyl-CoAs are condensed into acetoacetyl-CoA by acetoacetyl-CoA thiolase (AACT), followed by synthesizing 3-hydroxy-3-methyl-glutarylCoA (HMG-CoA) through HMG-CoA synthase (HMGS). Subsequently, HMG-CoA is reduced to form MVA by HMG-CoA reductase (HMGR). HMG-CoA reductase is encoded by the HMGR gene, of which the upregulated expression will significantly increase the carbon flux of isoprene pathway, so in the MVA pathway, this reaction (HMGR) is the rate-limiting step [46]. Thereafter, MVA is converted into IPP (C5) by three enzymes of mevalonate kinase (MK), phosphomevalonate kinase (PMK), and mevalonate pyrophosphate decarboxylase (MPDC) [46], followed by forming DMAPP through IPP isomerase (encoded by the idi gene). At this point the carotenoid precursors are formed. The second stage is the sequential addition of IPP on the basis of precursors to condense and finally produce phytoene. First, one molecule of IPP and one molecule of DMAPP are combined into geranyl pyrophosphate (GPP, C10) by prenyltransferase (ispA) [16]. Subsequently, GPP is added one molecule of IPP in turn by farnesyl pyrophosphate synthase (FPP synthase, ispA) and geranylgeranyl pyrophosphate synthase (GGPP synthase, crtE), firstly synthesizing FPP (C15) and next producing GGPP (C20). Thereafter, phytoene synthase (CrtB or PSY, crtYB) ligates two GGPP by tail to tail to form the first colorless carotenoid phytoene (C40) containing only three conjugated double bonds [7]. In the third stage, various carotenoids are produced by further structural modification, including desaturation, cyclization, hydroxylation, and ketolation [47]. First the phytoene produced in the last stage is gradually dehydrogenated to form red lycopene by the phytoene desaturase encoded by crtI gene. The intermediate of this step from phytoene to the final lycopene with 13 double bonds is different in different organisms. In the alga such as H. pluvialis, yellow ζ-carotene is produced [45], while in most bacteria and fungi, yellow neurosporene is produced [16]. Next step is the cyclization modification process of carotenoids, during which lycopene forms γ-carotene through the cyclization of one terminal isoprene group by lycopene cyclase (crtYB) [48] and is further cyclized into β-carotene. There is a complex process from β-carotene to AX requiring the addition of a hydroxyl group and a keto group on each of β-ionone ring [7]. In most AX-producing microorganisms (such as algae), this process requires two separate enzymes, namely, β-carotene hydroxylase (CrtZ or BCH, crtZ) and β-carotene ketolase (CrtW or BKT, crtW) [49], while P. rhodozyma only needs one enzyme, AX synthase (CrtS or Asy, crtS), called cytochrome P450 monooxygenase [16]. In addition, Alcaino et al. [50] isolated the P450 reductase (crtR) in P. rhodozyma, which has been proven to provide the electrons needed for oxidation when the Asy catalyzes, playing a supporting role. To verify the important role of this enzyme in the conversion of β-carotene to AX, Alcaino et al. knocked out the crtR gene and found that the mutant could only accumulate β-carotene without AX, thus indicating that this enzyme is necessary [50]. Ukibe et al. [51] found that this was also the case in Saccharomyces cerevisiae engineering bacteria.

232 Chapter 12 In addition to AX as main end product, An et al. [52] also proposed a monocyclic pathway in the carotenoid synthetic pathway of P. rhodozyma, in which neurosporene or lycopene formed torulene through the enzyme encoded by crtI and crtYB and then produced HDCO (3hydroxy-30 ,40 -didehydro-β,ψ-carotene-4-one) and DCD (3,30 -dihydroxy-β,ψ-carotene4,40 -dione) through the combination of P450 monooxygenase and P450 reductase. It has been reported that the monocyclic pathway can eventually be converted into AX [53]. In general the entire pathway from carotenoid precursors to AX by P. rhodozyma involves six genes: ispA, crtE, crtYB, crtI, crtS, and crtR. Compared with the synthesis of AX from H. pluvialis, P. rhodozyma involves fewer enzymes and is more economical [42].

4 Accumulation strategies of astaxanthin from P. rhodozyma Wild P. rhodozyma can naturally synthesize AX, but the content is very low, leading to high cost, so it cannot well meet the demand of industrial production. Naturally derived AX is not economically competitive compared with synthetic AX, only niche applications, which is the main limiting factor for the commercial development of AX [16]. Therefore a large number of experiments to improve the AX content of P. rhodozyma have been carried out. Whether mutagenesis, genetic engineering, optimization of nutrients (including low-cost raw material), optimization of traditional culture conditions, or fermentation promoters (such as glutamate), all are considered in the following.

4.1 Strain improvement 4.1.1 Random mutagenesis The strain modification is to improve the productivity of the original strain. In general, strain modification can be carried out by random mutation, protoplast fusion, and genetic engineering, among which there are few researches on protoplast fusion of P. rhodozyma, so it is not summarized here (Table 12.2). Traditional random mutagenesis, that is, nontargeted mutagenesis, is one of the most direct and simple ways to increase the product yield of strain, which can increase the mutation rate of DNA molecules and accelerate the breeding process. Treated with physical methods or chemical reagents, cells in the logarithmic growth phase are expected to generate desirable traits. Common random mutagenesis methods include ethyl methane sulfonate (EMS), nitrosoguanidine (NTG) mutagenesis, ultraviolet (UV) radiation, and 60Co γ-radiation. UV radiation is a relatively common physical mutagenesis method. After UV radiation, AX content of P. rhodozyma changed from 0.19 to 0.36 mg/g DCW [57]. In 2016 Hina et al. [65] used UV radiation to irradiate P. rhodozyma, obtaining a strain of high-yield AX. Certainly, UV radiation is also common in other organisms for breeding [66]. In addition to UV radiation, it has been reported that a carotenoid-overproducing P. rhodozyma was obtained by low-dose 60Co γ-radiation below

Table 12.2: Examples summary of Phaffia rhodozyma (Xanthophyllomyces dendrorhous) strain improvement. Parent strain ZJB 00010 JCM9042 JMU-668 JMU-668 JMU-668 JMU-668 DSM 5626 DSM 5626 CBS6938 CBS6938 CGMCC2.1557 CBS6938

NBRC10129 NBRC10129 NBRC10129 CBS6938

MK19 NBRC10129 a

Mutant strain E5042

Method

Low-energy ion beam implantation MK19 NTG and 60Co γ-radiation JMU-MVP14 EMS JMU-MVP14 EMS JMU-MVP14 EMS JMU-MVP14 EMS 26 UV UV radiation 9 BE Ethidium bromide AXJ-20/crtYB/ NTG and crtYB, crtS asy overexpression AXJ-20/crtYB NTG and crtYB overexpression YZUXHONG UV, NTG and 60Co 686 γ-radiation AXG-13/#5221 NTG and stepwise transformation of key genes Padh4-crtE crtE overexpression by promoter Padh4 acaT/hmgS/ Triple hmgR overexpression acaT/hmgS/ Combinatorial hmgR/crtE/crtS overexpression CBS6938Three copies of (YB3asy) crtYB and one crtS gene CSR19 crtS overexpression ΔCYP61(, ) Double deletion of cyp61 genes

Scale

Biomass (g/L)

AX (mg/g)

AX (mg/L) a

50 L batch

30.7

2.510

77.06

Flask

19.85a

1.3

25.8

Flask 7 L batch 7 L fed-batch 1 m3 fed-batch Flask Flask Flask

2.75 11.2 32.81 85.11 3.6 4.23

6.01 6.47 4.94 3.29 0.36 0.37 4.7

16.53 72.47 155.99 279.96 1.29 1.58

1.3 L fed-batch Flask

9.7 10.25

Flask

2.24

References 2008 [54] 2010 [55] 2011 2011 2011 2011 2013 2013 2013

[56] [56] [56] [56] [57] [57] [58]

2013 [58] 22.96a

9.0

2014 [59] 2014 [53]

Flask

5.70a

0.43

2.45

2014 [60]

Flask

6.41a

0.39

2.5

2014 [61]

3.0

2014 [61]

Flask Flask

7.5 L batch Flask

0.53

25.8

0.98a

Data calculated from given data; AX, astaxanthin; NTG, nitrosoguanidine; EMS, ethyl methane sulfonate; UV, ultraviolet.

2014 [62]

25.3 1.65

2015 [63] 2016 [64]

234 Chapter 12 10 kilogray, whose carotenoid content can reach 3.3mg/g DCW and was 50% higher than parent strain [67]. Only if the yeast cells produced enough carotenoids can they survive by scavenging the oxygen radicals produced by the radioactive decomposition in the water [68]. Additionally, Liu et al. [54] used a low-energy ion beam implantation method to induce the original strain to obtain a genetically stable strain with an AX content from 1.114 to 2.512mg/g DCW, which was 125.5% higher than the original strain. In recent decades the emerging microbial breeding method, atmospheric and room temperature plasma (ARTP) mutagenesis has stimulated the research enthusiasm of researchers due to its convenient operation, short time, safety, and high mutation rate. It is widely used in bacteria, fungi, and plants to increase the yield of a target product or the tolerance of a strain to a substrate [69]. The present study has reported that AX accumulation in S. cerevisiae was promoted by a combination of metabolic engineering and ARTP mutagenesis, and the yield of AX obtained by fermentation in a 5-L bioreactor was 217.9 mg/L [70]. For chemical mutagenesis, EMS was used to treat P. rhodozyma, followed by producing free radicals to eliminate the low-yield strains using 0.5% H2O2 solution combined with UV irradiation. The result of the aforementioned method showed that AX content of the selected high-yield strain was 6.01mg/g, which was about nine times higher than that of the parent strain (0.59mg/g), but the biomass was decreased by 33.7% [56]. The problem of biomass decline is one of the problems of many mutagenesis methods. Other problems include the genetic instability of mutant strains, the accumulation of some useless intermediaries, and bad influence of normal physiological activity and metabolism [7]. As a single mutagen cannot achieve satisfactory results, it turns out that complex synergistic mutagenesis methods are sometimes more effective. For instance, it has been reported that NTG and 60Co γ-rays were combined to treat wild P. rhodozyma, of which the AX content was improved from 150 to 1300 μg/g [55]. In addition, treated by three types of mutagenesis methods (UV, NTG, and 60Co γ-radiation), P. rhodozyma accomplished an increase in AX content from 0.13 to 2.24 mg/g DCW [59]. Having been mutated, the high-yield AX strain needs to be screened out by screening agents. Generally an inhibitor that is not conducive to carotenoid synthesis is added to the plate medium, selecting out the strain that can resist the inhibitor according to colony color, size change, and the like. Screening agents used commonly are diphenylamine, 2-deoxyglucose, β-ionone, and antimycin A [7]. In addition, there are some high-throughput screening methods, such as the flow cytometer [71]. 4.1.2 Genetic engineering With the development of genetic and metabolic engineering, the AX synthesis pathway and the regulation mechanism of each gene are more and more clear. On the basis of the clear research of AX synthesis pathway in P. rhodozyma, most of the strain modification is carried out at the

Recent developments in astaxanthin production from Phaffia rhodozyma 235 gene level. In general, genetic modification enhances the metabolic flux of the precursors, directing it as far as possible toward the target product, mediating the metabolic flux to effectively convert the intermediate product to the final product [7]. Methods used commonly are (a) the overexpression of key enzyme genes (such as crtYB and crtS); (b) the supplementation with carotenoid-producing precursors (e.g., mevalonate); (c) the increase of metabolic flow toward precursors (e.g., GGPP); and (d) the downregulation of competitive synthetic pathways (such as ergosterol synthesis pathway). At the gene level, it is a key step to control the expression amount of key enzyme in controlling the entire metabolic flux. Generally speaking the target product can be increased by overexpressing the gene of the key enzyme, which can overcome key bottlenecks in the synthesis of AX. Using the strong promoter Padh4, the crtE gene encoding the rate-limiting enzyme (GGPP synthase) can be overexpressed, increasing the AX content in P. rhodozyma by 1.65 times compared with the control [60]. Also, it has been shown that phytoene synthase (crtYB) is a key enzyme in the carotenoid synthetic pathway, regulating the influx of carbon scaffolds, thereby controlling the synthesis of AX [72]. Furthermore the P450 monooxygenase (crtS) is the most important rate-limiting enzyme in the way from the β-carotene to AX, whose auxiliary enzyme is P450 reductase (crtR) [50]. The strain CSR19 (with ribosome binding site of P. rhodozyma crtS gene) overexpressing the crtS gene was constructed without antibiotic labeling, whose AX yield was 25.3mg/L and was 1.34 times that of the parent strain [63]. In addition, compared with the original strain, it was found that nearly 20% of the AX synthase gene (crtS) sequence from the two mutant strains mutated by 60 Co γ-ray was changed [73]. The overexpression of a single gene may not have a good effect, so a research report has developed a multigene expression system in P. rhodozyma with three strong promoters (Pact, Padh4, and Ptpi). With these, three key enzyme genes of the MVA synthetic pathway (acaT/ hmgS/hmgR) were overexpressed, and a strain was obtained with AX yield of 2.5mg/L, which was higher than each single gene overexpressing strain and was 1.6 times that of the parent strain [61]. In addition, the triple overexpressed genes combined with crtE and crtS genes can synergistically promote the accumulation of AX, and the AX yield was 2.1 times higher than the parent strain without reducing the biomass [61]. Except for single mutagenesis or genetic engineering, it has been reported that mutagenesis and genetic engineering were combined to treat P. rhodozyma, achieving a good result [53, 58]. In addition to the modification of related genes in the AX synthetic pathway, the anabolism of other products in vivo may also affect the synthesis of AX. It has been reported that Yamamoto et al. [64] knocked out the double CYP61 gene sequence encoding C-22 sterol desaturases in P. rhodozyma, which prevented FPP from flowing to the ergosterol synthesis direction and increased the metabolism of GGPP and carotene. Also the feedback inhibition of the MVA pathway by the enzyme was removed, and as a result, although the ergosterol yield was

236 Chapter 12 decreased, the AX yield was increased to 1.4 times that of the parent strain. Additionally, it has been reported that the synthesis of AX in P. rhodozyma was affected by the synthesis of proteins and fatty acids in vivo [74].

4.2 Medium components After the breeding of P. rhodozyma, some changes may occur due to the anabolic regulation of AX, which leads to different requirements for nutrient components and culture conditions, so these external conditions are required to be selected and optimized. In addition, the choice of medium components (such as carbon sources, nitrogen sources, minerals, and vitamins) affects AX yield and industrial production cost. More and more agroindustrial wastes are being studied as substrates for AX production, not only for P. rhodozyma but also for algae [75]. To some extent, it not only reduces the production cost of AX but also recycles wastes and protects the environment in a higher sense, creating a green and ecological benefit. As one of the essential nutrient components of the medium, carbon source provides a carbon frame material for cell growth, forming a structural substance of the cell, or utilized as a metabolic substrate. In many studies, it has been reported that the utilization of different kinds and concentrations of sugars by P. rhodozyma is different, and the AX yield is also different. Generally, high glucose concentration inhibits the synthesis of AX [55]. Recently a study analyzed the regulatory mechanism of high glucose concentration (>100 g/L) on cell metabolism and transcription levels [76]. The effects of glucose and nonfermentable carbon source succinate on the synthesis of AX in P. rhodozyma have been studied. This experiment has showed that when glucose was the sole carbon source, AX was induced to synthesize at the end of exponential growth phase, indicating that the growth of yeast is not related to AX synthesis in this case, and when succinate was the sole carbon source, the synthesis of AX proceeds with the growth of yeast until the end of the stable phase. As a result, both the AX content and biomass of the latter were higher than the former [77]. It has also been reported that glucose, xylose, and arabinose were added together to the medium, among which glucose was the first to be used by P. rhodozyma, followed by xylose and arabinose, but AX yield produced by P. rhodozyma (mg AX per gram of sugar consumed) was the highest in xylose, followed by arabinose, and the lowest in glucose [78]. These examples all suggest that glucose is not the most favorable carbon source for AX accumulation. Not long after discovering that P. rhodozyma has the ability to synthesize AX, scientists have been working on using low-cost agroindustrial wastes as carbon sources or nitrogen sources to cultivate yeast to produce AX [79, 80]. What’s more, some wastes are rich in trace elements, such as minerals and vitamins, which can promote the growth of yeast biomass and the synthesis of AX [78].

Table 12.3: Examples summary of optimization of Phaffia rhodozyma (Xanthophyllomyces dendrorhous) medium components. Strain

Carbon source

JTM 185 JTM 185 Unknown Unknown ATCC 24230 NRRL Y-17268 5308 YZUXHONG686 NRRL Y-17268 AS 2.1557 ATCC 74219 ATCC 74219 Y1655 Y119 YM119 ATCC 74219 ATCC 74219 ATCC 74219 ATCC 24202

Barley straw hydrolysate SCBH Cassava residues Pineapple Peel Succinate Glucose, raw glycerol Glucose Sucrose Rice parboiling wastewater SCBH PSM PSM Glucose Jerusalem artichoke extract Sugarcane molasses SSJ SSJ SSJ, detoxificated SSBH MPE

Nitrogen source YE

Scale Flask

YE Flask YE and (NH4)2SO4 Flask YE and ME Flask NH4NO3 12 L batch ME, YE, peptone Flask and PRE (NH4)2SO4 and 25 L fedKNO3 feeding, YE batch DCSLP Flask ME, peptone, and Flask sucrose Peptone, and YE Flask YE Flask YE and (NH4)2SO4 Flask Residual-brewery Flask YE and peptone Peptone 3L fed-batch

Biomass (g/L)

AX (mg/g)

AX (mg/L) a

References

12.78

0.23

2.94

7.16

0.36

12.8 5.39 8.3

0.34a 0.3041 0.2755

2.58a 2.98 4.39 1.64a 1.7

2011 2011 2011 2011 2012

5.1

0.969

4.94a

2013 [86]

16.36a 0.84a

2.56 6.288

41.89a,c 5.3

2014 [59] 2015 [87]

12.3 11.52a 6.31a

0.516b 2.256 3.565

6.10b 26 22.5 0.099

2015 2015 2015 2017

83.6

13.3b

982.5b

2017 [90]

45.55b

2017 [91]

3.86a

b

2011 [78] [78] [83] [84] [77] [85]

[88] [81] [81] [89]

YE

Flask

11.81

YE and urea YE, urea and (NH4)2SO4 YE and urea

2 L batch 2 L batch

29 34.9

2.49 1.53a

65.4 53.3

2018 [1] 2019 [92]

Flask

23.6

2.07

48.89

2019 [92]

CSL, YE

Flask

9.53a

0.22b

2.06b

2019 [82]

AX, astaxanthin; YE, yeast extract; ME, malt extract; SCBH, sugarcane bagasse hydrolysate; CSL, corn steep liquor; SSJ, sweet sorghum juice; SSBH, sweet sorghum bagasse hydrolysate; MPE, mesquite pods extract; PSM, partially saccharified mussel wastewater; DCSLP, dried corn steep liquor powder; PRE, parboiled rice effluent. a Data calculated from given data. b Data refer to carotenoid instead of astaxanthin. c Data based on the proportion of astaxanthin in carotenoids.

238 Chapter 12 There are numerous examples of the use of agroindustrial wastes as fermentation substrates for the culture of P. rhodozyma, such as partially saccharified mussel processing wastewater (twice AX yield that of control) [81], mesquite pods extract, and corn steep liquor (1.4 times AX yield that of control) [82] (Table 12.3). However, not all agricultural wastes have positive impact on the AX yield, for which one of the main reasons is that lignocellulosic hydrolysates often contain furfural, phenolic compounds or else that inhibit yeast growth or the AX synthesis [93, 94]. Therefore it is sometimes necessary to detoxify lignocellulosic hydrolysates [92]. The nitrogen source is the external source of nitrogen-containing substances such as proteins and nucleic acids in vivo, which is extremely important for cell growth. It has been reported that the cultivation of P. rhodozyma was carried out in a 25-L fermenter with the supplementation of (NH4)2SO4 and KNO3, increasing AX yield to 969μg/g, while the supplementation of glucose and peptone did not have much effect on AX yield [86]. Using low-cost dried corn steep liquor powder as nitrogen source and sucrose as carbon source, optimal conditions were obtained by central composite designed experiments and response surface methodology, under which AX yield was 41.89 mg/L [59]. Furthermore the C/N ratio of the medium is also a key factor in cell growth and AX accumulation. As stated in previous reports, high carbon loadings inhibited the growth of P. rhodozyma by reducing the amount of available nitrogen [95]. A C/N ratio below 5 has a negative effect on the AX generation of P. rhodozyma, indicating that abundant nitrogen promotes cell growth but inhibits the activity of enzyme that converts β-carotene to AX, and in contrast a high C/N ratio can affect cell growth, thus affecting the AX synthesis [95]. Thus there is a study reporting that the AX synthesis of P. rhodozyma was divided into two stages, maintaining the low C/N ratio in the first stage to promote cell growth and switching to high C/N ratio in the second stage to promote the synthesis of AX [96]. In addition, differential proteomics analysis of P. rhodozyma indicated that under different C/N ratios, proteins with the biggest expression change were mainly related to carotene generate metabolism and carbohydrate metabolic pathways [97].

4.3 Cultivation conditions Another effective strategy to increase AX yield is to optimize fermentation conditions such as temperature, pH, dissolved oxygen (DO), inoculum rate, and light (Table 12.4). Temperature is one of the most important culture conditions to control cell growth. P. rhodozyma is a moderate psychrophilic yeast that can grow at 0–27°C [16], and the appropriate temperature for cell growth and AX synthesis is generally at 18–22°C [105]. However, energy consumption of cooling process is high, resulting in high cost, which is not conducive to industrial production. Therefore it is necessary to study the effects of temperature on P. rhodozyma cell growth and AX generation, and what’s more, to study the process conditions that AX can be produced at a moderate temperature (25–37°C).

Recent developments in astaxanthin production from Phaffia rhodozyma 239 Table 12.4: Examples summary of Phaffia rhodozyma (Xanthophyllomyces dendrorhous) cultivation conditions and fermentation promoters. Strain ENM5 ENM5 VKPM Y2476 VKPM Y2476 VKPM Y2476 VKPM Y2476 13B

5308 10BE

ATCC 24060

AXJ-20 CBS 6938 MTCC 7536 UV3-721

Condition

Fermentation method

NFlask hexadecane H2O2 feeding Flask (SPCP) 1000 lx UV Flask light 1000 lx white Flask light White light 10 L (80 W) White light 800 L (324 W) Flask 670 lx illumination intensities 2500 lx/cm2 6 L fed-batch light intensity 707 lx Flask illumination intensities Solid state pH, temperature, fermentation moisture content, inoculation rate Low pH 20L fed-batch 1. 20°C, 18h; 5 L batch 2. 30°C, 30 h Sonication Flask Glutamate Flask feeding

Biomass (g/L) 9.2

AX (mg/g) 1.58

a b

AX (mg/L) 14.5

a

References 2006 [98]

54.8 53

1.06 4.4

58.3 235

2007 [99] 2010 [100]

55

4.0

221

2010 [100]

88

4.7

420

2010 [100]

86

4.1

350

2010 [100]

3.9

0.41

1.60

2013 [101]

0.93 5.66

0.49

2013 [86] 2.8

2014 [102]

109.23 μg/g wheat wastes

2016 [103]

114 g/kg CB 4.12

6.17 0.116

0.7 g/kg CB 0.48b

2017 [104] 2018 [105]

4.98b 3.1

1.728 1.14

8.6 3.53b

2018 [106] 2019 [107]

AX, astaxanthin; UV, ultraviolet; SPCP, semicontinuous perfusion culture process; CB, culture broth. a Data refers to carotenoid instead of astaxanthin. b Data calculated from given data.

The optimal growth temperature of some strains was changed after mutagenesis, genetic modification, or environmental induction, such as MK19 (25°C) [55], NRRL Y-17268 (25°C) [85], and YZUXHONG686 (23°C) [59]. P. rhodozyma was cultivated with a two-step process, first preincubated at 20°C for 18h and then transferred to 30°C for 30h. As a result, compared with the normal culture (20°C, 96h) (140.28μg/g DCW), the AX content (116.42μg/g DCW) of two-step process was lower. Nevertheless, the cell yield (Yx/s) and product yield (Yp/s) of two-step process did not decrease significantly, even higher, indicating that the moderate

240 Chapter 12 temperature did not have much effect on AX production. Additionally, the author and coworkers of the aforementioned experiment also monitored the transcription levels of AX-related synthetic genes at different culture times, finding that the expression levels of AX-related genes were negatively correlated with temperature in the cells cultured at two stages compared with normal culture [105]. In addition, pH is also a key factor. It has been reported that pH 5.0 was conducive to the accumulation of AX, while pH 6.0 was conducive to cell growth [108]. However, even belonging to the same genus, strains from different sources have different optimal pH for cultivation, such as YM119 (pH 5.0) [91], JMU-MVP14 (pH 6.0) [56], ATCC 74219 (pH 5.46) [1], Y119 (pH 5.0) [90], and CGMCC As 2.1557 (pH 9.0) [109]. In previous reports the cultivation of P. rhodozyma was divided into two phase, and when the pH was adjusted from 5.5 (growth phase) to 3.5 (maturation phase), AX yield can reach 0.7g/kg culture broth, achieving a AX accumulation rate of 3.3mg/kg culture broth/h [104]. Also, lowering the pH and increasing the concentration of trace elements and vitamins during the maturation phase can retard the significant decrease in cell concentration at this stage [104]. P. rhodozyma, isolated from the trunk secretions in the cold mountains, was exposed to intense UV radiation for a long time, producing ROS at the cellular level [101]. Also the trees colonized by P. rhodozyma contained some compounds that can form singlet oxygen under UV radiation [102]. These ROS can destroy the surrounding molecules by capturing hydrogen molecules or providing electrons or free radicals [110]. P. rhodozyma produced AX to protect itself from oxidative damage instead of synthesizing detoxifying enzymes against ROS [41]. AX can eliminate free radicals and singlet oxygen and also can induce the production of erythroid 2-related factor 2 (Nrf-2), which can prevent cellular oxidative stress damage [111]. Therefore light is very important for the synthesis of AX, H. pluvialis AX in particular. Numerous studies have reported the effects of light on AX synthesis, including light of different illumination intensity or types [112, 113]. At the 800-L bioreactor, treated with white light (324 W) and glucose feeding, P. rhodozyma achieved a maximum biomass (100g/L) and AX yield of 350 mg/L [100]. It has been reported that β-carotene yield was highest under UV light, while AX yield was highest under red/green light [110]. O2, as a substrate, is directly involved in the synthesis of AX by aerobic yeast P. rhodozyma. Therefore the amount of dissolved oxygen (DO) has a great influence on the accumulation of AX. Experiments have shown that the accumulation of AX is related to the rate of oxygen transfer. If DO concentration is lower than the critical DO concentration (with 10%–20% air saturation), cell growth and carotenoid production will be inhibited [114, 115]. Accordingly, higher O2 supply is beneficial to the biosynthesis of AX and the proportion of AX in the total amount of carotenoids. When the initial volumetric oxygen transfer coefficient was 148.5/h, P. rhodozyma obtained the maximal biomass (19 g/L) and AX yield (14 mg/L) [114].

Recent developments in astaxanthin production from Phaffia rhodozyma 241 For shake flask fermentation, medium volume and shaking speed are key parameters for DO. The results showed that during the 250-mL flask fermentation, the 25-mL loading volume was most conducive to the synthesis of AX with the biomass of 11.81g/L and the carotenoid yield of 45.55mg/L, which was 31.08 times higher than that of 95-mL loading volume [91]. Optimized by statistical central composite experimental design, the optimal condition for AX production was pH 4.4, temperature 21°C, 4% v/v inoculum, and 205-rpm shaking speed [106].

4.4 Fermentation promoters H. pluvialis can induce the synthesis of AX by adding some fermentation promoters through abiotic stress conditions [30]. Similarly, there are some examples of P. rhodozyma using some fermentation promoters or some additional environmental stress to promote AX accumulation (Table 12.4). For example, adding plant extracts (carrot, potato, and barley malt broth) to medium, it was found that carrot extract was the most effective to improve AX yield [116], which may be attributed to the presence of some trace elements or growth factor. In addition, the addition of 9% (v/v) n-hexadecane increased oxygen transfer rate by 90% and carotenoid yield by 57.6% [98], which the main promotion mechanism is that n-hexadecane is an oxygen carrier, promoting oxygen transfer and consequently promoting AX synthesis [117]. Besides, H2O2 can stimulate AX synthesis, increasing AX yield to 58.3mg/L, which was mainly related to the oxidative stress response of yeast to H2O2 [99]. Fed with glutamate during fermentation, P. rhodozyma obtained the maximum AX content of 1.14 mg/g, which were 40.7% higher than those of the control with little effect on cell growth, suggesting that nitrogen supplementation is beneficial to the accumulation of AX during cell growth [107]. Later the authors investigated the regulatory mechanisms of glutamate promoting AX synthesis through comparative proteomics. Glutamate, a key metabolite linking carbon and nitrogen metabolism, not only is a nitrogen source in cells but also participates in the tricarboxylic acid (TCA) cycle in the form of 2-keoglutarate. The addition of glutamate can promote the consumption of glucose, causing fluctuations in ROS production and TCA cycle and amino acid metabolism and then channeling isocitrate flux into the glyoxylate cycle, thus increasing the utilization of acetyl-CoA. Therefore more acetyl-CoA synthesizes carotenoid precursors via MVA pathway and accumulates AX [107]. In recent years, ultrasound not only has achieved good results in mutagenic breeding but also has been effective in improving the kinetics and productivity of biochemical processes. The mechanism of ultrasonic wave is very complicated, mainly attributed to the microturbulence formed by cavitation bubbles. Cavitation bubbles change the structure of the membrane in the cell, and the microturbulence not only enhances mass transfer in the system but also causes conformational changes in the secondary structure of the intracellular enzyme. These effects are manifested in an increase in cellular metabolism and an increase in product yield; thus

242 Chapter 12 ultrasound can be used in breeding, promoting biochemical processes, or obtaining a high-titer product. Induced by ultrasound during fermentation, P. rhodozyma generated more AX, of which the content increased from 1.36 to 1.728 mg/g [106].

5 Potential applications of astaxanthin from P. rhodozyma AX is a natural carotenoid, having abundant physiological effects, such as protecting vision, lowering blood fat, lowering blood sugar, lowering blood pressure, fighting inflammation, preventing heart disease, treating cardiovascular disease, fighting cancer, delaying aging, and enhancing immunity. With these physiological effects, AX is widely used in food, health care products, cosmetics, and pharmaceutical industries (Table 12.5). Furthermore, it is widely used in the aquaculture industry due to its effect of pigment deposition and its improvement of animal reproduction and growth performance. The market value of AX reached $288.7 million Table 12.5: Examples summary of patent applications for astaxanthin. Patent no. US20180098549 AU2019100450 CN109820746

CN109820837

CN109820905

US20190174797

WO2019138995 WO2019111896 CN107029020 CN105503383 CN108208829 US20170266095

Title

Application field

Baked food produced from Food astaxanthin containing dough Dog feed with astaxanthin and Dog feed preparation method thereof Food/beverages/ Lotion containing natural cosmetics/skincare astaxanthin ester and preparation products/medicines method thereof Microcapsule containing natural Food/beverages/ astaxanthin ester and preparation cosmetics/skincare method thereof products/medicines Pharmaceutical composition for Medicines/healthcare treating insomnia and application products/food of pharmaceutical composition Method for increasing the Fish feed utilization of soybean protein by salmonid fish Dry eye remedy Medicines Antipruritic agent Medicines Astaxanthin health product having Health product antioxidation function Application of astaxanthin to water- Agricultural fertilizer soluble fertilizer Astaxanthin nutrition supplement Nutrition supplement Cosmetics/health Method for extracting and supplement producing useful substance and soap, cosmetics, or health supplement having said useful substance as main component

References [118] [119] [120]

[121]

[122]

[123]

[124] [125] [126] [127] [128] [129]

Recent developments in astaxanthin production from Phaffia rhodozyma 243 in 2017 and is expected to reach $426.9 million in 2022 with a compound annual growth rate of 8.1% [130]. Added as a food additive to yogurt [131], orange juice [132], skimmed milk [132], and vinegar [133], AX makes foods show bright colors and more nutrients. In the market, there are many health products about AX, such as the natural AX soft capsule BioAstin produced by Cyanotech Corporation, which can prevent diseases caused by organ aging and prevent middle-aged and elderly people from cardiovascular disease and neurological diseases. AX can also be added to health care products or medicines to protect vision, preventing and treating various types of eye damage, such as age-related macular degeneration, a progressive eye disease that is the leading cause of severe vision loss and blindness in adults over 60years old [134]. In addition, there are various experiments showing that AX has certain effects on treating skin diseases [135], obesity [136], gastric ulcers [137], diabetes [138], cardiovascular diseases [139], and neurodegenerative diseases [140], laying the important position of AX in the pharmaceutical industry. In the cosmetics industry, AX is popular among people, especially women, for its unique antioxidation, anti-UV, and antiaging properties. So far, AX has been added to masks, lipsticks, sunscreens, and essences [90, 141], developed into many functional cosmetics. Approved by the FDA for use as feed additives, AX has also been used in aquaculture, poultry farming, and pet industry. In addition to coloring, AX can also be used as a nutrient to enhance the immunity of aquatic animals, improve the survival rate of aquatic animals in early childhood, and enhance fertility [6]. These aquatic products that have accumulated AX can be absorbed by human body through the food chain, which can improve sleep, protect eyesight, and resist fatigue. AX was used as a feed additive to feed chickens, significantly improving the laying rate and immunity and decreasing mortality, making egg yolk color more attractive [142].

6 Conclusions AX is one of the most important carotenoids, whose current market value ($288.7 million in 2017) is only slightly lower than capsanthin ($300.0 million in 2017), and with the expansion of AX application and market demand, it is expected to surpass capsanthin market by 2020 [106]. However, natural AX, especially H. pluvialis AX, has only a niche market instead of economic competitive advantage over synthetic AX because of its high price. Compared with the harsh cultivation conditions of H. pluvialis, P. rhodozyma as the sole source of yeast for AX production has become a promising source of AX due to its clear metabolic pathways and simple cultivation conditions. Up to now a lot of studies have been done on the production and application of P. rhodozyma AX, making some progress, which greatly increased AX yield and reduced the production cost.

244 Chapter 12 However, compared with H. pluvialis, the content and yield of AX from P. rhodozyma still cannot meet the needs of large-scale industrial production. Therefore future studies are expected to make more use of the means of omics (genomics, transcriptomics, proteomics, and metabolomics) to analyze the mechanism of various regulatory factors promoting or inhibiting AX synthesis at the microlevel, so as to rationally design experiments to regulate the synthesis of AX at the macrolevel. Not only the modification of strains but also the promotion of external conditions, or the synergistic effect of both, are expected to be closely around the metabolic pathway of P. rhodozyma AX, so as to make the metabolic flux flow to the direction of AX synthesis as much as possible. In addition, the downstream processing technology of AX production is another important technology to increase AX yield and reduce production cost, playing an important role in the application field of AX. Unfortunately the safety of P. rhodozyma AX for human consumption has not been directly proven [9], which is a major stumbling block to the development of the P. rhodozyma AX market. Therefore there is still plenty of research space and challenges for the market development of P. rhodozyma AX.

Acknowledgments This study gratefully acknowledges the financial support of the National Natural Science Foundation of China, China [grant no. 51878291], and Guangzhou Science and Technology Program, China [grant no. 2014 Y2-00515].

References [1] Stoklosa RJ, Johnston DB, Nghiem NP. Utilization of sweet Sorghum juice for the production of Astaxanthin as a biorefinery co-product by Phaffia rhodozyma. ACS Sustain Chem Eng 2018;6:3124–34. https://doi.org/ 10.1021/acssuschemeng.7b03154. [2] Anuradha CV. Astaxanthin, a marine carotenoid against hepatic oxidative stress: a systematic review. In: Masucci S, editor. The liver. Amsterdam: Elsevier; 2018. p. 211–28. https://doi.org/10.1016/B978-0-12803951-9.00018-5. [3] Kurashige M, Okimasu E, Inoue M, Utsumi K. Inhibition of oxidative injury of biological membranes by astaxanthin. Physiol Chem Phys Med NMR 1990;22:27–38. https://doi.org/10.1016/0098-2997(90)90003-K. [4] Fakhri S, Abbaszadeh F, Dargahi L, Jorjani M. Astaxanthin: a mechanistic review on its biological activities and health benefits. Pharmacol Res 2018;136:1–20. https://doi.org/10.1016/j.phrs.2018.08.012. [5] Ambati RR, Moi PS, Ravi S, Aswathanarayana RG. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications - a review. Mar Drugs 2014;12:128–52. https://doi.org/10.3390/ md12010128. [6] Lim KC, Yusoff FM, Shariff M, Kamarudin MS. Astaxanthin as feed supplement in aquatic animals. Rev Aquac 2018;10:738–73. https://doi.org/10.1111/raq.12200. [7] Barredo JL, Garcı´a-Estrada C, Kosalkova K, Barreiro C. Biosynthesis of astaxanthin as a main carotenoid in the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous. J Fungi 2017;3:44. https://doi.org/ 10.3390/jof3030044. [8] Lorenz RT, Cysewski GR. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol 2000;18:160–7. https://doi.org/10.1016/S0167-7799(00)01433-5. [9] Capelli B, Talbott S, Ding L. Astaxanthin sources: suitability for human health and nutrition. Funct Foods Health Dis 2019;9:430–45. https://doi.org/10.31989/ffhd.v9i6.584. [10] Xiao AF, Ni H, Cai HN, Li LJ, Su WJ, Yang QM. An improved process for cell disruption and astaxanthin extraction from Phaffia rhodozyma. World J Microbiol Biotechnol 2009;25:2029–34. https://doi.org/ 10.1007/s11274-009-0104-5.

Recent developments in astaxanthin production from Phaffia rhodozyma 245 [11] Visser H, Van Ooyen AJJ, Verdoes JC. Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous. FEMS Yeast Res 2003;4:221–31. https://doi.org/10.1016/S1567-1356(03) 00158-2. [12] Higuera-Ciapara I, F
elix-Valenzuela L, Goycoolea FM. Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr 2006;46:185–96. https://doi.org/10.1080/10408690590957188. [13] Davinelli S, Nielsen ME, Scapagnini G. Astaxanthin in skin health, repair, and disease: a comprehensive review. Nutrients 2018;10:1–12. https://doi.org/10.3390/nu10040522. [14] Yang Y, Kim B, Lee J. Astaxanthin structure, metabolism, and health benefits. J Hum Nutr Food Sci 2013;1:1–1003. [15] Capelli B, Bagchi D, Cysewski GR. Synthetic astaxanthin is significantly inferior to algal-based astaxanthin as an antioxidant and may not be suitable as a human nutraceutical supplement. Forum Nutr 2013;12:145–52. https://doi.org/10.1007/s13749-013-0051-5. [16] Schmidt I, Schewe H, Gassel S, Jin C, Buckingham J, H€ umbelin M, et al. Biotechnological production of astaxanthin with Phaffia rhodozyma/Xanthophyllomyces dendrorhous. Appl Microbiol Biotechnol 2011;89:555–71. https://doi.org/10.1007/s00253-010-2976-6. [17] Li J, Zhu D, Niu J, Shen S, Wang G. An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis. Biotechnol Adv 2011;29:568–74. https://doi.org/10.1016/j. biotechadv.2011.04.001. [18] Nguyen K. Astaxanthin: a comparative case of synthetic vs. natural production. Chem Biomol Eng Publ Other Work 2013;1:1–11. [19] Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H. Astaxanthin, a carotenoid with potential in human health and nutrition. J Nat Prod 2006;69:443–9. https://doi.org/10.1021/np050354+. [20] Niamnuy C, Devahastin S, Soponronnarit S, Vijaya Raghavan GS. Kinetics of astaxanthin degradation and color changes of dried shrimp during storage. J Food Eng 2008;87:591–600. https://doi.org/10.1016/j. jfoodeng.2008.01.013. [21] Chen CS, Wu SH, Wu YY, Fang JM, Wu TH. Properties of astaxanthin/Ca2+ complex formation in the deceleration of Cis/trans isomerization. Org Lett 2007;9:2985–8. https://doi.org/10.1021/ol0709533. [22] Liu X, Osawa T. Cis astaxanthin and especially 9-cis astaxanthin exhibits a higher antioxidant activity in vitro compared to the all-trans isomer. Biochem Biophys Res Commun 2007;357:187–93. https://doi.org/10.1016/ j.bbrc.2007.03.120. [23] Kaletta T, Hengartner MO. Finding function in novel targets: C. elegans as a model organism. Nat Rev Drug Discov 2006;5:387–99. https://doi.org/10.1038/nrd2031. [24] G€oc¸er M, Yanar M, Kumlu M, Yanar Y. The effects of red pepper, marigold flower, and synthetic astaxanthin on pigmentation, growth, and proximate composition of Penaeus semisulcatus. Turkish J Vet Anim Sci 2006;30:359–65. [25] Gong M, Bassi A. Carotenoids from microalgae: a review of recent developments. Biotechnol Adv 2016;34:1396–412. https://doi.org/10.1016/j.biotechadv.2016.10.005. [26] Ben-Amotz A, Levy Y. Bioavailability of a natural isomer mixture compared with synthetic all-trans β-carotene in human serum. Am J Clin Nutr 1996;63:729–34. https://doi.org/10.1093/ajcn/63.5.729. [27] Zhao Y, Yue C, Ding W, Li T, Xu JW, Zhao P, et al. Butylated hydroxytoluene induces astaxanthin and lipid production in Haematococcus pluvialis under high-light and nitrogen-deficiency conditions. Bioresour Technol 2018;266:315–21. https://doi.org/10.1016/j.biortech.2018.06.111. [28] Renuka N, Guldhe A, Singh P, Ansari FA, Rawat I, Bux F. Evaluating the potential of cytokinins for biomass and lipid enhancement in microalga Acutodesmus obliquus under nitrogen stress. Energ Conver Manage 2017;140:14–23. https://doi.org/10.1016/j.enconman.2017.02.065. [29] Kim JY, Lee C, Jeon MS, Park J, Choi YE. Enhancement of microalga Haematococcus pluvialis growth and astaxanthin production by electrical treatment. Bioresour Technol 2018;268:815–9. https://doi.org/10.1016/j. biortech.2018.08.014. [30] Ding W, Peng J, Zhao Y, Zhao P, Xu JW, Li T, et al. A strategy for boosting astaxanthin accumulation in green microalga Haematococcus pluvialis by using combined diethyl aminoethyl hexanoate and high light. J Appl Phycol 2019;31:171–81. https://doi.org/10.1007/s10811-018-1561-8.

246 Chapter 12 [31] Christian D, Zhang J, Sawdon AJ, Peng CA. Enhanced astaxanthin accumulation in Haematococcus pluvialis using high carbon dioxide concentration and light illumination. Bioresour Technol 2018;256:548–51. https:// doi.org/10.1016/j.biortech.2018.02.074. [32] Li D, Zhao Y, Ding W, Zhao P, Xu JW, Li T, et al. A strategy for promoting lipid production in green microalgae Monoraphidium sp. QLY-1 by combined melatonin and photoinduction. Bioresour Technol 2017;235:104–12. https://doi.org/10.1016/j.biortech.2017.03.114. [33] Sharon-Gojman R, Maimon E, Leu S, Zarka A, Boussiba S. Advanced methods for genetic engineering of Haematococcus pluvialis (Chlorophyceae, Volvocales). Algal Res 2015;10:8–15. https://doi.org/10.1016/j. algal.2015.03.022. [34] Hong ME, Choi HI, Kwak HS, Hwang SW, Sung YJ, Chang WS, et al. Rapid selection of astaxanthin-hyperproducing Haematococcus mutant via azide-based colorimetric assay combined with oil-based astaxanthin extraction. Bioresour Technol 2018;267:175–81. https://doi.org/10.1016/j. biortech.2018.07.033. [35] Dogaris I, Welch M, Meiser A, Walmsley L, Philippidis G. A novel horizontal photobioreactor for high-density cultivation of microalgae. Bioresour Technol 2015;198:316–24. https://doi.org/10.1016/j. biortech.2015.09.030. [36] Zhang Z, Huang JJ, Sun D, Lee Y, Chen F. Two-step cultivation for production of astaxanthin in Chlorella zofingiensis using a patented energy-free rotating floating photobioreactor (RFP). Bioresour Technol 2017;224:515–22. https://doi.org/10.1016/j.biortech.2016.10.081. [37] Khoo KS, Lee SY, Ooi CW, Fu X, Miao X, Ling TC, et al. Recent advances in biorefinery of astaxanthin from Haematococcus pluvialis. Bioresour Technol 2019;288:121606. https://doi.org/10.1016/j. biortech.2019.121606. [38] Wu S, Li W, Zhou W, Zhan Y, Hu C, Zhuang J, et al. Large-scale one-step synthesis of carbon dots from yeast extract powder and construction of carbon dots/PVA fluorescent shape memory material. Adv Opt Mater 2018;6:1–8. https://doi.org/10.1002/adom.201701150. [39] Golubev WI. Perfect state of Rhodomyces dendrorhous (Phaffia rhodozyma). Yeast 1995;11:101–10. https:// doi.org/10.1002/yea.320110202. [40] Miller MW, Yoneyama M, Soneda M. Phaffia, a new yeast genus in the Deuteromycotina (Blastomycetes). Int J Syst Bacteriol 1976;26:286–91. https://doi.org/10.1099/00207713-26-2-286. [41] Andrewes AG, Phaff HJ, Starr MP. Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast. Phytochemistry 1976;15:1003–7. https://doi.org/10.1016/S0031-9422(00)84390-3. [42] Ojima K, Breitenbach J, Visser H, Setoguchi Y, Tabata K, Hoshino T, et al. Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a β-carotene 3-hydroxylase/ 4-ketolase. Mol Genet Genomics 2006;275:148–58. https://doi.org/10.1007/s00438-005-0072-x. [43] Bhosale P, Bernstein PS. Microbial xanthophylls. Appl Microbiol Biotechnol 2005;68:445–55. https://doi. org/10.1007/s00253-005-0032-8. [44] Misawa N. Pathway engineering for functional isoprenoids. Curr Opin Biotechnol 2011;22:627–33. https:// doi.org/10.1016/j.copbio.2011.01.002. [45] Lee PC, Schmidt-Dannert C. Metabolic engineering towards biotechnological production of carotenoids in microorganisms. Appl Microbiol Biotechnol 2002;60:1–11. https://doi.org/10.1007/s00253-002-1101-x. [46] Ye VM, Bhatia SK. Pathway engineering strategies for production of beneficial carotenoids in microbial hosts. Biotechnol Lett 2012;34:1405–14. https://doi.org/10.1007/s10529-012-0921-8. [47] Ajikumar PK, Tyo K, Carlsen S, Mucha O, Phon TH, Stephanopoulos G. Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. Mol Pharm 2008;5:167–90. https:// doi.org/10.1021/mp700151b. [48] Krubasik P, Sandmann G. A carotenogenic gene cluster from Brevibacterium linens with novel lycopene cyclase genes involved in the synthesis of aromatic carotenoids. Mol Gen Genet 2000;263:423–32. https:// doi.org/10.1007/s004380051186. [49] Misawa N. Carotenoid β-ring hydroxylase and ketolase from marine bacteria - promiscuous enzymes for synthesizing functional xanthophylls. Mar Drugs 2011;9:757–71. https://doi.org/10.3390/md9050757.

Recent developments in astaxanthin production from Phaffia rhodozyma 247 [50] Alcaı´no J, Barahona S, Carmona M, Lozano C, Marcoleta A, Niklitschek M, et al. Cloning of the cytochrome p450 reductase (crtR) gene and its involvement in the astaxanthin biosynthesis of Xanthophyllomyces dendrorhous. BMC Microbiol 2008;8:169. https://doi.org/10.1186/1471-2180-8-169. [51] Ukibe K, Hashida K, Yoshida N, Takagi H. Metabolic engineering of Saccharomyces cerevisiae for astaxanthin production and oxidative stress tolerance. Appl Environ Microbiol 2009;75:7205–11. https://doi. org/10.1128/AEM.01249-09. [52] An GH, Cho MH, Johnson EA. Monocyclic carotenoid biosynthetic pathway in the yeast Phaffia rhodozyma (Xanthophyllomyces dendrorhous). J Biosci Bioeng 1999;88:189–93. https://doi.org/10.1016/S1389-1723 (99)80200-X. [53] Gassel S, Breitenbach J, Sandmann G. Genetic engineering of the complete carotenoid pathway towards enhanced astaxanthin formation in Xanthophyllomyces dendrorhous starting from a high-yield mutant. Appl Microbiol Biotechnol 2014;98:345–50. https://doi.org/10.1007/s00253-013-5358-z. [54] Liu ZQ, Zhang JF, Zheng YG, Shen YC. Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation. J Appl Microbiol 2008;104:861–72. https://doi. org/10.1111/j.1365-2672.2007.03603.x. [55] Miao L, Wang Y, Chi S, Yan J, Guan G, Hui B, et al. Reduction of fatty acid flux results in enhancement of astaxanthin synthesis in a mutant strain of Phaffia rhodozyma. J Ind Microbiol Biotechnol 2010;37:595–602. https://doi.org/10.1007/s10295-010-0706-9. [56] Ni H, Hong Q, Xiao A, Li L, Cai H, Su W. Characterization and evaluation of an astaxanthin over-producing Phaffia rhodozyma. Shengwu Gongcheng Xuebao/Chin J Biotechnol 2011;27:1065–75. https://doi.org/ 10.13345/j.cjb.2011.07.009. [57] Stachowiak B. Efficiency of selected mutagens in generating Xanthophyllomyces dendrorhous strains hyperproducing astaxanthin. Polish J Microbiol 2013;62:67–72. https://doi.org/10.33073/pjm-2013-008. [58] Gassel S, Schewe H, Schmidt I, Schrader J, Sandmann G. Multiple improvement of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous by a combination of conventional mutagenesis and metabolic pathway engineering. Biotechnol Lett 2013;35:565–9. https://doi.org/10.1007/s10529-012-1103-4. [59] Xie H, Zhou Y, Hu J, Chen Y, Liang J. Production of astaxanthin by a mutant strain of Phaffia rhodozyma and optimization of culture conditions using response surface methodology. Ann Microbiol 2014;64:1473–81. https://doi.org/10.1007/s13213-013-0790-y. [60] Hara KY, Morita T, Endo Y, Mochizuki M, Araki M, Kondo A. Evaluation and screening of efficient promoters to improve astaxanthin production in Xanthophyllomyces dendrorhous. Appl Microbiol Biotechnol 2014;98:6787–93. https://doi.org/10.1007/s00253-014-5727-2. [61] Hara KY, Morita T, Mochizuki M, Yamamoto K, Ogino C, Araki M, et al. Development of a multi-gene expression system in Xanthophyllomyces dendrorhous. Microb Cell Fact 2014;13:175. https://doi.org/ 10.1186/s12934-014-0175-3. [62] Ledetzky N, Osawa A, Iki K, Pollmann H, Gassel S, Breitenbach J, et al. Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous. Arch Biochem Biophys 2014;545:141–7. https://doi.org/10.1016/j. abb.2014.01.014. [63] Chi S, He Y, Ren J, Su Q, Liu X, Chen Z, et al. Overexpression of a bifunctional enzyme, CrtS, enhances astaxanthin synthesis through two pathways in Phaffia rhodozyma. Microb Cell Fact 2015;14:1–12. https:// doi.org/10.1186/s12934-015-0279-4. [64] Yamamoto K, Hara KY, Morita T, Nishimura A, Sasaki D, Ishii J, et al. Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes. Microb Cell Fact 2016;15:1–8. https://doi.org/10.1186/s12934-016-0556-x. [65] Nangia H, Hasan M, Azhar M, Bhatt P, Panda B. Strain improvement of Phaffia rhodozyma for astaxanthin production. Drug Dev Ther 2016;7:63–8. https://doi.org/10.4103/2394-6555.180169. [66] Yolmeh M, Khomeiri M. Effect of mutagenesis treatment on antimicrobial and antioxidant activities of pigments extracted from Rhodotorula glutinis. Biocatal Agric Biotechnol 2017;10:285–90. https://doi.org/ 10.1016/j.bcab.2017.04.007.

248 Chapter 12 [67] Sun N, Lee S, Song KB. Characterization of a carotenoid-hyperproducing yeast mutant isolated by low-dose gamma irradiation. Int J Food Microbiol 2004;94:263–7. https://doi.org/10.1016/S0168-1605(03) 00311-8. [68] Lee SH, Sun NK, Jang JW, An GH, Won M, Song KB. Characterization of Phaffia rhodozyma 3A 4-8 generated by low-dose γ-irradiation. J Food Sci 2006;69:M258–61. https://doi.org/10.1111/j.13652621.2004.tb09939.x. [69] Ottenheim C, Nawrath M, Wu JC. Microbial mutagenesis by atmospheric and room-temperature plasma (ARTP): the latest development. Bioresour Bioprocess 2018;5:12. https://doi.org/10.1186/s40643-018-0200-1. [70] Jin J, Wang Y, Yao M, Gu X, Li B, Liu H, et al. Astaxanthin overproduction in yeast by strain engineering and new gene target uncovering. Biotechnol Biofuels 2018;11:230. https://doi.org/10.1186/s13068-0181227-4. [71] Ukibe K, Katsuragi T, Tani Y, Takagi H. Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry. FEMS Microbiol Lett 2008;286:241–8. https:// doi.org/10.1111/j.1574-6968.2008.01278.x. [72] Pollmann H, Breitenbach J, Sandmann G. Development of Xanthophyllomyces dendrorhous as a production system for the colorless carotene phytoene. J Biotechnol 2017;247:34–41. https://doi.org/10.1016/j. jbiotec.2017.02.027. [73] Najafi N, Hosseini R, Ahmadi AR. The effect of gamma irradiation on astaxanthin synthetase encoding gene in two mutant strains of Phaffia rhodozyma. Iran J Microbiol 2013;5:293–8. [74] Xiao A, Jiang X, Ni H, Yang Q, Cai H. Study on the relationship between intracellular metabolites and astaxanthin accumulation during Phaffia rhodozyma fermentation. Electron J Biotechnol 2015;18:148–53. https://doi.org/10.1016/j.ejbt.2015.02.002. [75] Xiao R, Li X, Leonard E, Tharayil N, Zheng Y. Investigation on the effects of cultivation conditions, fed-batch operation, and enzymatic hydrolysate of corn Stover on the astaxanthin production by Thraustochytrium striatum. Algal Res 2019;39:101475. https://doi.org/10.1016/j.algal.2019.101475. [76] Miao L, Chi S, Wu M, Liu Z, Li Y. Deregulation of phytoene-β-carotene synthase results in derepression of astaxanthin synthesis at high glucose concentration in Phaffia rhodozyma astaxanthin-overproducing strain MK19. BMC Microbiol 2019;19:1–11. https://doi.org/10.1186/s12866-019-1507-6. [77] Wozniak A, Lozano C, Barahona S, Niklitschek M, Marcoleta A, Alcaı´no J, et al. Differential carotenoid production and gene expression in Xanthophyllomyces dendrorhous grown in a nonfermentable carbon source. FEMS Yeast Res 2011;11:252–62. https://doi.org/10.1111/j.1567-1364.2010.00711.x. [78] Montanti J, Nghiem NP, Johnston DB. Production of astaxanthin from cellulosic biomass sugars by mutants of the yeast Phaffia rhodozyma. Appl Biochem Biotechnol 2011;164:655–65. https://doi.org/10.1007/ s12010-011-9165-7. [79] Haard NF. Astaxanthin formation by the yeast Phaffia rhodozyma on molasses. Biotechnol Lett 1988;10:609–14. https://doi.org/10.1007/BF01024710. [80] Meyer PS, du Preez JC. Astaxanthin production by a Phaffia rhodozyma mutant on grape juice. World J Microbiol Biotechnol 1994;10:178–83. https://doi.org/10.1007/BF00360882. [81] Amado IR, Va´zquez JA. Mussel processing wastewater: a low-cost substrate for the production of astaxanthin by Xanthophyllomyces dendrorhous. Microb Cell Fact 2015;14:177. https://doi.org/10.1186/s12934-0150375-5. ´ , Aguilar-Machado DE, Balagurusamy N, Montan˜ez J, Morales-Oyervides L. [82] Villegas-M
endez MA Agro-industrial wastes for the synthesis of carotenoids by Xanthophyllomyces dendrorhous: mesquite pods-based medium design and optimization. Biochem Eng J 2019;150:107260. https://doi.org/10.1016/j. bej.2019.107260. [83] Yang J, Tan H, Yang R, Sun X, Zhai H, Li K. Astaxanthin production by Phaffia rhodozyma fermentation of cassava residues substrate. CIGR J 2011;13:1–9. [84] Yang J, Yang R, Tan H, Zhai H, Sun X. Astaxanthin production by Phaffia rhodozyma fermentation of pineapple peel substrate. Adv Mater Res 2011;233:2962–9. https://doi.org/10.4028/www.scientific.net/ AMR.233-235.2962.

Recent developments in astaxanthin production from Phaffia rhodozyma 249 [85] Silva CM, Borba TM, Burkert CAV, Burkert JFM. Carotenoid production by Phaffia rhodozyma using raw glycerol as an additional carbon source. Int J Food Eng 2012;8:18. https://doi.org/10.1515/15563758.2843. [86] Tropea A, Gervasi T, Melito MR, Curto AL, Curto RL. Does the light influence astaxanthin production in Xanthophyllomyces dendrorhous? Nat Prod Res 2013;27:648–54. https://doi.org/ 10.1080/14786419.2012.688045. [87] Rios DAdS, Borba TDMD, Kalil SJ, Burkert JFDM. Rice parboiling wastewater in the maximization of carotenoids bioproduction by Phaffia rhodozyma. Ci^enc Agrotecnol 2015;39:401–10. https://doi.org/ 10.1590/s1413-70542015000400011. [88] Zhou LY, Zhu MJ. Sugarcane bagasse hydrolysate-based growth and production of carotenoids by yeast Phaffia rhodozyma. Int J Mod Biol Med 2015;6:136–45. [89] Irtiza A, Shatunova S, Glukhareva T, Kovaleva E. Production of astaxanthin rich feed supplement for animals from Phaffia rhodozyma yeast at low cost. AIP Conf Proc 2017;1886:020105. https://doi.org/ 10.1063/1.5003002. [90] Jiang GL, Zhou LY, Wang YT, Zhu MJ. Astaxanthin from Jerusalem artichoke: production by fed-batch fermentation using Phaffia rhodozyma and application in cosmetics. Process Biochem 2017;63:16–25. https://doi.org/10.1016/j.procbio.2017.08.013. [91] Liu F, Zhu MJ. Investigation on the production of carotenoid from molasses by Phaffia rhodozyma. Int J Mod Biol Med 2017;8:1–13. [92] Stoklosa RJ, Johnston DB, Nghiem NP. Phaffia rhodozyma cultivation on structural and non-structural sugars from sweet sorghum for astaxanthin generation. Process Biochem 2019;83:9–17. https://doi.org/10.1016/j. procbio.2019.04.005. [93] Xue S, Jones AD, Sousa L, Piotrowski J, Jin M, Sarks C, et al. Water-soluble phenolic compounds produced from extractive ammonia pretreatment exerted binary inhibitory effects on yeast fermentation using synthetic hydrolysate. PLoS One 2018;13:e0194012. https://doi.org/10.1371/journal.pone.0194012. [94] Mussatto SI, Roberto IC. Alternatives for detoxification of diluted-acid lignocellulosic hydrolyzates for use in fermentative processes: a review. Bioresour Technol 2004;93:1–10. https://doi.org/10.1016/j. biortech.2003.10.005. [95] Vustin MM, Belykh EN, Kishilova SA. Relationship between astaxanthin production and the intensity of anabolic processes in the yeast Phaffia rhodozyma. Mikrobiologiya 2004;73:643–9. https://doi.org/10.1007/ s11021-005-0004-0. [96] de la Fuente JL, Peiro E, Diez B, Marcos AT, Schleissner C, Rodriguez Saiz M, et al. Method of Production of Astaxanthin by Fermenting Selected Strains of Xanthophyllomyces dendrorhous. United States Patent US20050124032. [97] Pan X, Wang B, Gerken HG, Lu Y, Ling X. Proteomic analysis of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous in response to low carbon levels. Bioprocess Biosyst Eng 2017;40:1091–100. https://doi.org/10.1007/s00449-017-1771-5. [98] Liu YS, Wu JY. Use of n-hexadecane as an oxygen vector to improve Phaffia rhodozyma growth and carotenoid production in shake-flask cultures. J Appl Microbiol 2006;101:1033–8. https://doi.org/10.1111/ j.1365-2672.2006.03009.x. [99] Yuan SL, Wu JY. Perfusion culture process plus H2O2 stimulation for efficient astaxanthin production by Xanthophyllomyces dendrorhous. Biotechnol Bioeng 2007;97:568–73. https://doi.org/10.1002/bit.21256. [100] de la Fuente JL, Rodrı´guez-Sa´iz M, Schleissner C, Dı´ez B, Peiro E, Barredo JL. High-titer production of astaxanthin by the semi-industrial fermentation of Xanthophyllomyces dendrorhous. J Biotechnol 2010;148:144–6. https://doi.org/10.1016/j.jbiotec.2010.05.004. [101] Stachowiak B. Effect of illumination intensities on astaxanthin synthesis by Xanthophyllomyces dendrorhous and its mutants. Food Sci Biotechnol 2013;22:1033–8. https://doi.org/10.1007/s10068-013-0180-z. [102] Stachowiak B. Astaxanthin synthesis by Xanthophyllomyces dendrorhous DSM 5626 and its mutants on carrot extract medium under different illumination intensity. Appl Biochem Microbiol 2014;50:471–6. https://doi.org/10.1134/S000368381405010X.

250 Chapter 12 [103] Dursun D, Dalgic¸ AC. Optimization of astaxanthin pigment bioprocessing by four different yeast species using wheat wastes. Biocatal Agric Biotechnol 2016;7:1–6. https://doi.org/10.1016/j.bcab.2016.04.006. [104] Schewe H, Kreutzer A, Schmidt I, Schubert C, Schrader J. High concentrations of biotechnologically produced astaxanthin by lowering pH in a Phaffia rhodozyma bioprocess. Biotechnol Bioprocess Eng 2017;22:319–26. https://doi.org/10.1007/s12257-016-0349-4. [105] Liu S, Liu B, Wang H, Xiao S, Li Y, Wang J. Production of astaxanthin at moderate temperature in Xanthophyllomyces dendrorhous using a two-step process. Eng Life Sci 2018;18:706–10. https://doi.org/ 10.1002/elsc.201800065. [106] Batghare AH, Singh N, Moholkar VS. Investigations in ultrasound-induced enhancement of astaxanthin production by wild strain Phaffia rhodozyma MTCC 7536. Bioresour Technol 2018;254:166–73. https://doi. org/10.1016/j.biortech.2018.01.073. [107] Wang B, Pan X, Jia J, Xiong W, Manirafasha E, Ling X, et al. Strategy and regulatory mechanisms of glutamate feeding to enhance astaxanthin yield in Xanthophyllomyces dendrorhous. Enzyme Microb Technol 2019;125:45–52. https://doi.org/10.1016/j.enzmictec.2019.02.010. [108] Hu ZC, Zheng YG, Wang Z, Shen YC. pH control strategy in astaxanthin fermentation bioprocess by Xanthophyllomyces dendrorhous. Enzyme Microb Technol 2006;39:586–90. https://doi.org/10.1016/j. enzmictec.2005.11.017. [109] Guo X, Li X, Xiao D. Optimization of culture conditions for production of astaxanthin by Phaffia rhodozyma, In: 2010 4th Int Conf Bioinforma Biomed Eng ICBBE 2010; 2010. p. 1–4. https://doi.org/10.1109/ ICBBE.2010.5516101. [110] Marsan C. Light activation of Xanthophyllomyces dendrorhous metabolism. Major Qualif Proj 2019;. [111] Inoue Y, Shimazawa M, Nagano R, Kuse Y, Takahashi K. Astaxanthin analogs, adonixanthin and lycopene, activate Nrf2 to prevent light-induced photoreceptor degeneration. J Pharmacol Sci 2017;134:147–57. https:// doi.org/10.1016/j.jphs.2017.05.011. [112] Stachowiak B. Astaxanthin synthesis by Xanthophyllomyces dendrorhous DSM 5626 and its astaxanthin overproducing mutants on xylose media under different illumination. Acta Sci Pol Technol Aliment 2014;13:279–88. https://doi.org/10.17306/J.AFS.2014.3.6. [113] Va´zquez M. Effect of the light on carotenoid rofiles of Xanthophyllomyces dendrorhous strains (formerly Phaffia rhodozyma). Food Technol Biotechnol 2001;39:123–8. [114] Wang W, Yu L. Effects of oxygen supply on growth and carotenoids accumulation by Xanthophyllomyces dendrorhous. Zeitschrift Naturforsch C J Biosci 2010;64:853–8. https://doi.org/10.1515/znc-2009-11-1216. [115] Liu YS, Wu JY, Ho KP. Characterization of oxygen transfer conditions and their effects on Phaffia rhodozyma growth and carotenoid production in shake-flask cultures. Biochem Eng J 2006;27:331–5. https:// doi.org/10.1016/j.bej.2005.08.031. [116] Stachowiak B. Astaxanthin synthesis by yeast Xanthophyllomyces dendrorhous and its mutants on media based on plant extracts. Indian J Microbiol 2012;52:654–9. https://doi.org/10.1007/s12088-012-0306-7. [117] Abdullah S, Poomputsa K, Mekvichitsaeng P, Ruanglek V, Akeprathumchai S. Identifying parameters influencing growth and astaxanthin production by Xanthophyllomyces dendrorhous cultivated in pineapple juice concentrate base low cost medium. Thai J Agric Sci 2011;44:89–94. [118] Ooi Y, Kitamura A, Yamashita E. Baked Food Produced From Astaxanthin Containing Dough. United States Patent US20180098549. [119] Yu J. Dog Feed With Astaxanthin and Preparation Method Thereof. Australia Patent AU2019100450. [120] Li T, Xu YG, Lyu ZC. Lotion Containing Natural Astaxanthin Ester and Preparation Method Thereof. Chinese Patent CN109820746. [121] Li T, Xu YG, Lyu ZC. Microcapsule Containing Natural Astaxanthin Ester and Preparation Method Thereof. Chinese Patent CN109820837. [122] Tao R. Pharmaceutical Composition for Treating Insomnia and Application of Pharmaceutical Composition. Chinese Patent CN109820905. [123] Harris HW, Craig SR. Method for Increasing the Utilization of Soybean Protein by Salmonid Fish. United States Patent US20190174797.

Recent developments in astaxanthin production from Phaffia rhodozyma 251 [124] Yasuhiro N, Tomihisa Y. Dry Eye Remedy. Japanese Patent WO2019138995. [125] Yasuhiro N, Kaoru K. Antipruritic Agent. Japanese Patent WO2019111896. [126] Wang MH, Zhu ZY, Wang JK, Li M, Zha YF, Sun M, et al. Astaxanthin Health Product Having Antioxidation Function. Chinese Patent CN107029020. [127] Wang LW. Application of Astaxanthin to Water-Soluble Fertilizer. Chinese Patent CN105503383. [128] Wang YB. Astaxanthin Nutrition Supplement. Chinese Patent CN108208829. [129] Hirao C. Method for Extracting and Producing Useful Substance, and Soap, Cosmetics, or Health Supplement Having Said Useful Substance as Main Component. United States Patent US20170266095. [130] McWilliams A. FOD025F the Global Market for Carotenoids BCC Research Report Overview the Global Market for Carotenoids. [131] Feng ZZ, Li MY, Wang YT, Zhu MJ. Astaxanthin from Phaffia rhodozyma: microencapsulation with carboxymethyl cellulose sodium and microcrystalline cellulose and effects of microencapsulated astaxanthin on yogurt properties. LWT Food Sci Technol 2018;96:152–60. https://doi.org/10.1016/j.lwt.2018.04.084. [132] Anarjan N, Tan CP. Chemical stability of astaxanthin nanodispersions in orange juice and skimmed milk as model food systems. Food Chem 2013;139:527–31. https://doi.org/10.1016/j.foodchem.2013.01.012. [133] Jiang GL, Zhu MJ. Preparation of astaxanthin-encapsulated complex with zein and oligochitosan and its application in food processing. LWT Food Sci Technol 2019;106:179–85. https://doi.org/10.1016/j. lwt.2019.02.055. [134] Satoh T. Astaxanthin: health benefits and toxicity. In: Gupta RC, editor. Nutraceuticals: efficacy, safety and toxicity. Amsterdam: Elsevier; 2016. p. 531–9. https://doi.org/10.1016/B978-0-12-802147-7.00038-3. [135] Fang Q, Guo S, Zhou H, Han R, Wu P, Han C. Astaxanthin protects against early burn-wound progression in rats by attenuating oxidative stress-induced inflammation and mitochondria-related apoptosis. Sci Rep 2017;7:1–13. https://doi.org/10.1038/srep41440. [136] Murai T, Kawasumi K, Tominaga K, Okada Y, Kobayashi M, Arai T. Effects of astaxanthin supplementation in healthy and obese dogs. Vet Med Res Rep 2019;10:29–35. https://doi.org/10.2147/vmrr.s186202. [137] Kamath BS, Srikanta BM, Dharmesh SM, Sarada R, Ravishankar GA. Ulcer preventive and antioxidative properties of astaxanthin from Haematococcus pluvialis. Eur J Pharmacol 2008;590:387–95. https://doi.org/ 10.1016/j.ejphar.2008.06.042. [138] Sila A, Kamoun Z, Ghlissi Z, Makni M, Nasri M, Sahnoun Z, et al. Ability of natural astaxanthin from shrimp by-products to attenuate liver oxidative stress in diabetic rats. Pharmacol Rep 2015;67:310–6. https://doi.org/ 10.1016/j.pharep.2014.09.012. [139] Visioli F, Artaria C. Astaxanthin in cardiovascular health and disease: mechanisms of action, therapeutic merits, and knowledge gaps. Food Funct 2017;8:39–63. https://doi.org/10.1039/c6fo01721e. [140] Wu H, Niu H, Shao A, Wu C, Dixon BJ, Zhang J, et al. Astaxanthin as a potential neuroprotective agent for neurological diseases. Mar Drugs 2015;13:5750–66. https://doi.org/10.3390/md13095750. [141] Cheng XY, Xiong YJ, Yang MM, Zhu MJ. Preparation of astaxanthin mask from Phaffia rhodozyma and its evaluation. Process Biochem 2019;79:195–202. https://doi.org/10.1016/j.procbio.2018.12.027. [142] Elwan HAM, Elnesr SS, Abdallah Y, Hamdy A, El-Bogdady AH. Red yeast (Phaffia rhodozyma) as a source of Astaxanthin and its impacts on productive performance and physiological responses of poultry. Worlds Poult Sci J 2019;75:273–84. https://doi.org/10.1017/S0043933919000187.

This page intentionally left blank

CHAPTER 13

Turning leftover to treasure: An overview of astaxanthin from shrimp shell wastes J.Y. Cheong and M. Muskhazli Department of Biology, Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Abbreviations CC FAO FTIR GRAS HPE HPLC HPLC-MS HPTLC MAE MALDI-TOF NMR OCC SFE TLC UAE US FDA USD

column chromatography Fisheries and Agriculture Organization Fourier transform infrared spectroscopy generally recognize as safe high-pressure extraction high-pressure liquid chromatography high-pressure liquid chromatography mass spectrometer high-pressure thin-layer chromatography microwave-assisted extraction matrix-assisted laser desorption ionization-time of flight nuclear magnetic resonance open column chromatography supercritical fluid extraction thin-layer chromatography ultrasound-assisted extraction United States Food and Drug Analysis United States Dollar

1 Introduction For decades the world consumption of crustaceans has increased tremendously be it freshly caught, processed foods, or frozen foods. According to data collected by the FAO in 2018, for over a decade, three giant countries have dominated the total capturing of fishes, crustaceans, and mollusks. In 2007 China has recorded the highest capturing of fishes, crustaceans, and mollusks with 14 million tons and is increasing yearly up to a total of 17.5 million tons in 2016. Meanwhile, Indonesia and India both rank second and third, with a total capture of 5 million and 3.8 million tons in 2007 increasing to 6.5 million and 5 million tons in 2010, respectively. However, in recent years, the total world capture of crustaceans has remained consistent at around 500,000 tons per year [1]. Although there is not much increase in total crustacean capturing, the demand for these few species (Portunus trituberculatus, Euphausia superba, Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00022-2 Copyright # 2021 Elsevier Inc. All rights reserved.

253

254 Chapter 13 Portunus pelagicus, Pleoticus muelleri, Penaeus chinensis, and Homarus americanus) is increasing over a 5-year period (2012–16). It is possible that the increase in demand for these crustaceans is due to its attractive coloration and higher nutritional value due to the presence of astaxanthin [2, 3]. As the crustacean capture yield is around 500,000 tons yearly, a huge amount of crustacean waste is produced from seafood processing. The amount of raw wastes discarded varies according to species and may accumulate up to 45%–60% of the crustacean’s total weight [4, 5]. This has inevitably resulted in a minimum of 225,000 tons of crustacean wastes being produced yearly, and this amount is accumulated throughout the years. Crustacean wastes are classified as unconsumed parts of a crustacean, which include the shell part (head, carapace, tail, legs, and antennae) and the organ parts (gills and organs). The accumulation of crustacean shell wastes poses a big threat to the environment due to its low biodegradation rate [6], and the biggest coastal pollution arises when these wastes are dumped into the sea [7]. Additionally, some countries like Australia charge a fee of USD 150 per ton for the disposal of these wastes [8]. One of the approaches for reducing pollution caused by crustacean wastes was by recycling them and utilizing it as chicken feed [9], bait for fisheries, fertilizer, and as a primary chitin source whereby these shells can potentially fetch a high value of at least USD 100–120 per ton [8]. Previously the recycling of crustacean waste is generally carried out by sun-drying of shells. This is indeed a cheap processing method, although it causes environmental pollution, produces an unpleasant smell and surface pollution, and is unhygienic for medical uses [10, 11]. Moreover, it should be considered that crustacean waste especially from shrimp, crab, lobster, krill, prawns, and crayfishes contains various biological compounds and amino acids and is a rich source of carotenoid pigments especially astaxanthin [12]. Various carotenoids are present in the crustacean exoskeleton, including β-carotene, lutein, zeaxanthin, and astaxanthin. Of all the carotenoids present, astaxanthin was given the greatest attention due to its multipotentiality and uses. According to Saranya et al. [13], astaxanthin has been used as a food coloring agent for aesthetic purposes since the tribal and ancestral ages. This trend has persisted to the present day, whereby astaxanthin is used in aquaculture [14, 15], cosmetics [16, 17] and food industries [18] to provide coloration, medical industries [19, 20], and pharmaceutical industries [21]. Depending on crustacean species, astaxanthin represents an amount between 74% and 98% of the total pigments present [22]. One of the main reasons that make astaxanthin popular was its enormous antioxidant activity, 14.3 times stronger than vitamin E, 53.7 times stronger than β-carotene, and 64.9 times than that of vitamin C [23]. Generally, crustaceans can directly produce astaxanthin only when provided with the precursor carotene molecules and carotenoid precursors from microalgae and phytoplanktons [24]. The accumulation of β-carotene is required as a precursor carotenoid molecule for the conversion of astaxanthin. Two intermediates taking part in the pathway have been proposed as echinenone and canthaxanthin, as most crustaceans rely on it to form astaxanthin [25]. The pathway suggested in the bioconversion of β-carotene to astaxanthin is presented in Fig. 13.1. Alternatively, there are two pathways that rely on β-doradexanthin as an intermediate; however, one of it has not

Turning leftover to treasure 255

3-Hydroyechineone

Fig. 13.1 Bioconversion pathway suggested for astaxanthin from β-carotene. Crustaceans rely on these three important intermediates, β-doradexanthin, canthaxanthin, and 3-hydroxyechinenone in three pathways shown earlier to obtain astaxanthin. Arrows in black denotes the most common conversion pathway proposed for most crustaceans. Arrows shaded in gray proposes the pathway that relies on the less common β-doradexanthin during the conversion.

been demonstrated [26]. As canthaxanthin and β-doradexanthin have been found as an intermediate in certain copepod, there has not been a single pathway that applies to all [27]. However, in certain crustaceans such as hermit crab, red king crab, clawed lobster, spiny lobster, and shrimp, pigment coloration has to be supplied consistently because they are not able to retain or produce astaxanthin from any precursor pigment molecules [28–30], and a report shows that pigmentation in banana shrimp is heritable, suggesting a genetic basis to the retention of carotenoids [31]. Astaxanthin (3,30 -dihydroxy-β,β0 -carotene-4,40 -dione) is naturally biosynthesized by plants, microalgae, yeasts, and marine bacterium. In freshwater environment, astaxanthin is naturally produced by microalgae, especially Haematococcus pluvialis [32]. The amount of natural astaxanthin produced by these microalgae goes up to 80%–99% of total carotenoids and up to 3%–5% of its dry weight [12,33–35]. Other species of algae such as Chlorella zofingiensis [36] and Chromochloris zofingiensis [37] were also known to produce astaxanthin and are actively being studied. Upon ingestion of these pigmented algae by crustaceans, astaxanthin pigmentation is deposited in the muscles, eggs, organs, or carapace where the complexity of astaxanthin can occur in three different forms: free molecules, forming complexes with protein (carotenoprotein) or lipid (carotenolipoproteins), or esterified (esters) [38]. Studies have identified marine and freshwater bacteria that are able to produce astaxanthin, such as the marine bacterium, Agrobacterium aurantiacum [39] and the freshwater bacterium Sphingomonas astaxanthinifaciens sp. [40]. However, only Xanthophyllomyces dendrorhous and H. pluvialis accumulate the highest astaxanthin content in nature [41]. Elsewhere,

256 Chapter 13 genetically engineered bacteria that are capable of producing astaxanthin are common and were pioneered by the Escherichia coli [42]. Presently, genetic modifications have been performed on various bacterial and fungal species such as Corynebacterium glutamicum [43], Methylomonas sp. [44], Yarrowia lipolytica [45], and Mucor circinelloides [46].

2 Properties of astaxanthin Astaxanthin, also known as 3,30 -dihydroxy-β-carotene-4,40 -dione, is a pigment classified under the carotenoid class. The presence of oxygen in astaxanthin allows it to be subclassified under xanthophylls together with β-carotene, cantaxanthin, lutein, and zeaxanthin. Members of the xanthophyll subclass share a common semisymmetric layout with two terminal carbon rings attached to a polyene chain [38]. The uniqueness of astaxanthin compared with other members of the subclass is the presence of hydroxyl and keto moieties on both of its ends [47]. Although most of the xanthophylls contain oxygenated groups in their terminal carbon rings, they do not have structural requirements to perform the activities of vitamin A, which makes them less effective as a human health dietary supplement [48]. The terminal ring moiety and the polyene chain function to trap radicals at which the hydrogen on the C3 methine is suggested as a trapping site [49]. Owning a polar-nonpolar-polar structure, astaxanthin places itself within the plasma membrane of eukaryote cells [50]. Both the polar ends (Fig. 13.2) that possess the terminal carbon rings are hydrophilic and can scavenge radicals at both surfaces and in the interior phospholipid membrane [51].

Fig. 13.2 By positioning its structure across the phospholipid bilayer, the astaxanthin’s terminal rings effectively scavenge reactive oxygen species (ROS) on the outer membrane surface and polyene chain trapping ROS in the cytoplasm side. Its unique structure with 13 conjugated double bonds, the hydroxyl groups at the 3 and 30 position, and oxo groups at 4 and 40 position, astaxanthin prevents free radical chain reactions, scavenging lipid peroxyl radicals, trapping energy (quenching), and the transfer of electrons, or through hydrogen abstraction (scavenging).

Turning leftover to treasure 257 The presence of a hydroxyl group (dOH) at either end of the astaxanthin chain esterifies itself with fatty acid, forming astaxanthin monoester or diester. Mono- and diesters of astaxanthin are easily found in algae and crustaceans. Gomez-Estaca et al. [52] reported the presence of five astaxanthin monoesters and 10 diesters found in the shrimp Litopenaeus vannamei. Meanwhile, astaxanthin mono- and diesters are reported to be predominant in shrimp waste, and the encapsulation of astaxanthin allows it to be a better source of food colorant and functional ingredient [53]. The process of astaxanthin esterification is common in naturally free astaxanthin due to its vulnerability toward oxidation, and this changes the ratio of its cis and trans isomers [54]. Stereoisomerism in astaxanthin is common due to the presence of double bonds that create a geometric isomer of cis and trans. The trans isomer is more predominant in nature due to its stability, while the cis are thermodynamically less stable in nature [55]. In the microalgae H. pluvialis, geometric isomers trans-astaxanthin and cis-astaxanthin exists in a ratio of 3:1 [56]. Nevertheless, trans isomers can be transformed into cis with the presence of light, acid, heat, or metal ions, and studies have revealed that 9-cis astaxanthin has the highest antioxidant potency than that of trans [57]. On the other side, astaxanthin consist of three types of R/S chiral isomers and 272 possible stereoisomers of E/Z formation with the commonly important isomers for quantitative measures being an all-E, 9Z, 13Z, and 15Z [58]. In nature the stereoisomers 3R, 30 R and 3S, 30 S were the most abundant and were biosynthesized by Haematococcus (3S, 30 S) and X. dendrorhous (3R, 30 R) [59]. As for shrimps, free astaxanthin can accumulate up to 4.44%–52.31% and astaxanthin ester up to 47.69%–95.56% of the total carotenoid present [60]. Meanwhile, synthetically produced astaxanthin consists of all three mixtures of isomers (3R, 30 R, 3R, 30 S, and 3S, 30 S) in a ratio of 1:2:1 [38].

3 The color of astaxanthin Astaxanthin is the main carotenoid in crustaceans, and the predominant color of astaxanthin is red. It commonly appears as a free form molecule, but occasionally, it is associated with esters and/or some macromolecules. The different kinds of structure present allow the king of carotenoids to appear in various colors ranging from hues of dark blue, orange, and slight brownish red. The color of this pigment is very dependent on the molecules attached to it. For instance, astaxanthin appears purple, blue, and yellow in raw crustaceans. This is due to structural changes in the pigment that is attached to a protein forming carotenoprotein such as crustacyanin [61]. Ovoverdin, a carotene-protein gives green coloration to lobster eggs when bonded with astaxanthin [62]. In crustacean shells, another commonly known protein bound to astaxanthin, which produces the color purple blue, is α- and β-crustacyanin [63]. The carotene– protein form provides extra stability to astaxanthin. Thermal processing of seafood such as cooking, boiling, microwaving, frying, and baking alters the color of astaxanthin. The color of raw crustaceans and cooked crustaceans is simply extravagant as the dark blue color now changes to bright red or orange when exposed to heat [64]. This is because the binding protein degrades when heat is applied and therefore reveals the

258 Chapter 13 natural color of astaxanthin [65]. On the other hand, when heat is substituted with alkaline solutions, the shrimps appear to be red, signifying the denaturation of carotenoproteins and revealing the true color of astaxanthin (in its free form), which appears to be red-orange [66]. An important quality criterion in determining the seafood price lies on the reddish hue displayed by astaxanthin [67]. According to Parisenti et al. [3], hues of gray in raw shrimps and bright orange color of cooked shrimp are much preferred by consumers.

4 Managing crustacean wastes and turning it into a gem In the early 1950s the fishery processing industry was relatively small, and therefore less waste was produced. At that time, fisheries products were much more focused on fresh produce and less on processed seafood [68]. It was not until the 1970s that the processing industries bloomed, and as the supply of fisheries products became larger, so did the wastes. Krill waste was estimated to be 800,000 dry tons per year in 1978 [69]. In 2016 Antarctic krill (Euphausia superba) captured was around 270 million tons [1], and of this the head and shell comprise 70% of its body weight making a total of 191 million tons of wastes. As time passes, the blooming of factories processing shellfishes inevitably produced a large amount of waste, which ultimately leads to pollution.

4.1 A better solution to manage crustacean waste As crustacean waste pollution starts to increase and significant biomolecules are discovered, another solution used to reduce the waste and obtain valuable biomolecules is by treating them with strong chemicals (acid and alkali). Deproteinizing and demineralizing crustacean wastes are crucial steps before the extraction of biomolecules can take place. Alkalis used in deproteinization includes NaOH, Na2CO3, NaHCO3, KOH, K2CO3, Ca(OH)2, Na2SO3, NaHSO3, CaHSO3, Na3PO4, and Na2S, whereby the concentrations used can range from 0.125 to 5.0 M [70]. Acid plays a role in demineralizing crustacean shell wastes by reacting with calcium carbonate to produce calcium chloride, water, and carbon dioxide gas. Generally, diluted hydrochloric acid works best and is used in demineralization, although other acids such as nitric acid, sulfuric acid, ethanoic acid, and methanoic acid were also used [71]. Chemical degradation is a common and popular method of practice especially in industries due to its efficiency and fast results [72] as it allows the recovery of chitin and the production of chitosan from it, but not carotenoids [73]. This is a huge loss as carotenoids are valuable and play a versatile role in various industries. Moreover the usage of strong acid and alkali not only causes pollution but also destroys the properties of crustacean shells, such as the depolymerization of the shells, changes to molecular weight, structure, viscosity, and destruction of native proteins [74].

Turning leftover to treasure 259

4.2 The modern trend in managing crustacean wastes A modern idea of utilizing commercial enzymes such as Alcalases and Delvolase to replace chemical processing was brought up. The action of enzymes toward crustacean wastes is less harmful and works similarly with chemical solutions. Enzymatic deproteinization and demineralization are environmentally friendly and retain the native properties of the biomolecules, the production of quality chitin, and protein residues and major carotenoids [75]. The use of protease enzyme disrupts the carotene–protein bond, hence releasing the carotenoid. According to Sachindra and Mahendrakar [76], a longer time is required for protease treatment if the goal was to recover carotenoid by means of oil or solvent extraction; however, the treatment time should be shorter if the goal is to recover carotenoprotein, for fear that the protease activity would over disrupt the carotenoprotein bond. In a study by Jo et al. [77], various commercially available enzymes were compared for its efficiency to deproteinize crab shell wastes, with Delvolase highest deproteinizing activity with an 84% protein removal and 50% demineralization from the crab shell wastes. In another study, Alcalase was used to deproteinize shrimp shell wastes whereby an optimum concentration of 0.3% was found to yield maximum carotenoid recovery in a period of 4h under the temperature range of 25–30°C [78]. A high astaxanthin recovery (192 ppm) was obtained by Phuong et al. [79] when black tiger shrimp (Penaeus monodon) was treated with Alcalases. Hereby, enzymatic degradation of crustacean wastes is able to recover carotenoids and produces quality chitin and other biomolecules. When calculated and optimized, microorganism-mediated fermentation can be an economical approach with maximum gains. Microbial fermentation is forecasted as the most eco-friendly, safe, technologically flexible, and economical practice [6]. Microorganisms are long known to humankind, be it as friend or foe. As detritivores, bacteria can degrade almost all substances in this world. Since crustacean shells are generally made up of biomolecules and pigments, this kind of microorganism-mediated deproteinization and demineralization results in a liquor rich in biomolecules, especially carotenoids like astaxanthin. Anaerobic fermentation with lactic acid producing bacteria provides a certainty that the free astaxanthin would not be oxidized that easily. This was proven when shrimp shell wastes were fermented with lactic acid producing bacteria, a good amount of astaxanthin was recovered up to 115 μg/g [7]. Additionally, Pacheco et al. [80] investigated the effects of temperature on lactic acid fermentation and the recovery of astaxanthin and found that lower temperature (20–30°C) encourages the recovery of free astaxanthin. Prolonged exposure of astaxanthin toward high temperature causes it to degrade [81]. Elsewhere, Sachindra et al. [82] conducted an investigation into shrimp wastes of Penaeus indicus by comparing acid ensilage and lactic acid fermented ensilage and found that acid ensilage gave out a high initial carotenoid recovery of 43.092.84 μg/g compared with fermented ensilage, which only yielded 32.20  1.85 μg/g. However, as storage days increase for both ensilages, the amount of carotenoid extracted was higher in

260 Chapter 13 fermented ensilage (41.85  1.02 μg/g) compared with acid ensilage (26.76  1.16 μg/g). Acid has a degradative effect on the carotenoids and disturbs its stability, but through the ensilaging method, it stabilizes the carotenoid in shrimp waste [83]. However, this method reduces the amount of free astaxanthin. Aside from that, recycling of shrimp wastes was also attempted with aerobic bacteria but was mainly used to recover chitin. The genus Bacillus is popularly used as a deproteinizing agent to deproteinize crustacean wastes [84,85]. The enzyme from Bacillus cereus SV1 was used to treat shrimp waste to obtain chitin [75] whereby the enzyme avoid unwanted by-products and side effects, including heavy metal residues, and overhydrolysis. Nevertheless, none of these methods were used to recover astaxanthin or carotenoids from the wastes. The sensitivity of free astaxanthin toward light and oxygen that causes it to be oxidized poses a slight problem in microbial fermentation, especially if it involves aerobic fermentation. However, in a study conducted by Cheong [86], an aerobic bacteria species, Aeromonas hydrophila, was isolated and applied to shrimp shell wastes in a liquid state fermentation. Free astaxanthin was present at the end of the fermentation process, showing that aerobic fermentation is a viable, and astaxanthin was not fully oxidized by oxygen as most of them are present in the form of carotenoproteins and chitinocarotenoids. Alternatively, cocultured fermentation using different microorganisms within a system may increase deproteinization and demineralization efficiency. The combination of a proteolytic bacteria and a lactic acid-producing strain would result in a more efficient biomolecule recovery in crustacean wastes. This method is not popular among researches probably because of the selection of bacteria and the challenges in optimizing the experimental protocol. When Bacillus licheniformis 21,886 was firstly inoculated in a cofermented system before the inoculation of Gluconobacter oxydans DSM-2003 60h later, the deproteinization and demineralization efficiencies on shrimp head wastes were 87% and 93.5%, respectively [84]. Although the fermentation time was half of the cultivated cycle reported, the fermentation system took a longer time compared with the normal microbial fermentation time of 48 h. Meanwhile, another protease-producing bacteria Teredinobacter turnirae was experimented in a cocultivation with Lactococcus lactis. However, it was found that by inoculating T. turnirae first, followed by L. lactis at the end of the fourth day where protein content was at its minimum in the prawn waste, the demineralization process achieved was the highest at 95.5% [87]. Both studies showed that high deproteinization or demineralization was achieved when the cocultivation system was first inoculated with a protease-producing bacteria followed by lactic acid bacteria. Similarly, Francisco et al. [88] reported high deproteinization and demineralization using a cocultivation system of Lactobacillus plantarum and Lactococcus sp. These studies concluded that calcium carbonate was successfully removed from crustacean wastes under the cocultivation system due to the presence of lactic acid.

Turning leftover to treasure 261

4.3 Mechanical methods The exoskeleton of crustaceans is generally hardened by minerals, and the presence of chitin, protein, and carotenoids is intermingled between all these structures. Generally, mechanical cell disruption includes the process of grinding, homogenization, bead beater, milling, frozen cells (freeze drying), ultrasonication, and Hughes press, which increases the surface area and shears the cell apart. This approach is energy wasting as energy will be dissipated as heat and has a high capital cost [89]. Alternatively, grinding and crushing shrimp shell wastes with liquid nitrogen allow for rapid cooling and freezing, which also avoids heat being introduced into the system. Direct extraction of astaxanthin from it shows the highest recovery compared with other treatments [90]. Other systems introduced by the authors were prolonged heat and water treatments toward the shells, which may have degraded and oxidized the free astaxanthin present. Similarly, Mezzomo et al. [91] agreed that cooking, drying, and milling presented the best carotenoid recovery results.

5 Extraction of astaxanthin Before extracting astaxanthin or carotenoids from crustacean shells, pretreatments are mandatorily conducted to enhance the yield of astaxanthin (Table 13.1). Microwave- and ultrasound-assisted extraction (MAE and UAE) are methods used in disrupting the composition of crustacean shells whereby MAE holds the advantage of being a simple device with a wide area of application, high extraction efficiency, low consumption of organic solvents, and good reproducibility [18]. Meanwhile, ultrasound-assisted extraction uses a high-frequency sound (more than 16kHz) to disrupt the target compound in cells. Both methods are environmentally friendly, which increases productivity especially toward thermosensitive compounds [92]. In a comparison study by Tsiaka et al. [104], the shrimp waste of Aristeus antennatus was subjected to MAE and UAE disruption and solvent extraction. From their novel study, it was concluded that UAE gave a better recovery than MAE due to certain conditions used in MAE. Meanwhile the amount of carotenoid extracted using MAE and UAE was not significantly different and ranged between 60% and 105% per 100g of waste. But MAE significantly induces the conversion of all E-astaxanthin to a preferable 13 Z form, while UAE most probably degrades the pigment into a colorless compound [18]. Reports on utilizing MAE and UAE on crustacean wastes are rare, and this is a promising technique to explore as both techniques are fast, reliable, reproducible, and less laborious. This is an opportunity to lower the cost of the extractions of astaxanthin and other carotenoids from crustacean wastes. However, both techniques require a medium of extraction, usually an organic solvent that potentially poses a threat to the environment. Maceration is a technique used by many researchers in enhancing the extractability of astaxanthin from crustacean shells, whereby heat is not introduced and avoids thermal degradation of carotenoids. In comparison with MAE and UAE, both techniques generally

Table 13.1: Various extraction method and solvents used to recover astaxanthin from crustacean waste. Methods of extraction

Source of pigment

Solvents of extraction

Astaxanthin amount

Remarks

References

(i) Chemical 3% HCI and 4% NaOH 1.5 M HCl and 1.0 M NaOH 1.0 M HCl and 1.0 M NaOH

Shrimp waste

n/a

n/a

[93]

Litopenaeus vannamei

Ethanol and acetone

n/a

[94]

Litopenaeus vannamei Boone

n/a

n/a

Bleaching was done after acid and alkali treatment for 10 min

[95]

(ii) Enzymatic 3% Alcalase Deproteinizing enzyme Acetone extraction on wet samples Alcalase

Callinectes sapidus Aristeus alcocki

Acetone Acetone

97.7  14.3 μg/g 21.43  1.33 μg/g

[96] [97]

Aristeus alcocki

Acetone

70.27  2.58 μg/g

[97]

Xiphopenaeus kroyeri

Soy oil and petroleum ether/acetone/water (15:75:10 v/v)

5.7 mg/ 100 g

[98]

(iii) Microbial DMSO4 Lactobacillus plantarum Penaeus semisulcatus and Paralithodes brevipes and Lactobacillus rhamnosus Shrimp shell wastes Soybean oil Lactobacillus spp. Shrimp shell waste Chloroform-methanol Aeromonas hydrophila powder Penaeus semisulcatus Hexane-acetone (3:1 Lactobacillus spp. v/v)

n/a

5% CO2 was supplied

[13]

10.3 μg/g 2.2  0.119 μg/g

[99] [86]

23.128 mg/g

[100]

(iv) Mechanical Hobart grinder Commercial miller Cooking, drying, and milling Waring blender homogenization Waring blender homogenization

Litopenaeus setiferus Flaxseed oil Ground crayfish waste Acetone Shrimp waste Maceration in hexane/ isopropanol (1:1 v/v) Charybdis cruciate Acetone followed by petroleum ether Potamonpotamon Acetone followed by petroleum ether

4.83 mg/100 g 108 ppm 154.3  0.5 μg/g

[101] [71] [91]

23.6  1.4 g/100 g

[4]

7.2  1.0 g/100 g

[4]

(v) Others Wastewater of Penaeus vannamei Demineralization Note: n/a ¼ not applicable.

Penaeus vannamei

Sunflower oil

10–13 μg/mL

[102]

Callinectes sapidus

Supercritical CO2 ethanol extraction

n/a

[103]

264 Chapter 13 complete the extraction process rapidly, but a larger amount of solvent is required. In comparison, maceration may take up to 120 h upon completion, although it uses a minute amount of extraction solvent, typically 4 mL/g [91]. Mezzomo et al. [91] also showed that maceration of pink shrimp (P. brasiliensis and P. paulensis) with acetone, followed by Soxhlet extraction using hexane: isopropanol (1:1 v/v) was far more efficient in extracting astaxanthin compared with UAE. Unfortunately, maceration is not the “super method” because factors such as solvent system and source of pigment play a significant influence on the output. The easiest and most viable method to extract nonpolar carotenoids is by using edible oil. This could also be done in common households using cooking oil and crustacean wastes. The advantage of using oils, such as sunflower oil, is that the extracted carotenoid oil could be consumed directly and increases the quality of the oil itself. As an example, astaxanthin extracted from the shrimp Pandalus borealis can be consumed directly [105]. When heated between 40°C and 60°C, flaxseed oil containing astaxanthin from Litopenaeus setiferus is able to lower lipid oxidation compared with ordinary flaxseed oil [101], thus augmenting the quality of the oil. Other than extracting astaxanthin directly from crustacean shell wastes, the pigment can also be extracted from shrimp cooking wastewater, which appears mainly as free astaxanthin [102]. Supercritical fluid extraction (SFE) has been labeled as generally recognized as safe (GRAS). This technique is suitable to be used on thermolabile compounds and is considered environmentally safe as the usages of toxic solvents are avoided. Many studies opt for this technique to extract astaxanthin with carbon dioxide due to its nature of being nonflammable, inert, nontoxic, cheap, recyclable, environmentally friendly, and ideal for use in food industries although it requires a high pressure [106–108]. Under the conditions of 60°C and 20 Mpa with the presence of ethanol, the extracted astaxanthin yield was as high as 58.030.1μg/g, of which 12.25  0.9 μg/g was free astaxanthin [108]. Due to the chemical behavior of CO2, which is similar to lipophilic solvents, it is good in extracting nonpolar compounds, and cosolvents with high polarity such as water, ethanol, methanol, and other polar solvents are often used together [109]. Often, ethanol is a better cosolvent due to its GRAS designation. Elsewhere a study by Han et al. [110] demonstrated that an alternative fluid was used to extract astaxanthin from Euphausia pacific using subcritical 1,1,1,2-tetraflouroethane. The group managed to obtain a maximum astaxanthin yield of 87.74% under an optimized condition. Classically the extraction of astaxanthin and other carotenoids are carried out using organic nonpolar or semipolar solvents. The most popular solvents include acetone, hexane, ethanol, ethyl acetate, methylketone, and methanol [11]. Hamdi et al. [111] showed a high extractability of carotenoid when blue crab shells were macerated with hexane: isopropanol (1:1 v/v), but the extracted amount was even higher when enzymes were added to the extraction process. However, combining the enzyme and maceration systems reduced the amount of soluble protein and total polyphenol content by 45% and 78%, respectively, compared with hexane-isopropanol maceration alone. Meanwhile, dichloromethane was the most ideal solvent for the extraction of astaxanthin, with four times sample extraction giving the most satisfying

Turning leftover to treasure 265

(A)

(B) Fig. 13.3 An overview of astaxanthin extraction process in the (A) industry and (B) laboratory.

yields, and was also generally easy to conduct [112]; this comes with negative impacts such as large amount of solvent wastes, longer extraction time, and destruction of the structure and stability of astaxanthin. Due to that an alternative solvent extraction method was introduced, such as the high-pressure extraction (HPE) method that ensures fast, efficient, and low-temperature operation [113]. The application of high pressure onto the crustacean wastes not only promoted the destruction of the original micropore structure but also enlarges the diameter of micropores, which eventually joins with adjacent micropores to become a bigger pore, allowing the solvents to enter. According to Gamlath and Wakeling [114], the HPE method is claimed to be able to extract carotenoids with reduced structural damage, maintaining its color while providing high stability. Additionally, the end product of HPE is considered safe by the US FDA and is environmentally friendly [115]. A general overview of astaxanthin extraction is shown in Fig. 13.3.

6 Identification of astaxanthin Thin-layer chromatography (TLC) is the most basic and common chromatography technique used to identify carotenoids and astaxanthin. It is principally known to separate pigments according to its affinity toward the mobile and stationary phase, whereby higher affinity compounds will move slower than others, thus a lower Rf value [116]. TLC purifies the concentrated carotenoid extract through separation and allows for the identification of a pigment through a comparison with the retention factor (Rf) value. Additionally, TLC enables the visual monitoring of carotenoid separation; is considered cheap, reproducible, and fast; and has multifold detection [117]. The reported Rf values (Table 13.2) for astaxanthin have been internationally accepted and can be used as a reference provided the system of conduction is similar [97].

266 Chapter 13 Table 13.2: Internationally accepted Rf values for astaxanthin and its esters. Carotenoid Astaxanthin (free)

Astaxanthin monoesters

Astaxanthin diesters

Rf value

Mobile phase (v/v)

Reference

0.33 0.33 0.33 0.36 0.30 0.46 0.50 0.60 0.57–0.59 0.6 0.66 0.75 0.75 0.74–0.83 0.8 0.78 0.75

Hexane/acetone (70:30) Hexane/acetone (70:30) Hexane/acetone (3:1) Hexane/acetone (3:1) Isopropanol/hexane (1:1) Hexane/acetone (70:30) Hexane/acetone (70:30) Hexane/acetone (3:1) Hexane/acetone (70:30) Hexane/acetone (70:30) Hexane/acetone (3:1) Hexane/acetone (70:30) Hexane/acetone (3:1) Hexane/acetone (70:30) Hexane/acetone (70:30) Hexane/acetone (3:1) Isopropanol/hexane (1:1)

[118] [119] [86] [120] [121] [122] [118] [120] [122] [119] [86] [118] [120] [122] [119] [86] [121]

Generally, astaxanthin is extracted from crustacean wastes using a traditional solvent extraction before being subjected to TLC. The mixture of hexane/acetone (3:1 or 7:3 v/v) has been agreed by many to be the best mobile phase solvent in developing astaxanthin [86,123]. The Rf value for astaxanthin and its esters was reported by Lorenz Todd [124] when carotenoids were developed using hexane/acetone (3:1 ratio). Other researchers reported slightly different Rf values for the three astaxanthin forms. Generally an approximate Rf value of 0.3 is accepted for free astaxanthin, 0.6 for astaxanthin monoesters, and 0.8 for astaxanthin diesters. Classically, astaxanthin monoesters are identified using TLC together with the other carotenoids that are present in crustacean wastes. Limitations apply, especially if the amount of carotenoid present is too small that it does not produce a visible band and stereoisomers are unable to be identified. This could be overcome with some enhancements to the basic TLC method, whereby high pressure is applied. A more modern and technologically advanced method, the high-pressure TLC (HPTLC), is able to detect the presence of stereoisomers in a sample. In HPTLC the silica gel is manufactured in a smaller particle size compared with the traditional TLC silica gel. This ensures a higher packing density, which causes a compact band to be formed, which increases detection sensitivity. In a study by Zhang et al. [118], astaxanthin monoester stereoisomers were predetected as a single 3R, 30 R peak in the HPTLC chromatogram through artificial esterification and were later proven to consist of a single stereoisomer. Additionally, the authors suggested that the astaxanthin and astaxanthin

Turning leftover to treasure 267 monoester obtained from their study could be used as a standard for other related studies since there were no known standards for astaxanthin monoesters. Another alternative way of separating and identifying the extracted carotenoids is through column chromatography (CC). An open column chromatography (OCC) allows for the separation of carotenoids with similar polarity (Britton et al., 1995). In OCC the carotenoid fraction can be monitored visually based on the color differences [125]. Carotenoids are developed with CC, or OCC uses mixtures of polar and nonpolar solvents, such as hexane/ acetone mixtures [78] and dichloromethane/acetone mixtures [126] in different ratios to elute astaxanthin and its esters. Ultimately the fractions of column chromatography must be determined by high-pressure liquid chromatography (HPLC). Hence, column chromatography can be considered a purification method done before the identification of astaxanthin, astaxanthin esters, and other present carotenoids. HPLC is a method generally popular among researchers and is much more preferred than CC or OCC due to the sensitivity of the machine. However, the injected samples must be purified beforehand to avoid any complications during detection. The identification of a compound is solely based on the retention time, and this can vary among researchers, depending on the type of column and mobile phase used. Therefore there is no general retention time available for astaxanthin and its esters when using HPLC and a standard is required for identification. Carotenoids can firstly be separated using TLC before identified through HPLC [86]. Hereby, TLC acts as a semipurification step for the carotenoids as they are separated into respective bands. Nevertheless, limitation comes when identifying astaxanthin esters, and a HPLC-MS (HPLC with mass spectrometer) machine is required as there is no astaxanthin ester standard available in the market. A HPLC-MS machine is costly, and each broken molecular species of the MS is required to be analyzed [127]. Inevitably, chromatography techniques require a huge amount of solvents being used and low recovery for further characterizations, and certain chromatography techniques such as TLC may cause the degradation of astaxanthin. Other methods available to detect astaxanthin, astaxanthin esters, and carotenoids from crustaceans such as nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), and mass spectrometer. But these methods are best used for identifying new or unknown carotenoids, as a large amount of pure sample is required (10mg) and also involves a complex methodology to analyze the data obtained [125].

7 What comes next? Successfully extracted and identified astaxanthin is ready to be packaged and sold to the end consumer, either for consumption or laboratory use. Generally extracted astaxanthin can be directly encapsulated or spray dried before encapsulation. Encapsulation of astaxanthin

268 Chapter 13 provides it with a longer shelf life, preventing degradation and preserving their functions and benefits over a prolonged period [128]. Cashew gums were found to be able to incorporate with gelatine to form coacervates by encapsulating lipid extracts (containing astaxanthin) from shrimp wastes [129]. The encapsulation efficiency was reported to be 59.9%0.01% and was able to form multinucleated and polymorphic microcapsules, which are soluble in water and enhance the availability. They added that these microcapsules enable themselves to be dispersed in food such as yogurt, thus providing coloration and nutrition with no differences in odor. Meanwhile, Taksima et al. [130] examined the properties of encapsulating natural astaxanthin obtained from shrimp shells in alginate-chitosan beads when mixed into yogurt. The result revealed that the encapsulated astaxanthin beads were well accepted and have a high purchasing intent of 95.6%. The outcome of nanoencapsulation is beneficial to the food industry as astaxanthin can be used to increase the nutritional value in food and also provides natural coloring.

8 Contribution of astaxanthin to the economy In 2014 the estimated global market value for astaxanthin (synthetic and natural) was at USD 447 million [24]. In a recent market search, the global market value for astaxanthin reached a total of USD 369 million in 2014 and is expected to increase up to USD 423 million in 2019 [131]. According to BBC Research [131], astaxanthin’s sales volume is predicted to be 670 metric tons valued at USD 1.1 billion in 2020. With the high supply of astaxanthin in the market, most of it were dominated by those of synthetic origin. Synthetic astaxanthin is widely produced from petrochemical residues by giant companies such as BASF, Hoffman La-Roche, DSM, and NHU [132,133]. Chemically produced astaxanthin shares the same molecular formula with natural astaxanthin, but not its stereoisomers [132], and thus does not give the same effect as natural astaxanthin. Since there is a high demand for astaxanthin in the aquaculture industries, a cheaper option is usually preferred. Astaxanthin is used to provide coloration to aquaculture organisms, and if aquaculture products that rely on synthetic astaxanthin were consumed by humans, the effects of natural astaxanthin may not be released to its full potential and may negatively impact the human body, for example, increased carcinogenicity, hypersensitivity, and toxicity in the human body [134]. Nevertheless, in most cases, synthetic astaxanthin is often preferred due to its fast manufacturing, easy availability in a huge amount, and lower price compared with natural astaxanthin. The cost of production for synthetic astaxanthin is only about USD 2000/kg [135], while natural astaxanthin obtained from H. pluvialis biomass could cost up to USD 7000/kg [136]. Despite that, there are many ongoing researches that focus on developing cheaper and improved solutions to lower the cost of natural astaxanthin production, though there is still much optimization that needs to be done. Table 13.3 shows the utilization of astaxanthin in various products in different industries. Antarctic krill oil and Haematococcus algae dominate the supplement market especially for Antarctic krill oil as it is high in omega fatty acids and astaxanthin.

Table 13.3: Usage of astaxanthin in various products. Category Krill oil (Euphausia superba)

Product name

Dosage form/ astaxanthin content

Antartic krill oil (double strength)

1000 mg soft gel/500 μg per gel

Company

Purpose

Sports Research

General well-being

GNC

Support heart and joint health

Kirkland Signature

Supports healthy heart and provides antioxidant support to body Supports cardiovascular, brain and immune health. Other benefits include muscle recovery, eye and skin health Antioxidant, maintain a natural and healthy skin glow Supports beauty, improves skin condition, alleviates oxidative stress, reduces DNA damage in immune cell, and boosts health Benefits skin, immune system, brain, muscle and joints, vision, and vitality

AstaReal Natural astaxanthin 12 mg

Krill oil—with added omega 3 s EPA, DHA, phospholipids Soft gel/42.5μg per gel Krill oil—with phospholipids, phosphatidyl choline, choline, astaxanthin, Omega 3 s, EPA, DHA Soft gel/150 μg per gel Krill oil—with omega 3 fatty acids, phospholipids, and astaxanthin Soft gel capsules/12 Haematococcus pluvialis mg per capsule

Astaxanthin 10 mg

Soft gel/10 mg per gel

Haematococcus pluvialis

Solgar

Astaxanthin 6 mg

Capsule/4 mg per gel

Powderized Haematococcus pluvialis extract

Biolife

Astaxanthin 4 mg

Soft gel/4 mg per gel

Haematococcus pluvialis extract

Cell Labs

Krill oil 500 mg

Krill oil 500 mg

Algae (Haematococcus sp.)

Main ingredients

Kordel’s

Continued

Table 13.3: Category

Product name Asta-Glu-C

White shield drink

Astamate

Other sources

Jelly Aquarysta

Lighting perfection pressed powder SPF 18 PA ++ Golden astaxanthin for pets

Usage of astaxanthin in various products—cont’d Dosage form/ astaxanthin content

Main ingredients

Company

Purpose

Capsule/1 mg per capsule

Haematococcus pluvialis extract

Cell Labs

Protecting skin from UV rays and melanin activation Replenish skin

Liquid/4 mg per bottle Erythrito, collagen of 50 mL peptide (fish derived), N-acetylglucosamine, vitamin C, Haematococcus algae color (astaxanthin containing) Tablet/1 mg per tablet Hydrogenated maltose starch syrup, dextrin, vegetable oil containing vitamin E, crystalline cellulose, natural astaxanthin from Haematococcus pluvialis (1 mg) Jelly Collagen, human-type nanoceramide, nanoastaxanthin, nanolycopene, highly permeable resveratrol Pressed powder Astaxanthin, soluble collagen Powder

Seaweed, inulin, glucose, zinc, astaxanthin, probiotics, Norway golden seaweed and red astaxanthin

Fujiflim Corporation

Supports pet general health

Fujiflim Corporation

Enhances elasticity for youthful and radiant skin

Fujiflim Corporation

Moisturizes skin and flawless radiant appearance Improve fur, reduce discoloration of nose, strengthen wool, and enhance pigmentation

Sulfur Soap Trading

Salmon oil

Omega-3 krill bites

Asta Zan 14 turmeric and red algae

Liquid

EPA, DHA, astaxanthin, naturally occurring vitamin D3, vitamin B12 Soft chews/270 μg per Qrill pet meal, krill oil, chew astaxanthin, algae, organic hemp Powder Turmeric, almond powder, sunflower lecithin, astaxanthin

Goldfish pellet

Pellet

Super color flakes

Flakes

Astaxanthin

Pump/about 1 mg every 2 pumps

Salmon, whole herring, wheat flour, wheat germ, astaxanthin Salmon, whole herring, wheat flour, whole shrimp, astaxanthin Astaxanthin (1 mg), medium chain triglycerides, mixed tocopherols, silicon dioxide

Information is collected from the respective websites and/or the label on the product. The first five ingredients are listed as main ingredients or up till astaxanthin is stated.

Paws & Pals

Support flexible joints, a healthy heart, brain, and immune system

Zesty Paws

Support for the skin, coat, brain, heart, and joints Soothe joint pain and help support active dog’s stamina and recovery Overall health

Asta Zan 14

Omega One

Omega One

Color enhancement for fishes

Dr. Mercola

Antioxidant for dogs and cats

272 Chapter 13

9 Future prospects The best solution to recover astaxanthin from crustacean wastes without neglecting the environment and maintaining the pigment’s ability and structure is by far through enzymatic degradation. In modern crustacean waste management researches, this method remains as the most famous among others, with chemical treatment being the second most recognized. While enzymatic degradation conducted with lactic acid bacteria demonstrated high recovery, we should not forget that aerobic bacteria fermentation also allows for the recovery of astaxanthin. As it was previously shown by Cheong [86], A. hydrophila is able to deproteinize and dechitinize shrimp shell waste and successfully recover astaxanthin. This idea could be further enhanced by using bacteria from the genus Bacillus (which are famous for protease production) or other bacteria that are able to produce protease and chitinase simultaneously. Coculturing is also a viable method for astaxanthin recovery. In previous coculture studies, astaxanthin was not determined, although based on the successful deproteinization and demineralization records provided by researchers, it hints at the opportunity to study the recovery of astaxanthin via this method. The actual structures of the carotene-protein and chitin-carotenoids subjected to enzymatic degradation would require a detailed study to develop the best solution to extract astaxanthin from the shells, either through anaerobic, aerobic, single, or coculture methods. Additionally, there are many fungi that produce protease and chitinase, and these enzymes could be extracted to replace commercial enzymes used or applied directly onto crustacean shell wastes. To date, there is a paucity of studies involving fungi in deproteinization and/or dechitinization of crustacean wastes [137]. This is a realm that has yet to be fully explored and may provide additional knowledge to the research society. In terms of economy, it would be ideal if countries that primarily rely on the production of reliable and quality crustacean products invest in measures to recycle crustacean waste commercially and obtain biomolecules from it. This could potentially lower the cost of obtaining natural astaxanthin. Producing their own algae biomass, culturing astaxanthin-producing bacteria or genetically modified bacterial strains for the production of astaxanthin is another alternative [43,122]. The plant Adonis palestina Boiss is widely cultivated in Mongolia and China for astaxanthin [136]. Other species of plants under the same genus such as Asterias amurensis [138], Amanita aestivalis [139], and Artemisia annua [140], which produces astaxanthin could also be mass cultured to lower the cost of natural astaxanthin and subsequently made affordable to the public. Elsewhere the genetically modified Arabidopsis plants were found to be able to synthesize astaxanthin with more stability toward light and lipid peroxidation [141], which could potentially yield a more versatile product that may be used in various industries without fear of it being oxidized easily. These ultimately could be some solutions that lower the cost of production of astaxanthin and are considered safe to be consumed.

Turning leftover to treasure 273

10 Conclusion There are many sources of astaxanthin around us and recycling crustacean wastes is a solution to reduce environmental pollution caused by crustacean processing industries. With the advancement of the scientific community, the reliability toward chemicals that disrupt the shells, extract, and identify astaxanthin should be reduced, if not curbed. Since astaxanthin is ranked as the third most important carotenoid after β-carotene and lutein, consuming natural astaxanthin or using natural astaxanthin in animal and/or aquaculture feeds is highly recommended to prevent complications caused by synthetic ones. After all, astaxanthin is an organic molecule found in nature; its full potential can be realized with more in-depth research into the various organisms that are capable of synthesizing; it is without doubt vital to the scientific community, food industries, and the general public.

References [1] FAO Yearbook. Fisheries and aquaculture statistics 2016. Rome: Food and Agriculture Organization United Nation; 2018. [2] Niu J, Tian LX, Liu YJ, Yang HJ, Ye CX, Gao W. Effect of dietary astaxanthin on growth, survival and stress tolerance of post-larval shrimp, Litopenaeus vannamei. J World Aquac Soc 2009;40:795–802. [3] Parisenti J, Beirao LH, Tramonte VLCG, Ourique F, da Silveira Britto CC, Moreira CC. Preference ranking of color in raw and cooked shrimp. Int J Food Sci Technol 2011;46:2558–61. [4] Sachindra NM, Bhaskar N, Mahendrakar NS. Carotenoids in crabs from marine and fresh waters of India. LWT-Food Sci Technol 2005;38:221–5. [5] Islam MS, Khan S, Tanaka M. Waste loading in shrimp and fish processing effluents: potential source of hazards to the coastal and near shore environments. Mar Pollut Bull 2004;49:103–10. [6] Kaur S, Dhillon GS. Recent trends in biological extraction of chitin from marine shell wastes: a review. Crit Rev Biotechnol 2013;35:44–61. [7] Gimeno M, Ramı´rez-Herna´ndez JY, Ma´rtinez-Ibarra C, Pacheco N, Garcı´a-Arrazola R, Ba´rzana E, Shirai K. One-solvent extraction of astaxanthin from lactic acid fermented shrimp wastes. J Agric Food Chem 2007;55:10345–50. [8] Yan N, Chen X. Sustainability: don’t waste seafood waste. Nature 2015;524:155–7. [9] Xu Y, Gallert C, Winter J. Chitin purification from shrimp wastes by microbial deproteination and decalcification. Appl Microbiol Biotechnol 2008;79:687–97. [10] Nwanna LC, Ajenifuja AM, Enujiugha VN, Nwanna LC, Ajenifuja AM. Replacement of fish meal with chemically preserved shrimp head in the diets of African catfish, Clarias gariepinus. J Food Agric Environ 2004;2:79–83. [11] Mao Z, Guo N, Sun J, Xue C. Comprehensive utilization of shrimp waste based on biotechnological methods: a review. J Clean Prod 2017;143:814–23. [12] Saini RK, Keum YS. Carotenoid extraction methods: a review of recent developments. Food Chem 2018;240:90–103. [13] Saranya R, Ravi D, Vijayabharathi V. Extraction of astaxanthin from marine source by using Lactobacillus Spp. through fermentation technique. In: Ramanathan A, Senthivel T, editors. National seminar on probiotics in sustainable food production: current status and future prospects 2013. Tamil Nadu: Bonfring; 2013. p. 34–40. [14] Lim KC, Yusoff FM, Shariff M, Kamarudin MS. Astaxanthin as feed supplement in aquatic animals. Rev Aquac 2017;10:738–73. [15] Lam HS, Ngoc PT. Effect of dietary astaxanthin on pigment accumulation in the muscle-skin, resistance to salinity stress and copper toxicity of commercial clownfish, Amphipriono cellaris. Vietnam J Mar Sci Technol 2018;18:60–9.

274 Chapter 13 [16] Lee HS, Sung DK, Kim SH, Choi WI, Hwang ET, Choi DJ, Chang JH. Controlled release of astaxanthin from nanoporous silicified-phospholipids assembled boron nitride complex for cosmetic applications. Appl Surf Sci 2017;424:15–9. [17] Cheng XY, Xiong YJ, Yang MM, Zhu MJ. Preparation of astaxanthin mask from Phaffiarhodozyma and its evaluation. Process Biochem 2019;79:195–202. [18] Zhou T, Wang X, Ju Y, Shi C, Kan G. Stability application and research of astaxanthin integrated into food. IOP Conf Ser Mater Sci Eng 2018;394:1–5. [19] Bhuvaneswari S, Arunkumar E, Viswanathan P, Anuradha CV. Astaxanthin restricts weight gain, promotes insulin sensitivity and curtails fatty liver disease in mice fed a obesity-promoting diet. Process Biochem 2010;45:1406–14. [20] Grimmig B, Hudson C, Moss L, Peters M, Subbarayan M, Weeber EJ, Bickford PC. Astaxanthin supplementation modulates cognitive function and synaptic plasticity in young and aged mice. GeroScience 2019;41:77–87. [21] Sun R, Xiaa N, Xia Q. Non-aqueous nanoemulsions as a new strategy for topical application of astaxanthin. J Dispers Sci Technol 2019. https://doi.org/10.1080/01932691.2019.1635027. In press. [22] Shahidi F, Metusalach Brown JA. Carotenoids pigment in seafood and aquaculture. Crit Rev Food Sci Nutr 1998;38:1–67. [23] Capelli B, Cysewski G. Astaxanthin natural astaxanthin: king of carotenoids. Kailua Kona: Cyanotech Corporation; 2007. [24] Wade NM, Gabaudan J, Glencross B. A review of carotenoid utilisation and function in crustacean aquaculture. Rev Aquac 2015;1–16. [25] Goodwin TW. The biochemistry of the carotenoids. London: Chapman and Hall; 1984. [26] Ohkubo M, Tsushima M, Maoka T, Matsuno T. Carotenoids and their metabolism in the goldfish Carassius auratus (Hibuna). Comp Biochem Physiol B 1999;124:333–40. [27] Rhodes ACE. Dietary effects on carotenoid composition in the marine harpacticoid copepod Nitokra lacustris. J Plankton Res 2007;29:73–83. [28] Tlusty M, Hyland C. Astaxanthin deposition in the cuticle of juvenile American lobster (Homarus americanus): implications for phenotypic and genotypic coloration. Mar Biol 2005;147:113–9. [29] Barclay MC, Irvin SJ, Williams KC, Smith DM. Comparison of diets for the tropical spiny lobster Panulirusornatus: astaxanthin-supplemented feeds and mussel flesh. Aquac Nutr 2006;12:117–25. [30] Daly B, Swingle JS, Eckert GL. Dietary astaxanthin supplementation for hatchery-cultured red king crab, Paralithodescamtschaticus, juveniles. Aquac Nutr 2013;19:312–20. [31] Nguyen NH, Quinn J, Powell D, Elizur A, Thoa NP, Nocillado J, Lamont R, Remilton C, Knibb W. Heritability for body colour and its genetic association with morphometric traits in banana shrimp (Fenneropenaeus merguiensis). BMC Gen 2014;15:132–44. [32] Panis G, Carreon JR. Commercial astaxanthin production derived by green alga Haematococcus pluvialis: a microalgae process model and a techno-economic assessment all through production line. Algal Res 2016;18:175–90. [33] Ambati RR, Phang SM, Ravi S, Aswathanarayana R. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications—a review. Mar Drugs 2014;12:128–52. [34] Ambati RR, Gogisetty D, Aswathanarayana RG, Ravi S, Bikkina PN, Bo L, Yuepeng S. Industrial potential of carotenoid pigments from microalgae: current trends and future prospects. Crit Rev Food Sci Nutr 2019;59:180–1902. [35] Rao AR, Sindhuja HN, Dharmesh SM, Sankar KU, Sarada R, Ravishankar GA. Effective inhibition of skin cancer, tyrosinase, and antioxidative properties by astaxanthin and astaxanthin esters from the green alga Haematococcus pluvialis. J Agric Food Chem 2013;61:3842–51. [36] Liu J, Mao X, Zhou W, Guarnieri MT. Simultaneous production of triacylglycerol and high-value carotenoids by the astaxanthin-producing oleaginous green microalga Chlorella zofingiensis. Bioresour Technol 2016;214:319–27. [37] Roth MS, Cokus SJ, Gallaher SD, Walter A, Lopez D, Erickson E, Endelman B, Westcott D, Larabell CA, Merchant SS, Pellegrini M, Niyogi KK. Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production. Proc Natl Acad Sci 2017;114:4296–305.

Turning leftover to treasure 275 [38] Higuera-Ciapara I, F
elix-Valenzuela L, Goycoolea FM. Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr 2006;46:185–96. [39] Yokoyama A, Izumida H, Miki W. Production of astaxanthin and 4-ketozeaxanthin by the marine bacterium, agro-bacterium Aurantiacum. Biosci Biotechnol Biochem 1994;58:1842–4. [40] Asker D, Beppu T, Ueda K. Sphingomonas astaxanthinifaciens sp. nov., a novel astaxanthin-producing bacterium of the family Sphingomonadaceae isolated from Misasa, Tottori, Japan. FEMS Microbiol Lett 2007;273:140–8. [41] Schmidt I, Schewe H, Gassel S, Jin C, Buckingham J, H€ umbelin M, Sandmann G, Schrader J. Biotechnological production of astaxanthin with Phaffia rhodozyma/Xanthophyllomyces dendrorhous. Appl Microbiol Biotechnol 2011;89:555–71. [42] Lu Q, Bu YF, Liu JZ. Metabolic engineering of Escherichia coli for producing astaxanthin as the predominant carotenoid. Mar Drugs 2017;15:296–305. [43] Henke NA, Heider SAE, Peters-Wendisch P, Wendisch VF. Production of the marine carotenoid astaxanthin by metabolically engineered Corynebacterium glutamicum. Mar Drugs 2016;14:124–45. [44] Tao L, Sedkova N, Yao H, Ye RW, Sharpe PL, Cheng Q. Expression of bacterial haemoglobin genes to improve astaxanthin production in a methanotrophic bacterium Methylomonas sp. Appl Microbiol Biotechnol 2007;74:625–33. [45] Kildegaard KR, Adiego-Perez B, Belda DD, Khangura JK, Holkenbrink C, Irina B. Engineering of Yarrowia lipolytica for production of astaxanthin. Synth Syst Biotechnol 2017;2:287–94. [46] Papp T, Velayos A, Barto´k T, Eslava AP, Va´gv€ olgyi C, Iturriaga EA. Heterologous expression of astaxanthin biosynthesis genes in Mucor circinelloides. Appl Microbiol Biotechnol 2006;69:526–53. [47] Yang Y, Kim B, Lee JY. Asaxanthin structure, metabolism and health benefits. J Hum Nutr Food Sci 2013;1:1003–14. [48] Seabra LMAJ, Pedrosa LFC. Astaxanthin: structural and functional aspects. Rev Nutr 2010;23:1041–50. [49] Goto S, Kogure K, Abe K, Kimata Y, Kitahama K, Yamashita R, Terada H. Efficient radical trapping at the surface and inside the phospholipid membrane is responsible for highly potent antiperoxidative activity of the carotenoid astaxanthin. Biochim Biophys Acta 2001;1512:251–8. [50] Pashkow FJ, Watumull DG, Campbell CL. Astaxanthin: a novel potential treatment for oxidative stress and inflammation in cardiovascular disease. Am J Cardiol 2008;101:58D–68D. [51] Yamashita E. Astaxanthin as a medical food. Funct Foods Health Dis 2013;3:254–8. [52] Gomez-Estaca J, Calvo MM, Alvarez-Acero I, Montero P, Gomez-Guillen MC. Characterization and storage stability of astaxanthin esters, fatty acid profile and α-tocopherol of lipid extract from shrimp (L. vannamei) waste with potential applications as food ingredient. Food Chem 2017;216:37–44. [53] Montero P, Calvo MM, Gomez-Guillen MC, Gomez-Estaca J. Microcapsules containing astaxanthin from shrimp waste as potential food coloring and functional ingredient: characterization, stability, and bioaccessibility. LWT-Food Sci Technol 2016;70:229–36. [54] De Brujin WJC, Weesepoel Y, Vincken J-P, Gruppen H. Fatty acids attached to all-trans-astaxanthin alter its cis-trans equilibrium, and consequently its stability, upon light-accelerated autoxidation. Food Chem 2016;194:1108–15. [55] Britton G. Structure and properties of carotenoids in relation to function. FASEB J 1995;9:1551–8. [56] Yuan J-P, Chen F. Purification of trans-astaxanthin from a high-yielding astaxanthin ester-producing strain of the microalga Haematococcus pluvialis. Food Chem 2000;68:443–8. [57] Liu X, Osawa T. Cis astaxanthin and especially 9-cis astaxanthin exhibits a higher antioxidant activity in vitro compared to the all-trans isomer. Biochem Biophys Res Commun 2007;357:187–93. [58] Bjerkeng B. Carotenoid pigmentation of salmonid fishes—recent progress, In: Cruz-Sua´rez LE, Ricque-Marie D, Tapia-Salazar M, Olvera-Novoa MAY, Civera-Cerecedo R, editors. Avancesen Nutricio´n Acuı´cola V. Memorias del V Simposium Internacional de Nutricio´n Acuı´colaMexico: M
erida, Yucata´n; 2000. p. 71–89. [59] Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H. Astaxanthin, a carotenoid with potential in human health and nutrition. J Nat Prod 2006;69:443–9. [60] Su F, Huang B, Liu J. The carotenoids of shrimp (Decapoda: Caridea and Dendrobranchiata) cultured in China. J Crustacean Biol 2018;38:523–30.

276 Chapter 13 [61] Maoka T. Carotenoids in marine animals. Mar Drugs 2011;9:278–93. [62] Cheesman DF, Prebble J. Astaxanthin ester as a prosthetic group: a carotenoprotein from the hermit crab. Comp Biochem Physiol 1966;17:929–35. [63] Helliwell JR. The structural chemistry and structural biology of coloration in marine crustacean. Crystallogr Rev 2010;16:231–42. [64] Chen X, Yang H, Yan N. Shell biorefinery: dream or reality? Chem 2016;22:13402–21. [65] North A. Why do lobsters turn pink when they are cooked? Trends Biotechnol 2002;20:414–5. [66] Chantarasuwan C, Benjakul S, Visessanguan W. The effect of sodium carbonate and sodium bicarbonate on yield and characteristics of Pacific white shrimp (Litopenaeusvannamei). Food Sci Tech Int 2011;17:403–14. [67] Ju ZY, Deng DF, Dominy WG, Forster IP. Pigmentation of pacific white shrimp, Litopenaeus vannamei, by dietary astaxanthin extracted from Haematococcus pluvialis. J World Aquac Soc 2011;42:633–44. [68] Hansen ME, Illanes A. Applications of crustacean wastes in biotechnology. In: Martin AM, editor. Fisheries processing: biotechnological applications. London: Chapman and Hall; 1994. p. 174–205. [69] Allan GG, Fox JR, Kong N. A critical evaluation of the potential sources of chitin and chitosan. In: Muzzarelli RAA, Pariser ER, editors. Proceedings of the first international conference on chitin chitosan. Cambridge, MA: MIT Press; 1978. p. 64–78. [70] Younes I, Rinaudo M. Chitin and chitosan preparation from marine sources. Structure, properties and applications. Mar Drugs 2015;13:1133–74. [71] No HK, Meyers SP, Lee KS. Isolation and characterization of chitin from crawfish shell waste. J Agric Food Chem 1989;37:575–9. [72] Kandra P, Challa MM, Jyothi HKP. Efficient use of shrimp waste: present and future trends. Appl Microbiol Biotechnol 2012;93:17–29. [73] Healy M, Green M, Healy A. Bioprocessing of marine crustacean wastes. Acta Biotechnol 2003;23:151–60. [74] Gortari MC, Hours RA. Biotechnological processes for chitin recovery out of crustacean waste: a mini-review. Electron J Biotechnol 2013;16:1–14. [75] Manni L, Ghorbel-Bellaaj O, Jelloul IK, Younes I, Nasri M. Extraction and characterization of chitin, chitosan, and protein hydrolysates prepared from shrimp waste by treatment with crude protease from Bacillus cereus SV 1. Appl Biochem Biotechnol 2010;162:345–57. [76] Sachindra NM, Mahendrakar NS. Effect of protease treatment on oil extractability of carotenoids from shrimp waste. J Aquat Food Prod Technol 2011;20:22–31. [77] Jo GH, Jung WJ, Kuk JH, Oh KT, Kim YJ, Park RD. Screening of protease-producing Serratia marcescens FS-3 and its application to deproteinization of crab shell waste for chitin extraction. Carbohydr Polym 2008;74:504–8. [78] Sowmya R, Sachindra NM. Evaluation of antioxidant activity of carotenoid extract from shrimp processing byproducts by in vitro assays and in membrane model system. Food Chem 2012;134:308–14. [79] Phuong PTD, Minh NC, Cuong HN, Minh NV, Han NT, Hoa NV, Yen HTH, Trung TS. Recovery of protein hydrolysate and chitosan from black tiger shrimp (Penaeus monodon) heads: approaching zero waste process. J Food Sci Technol 2017;57:1850–6. [80] Pacheco N, Gamica-Gonzalez M, Ramirez-Hermandez JY, Flores-Albino B, Gimeno M, Barzana E, Shirai K. Effect of temperature on chitin and astaxanthin recoveries from shrimp waste using lactic acid bacteria. Bioresour Technol 2009;100:2849–54. [81] Ruen-Ngam D, Shotipruk A, Pavasant P. Comparison of extraction methods for recovery of astaxanthin from Haematococcus pluvialis. Sep Sci Technol 2011;46:64–70. [82] Sachindra NM, Bhaskar N, Siddegowda GS, Sathisha AD, Suresh PV. Recovery of carotenoids from ensilaged shrimp waste. Bioresour Technol 2007;98:1642–6. [83] Guillou A, Khalil M, Adambounou L. Effects of silagepreservation on astaxanthin forms and fatty acid profiles of processed shrimp (Pandalus borealis) waste. Aquaculture 1995;130:351–60. [84] Liu P, Liu S, Guo N, Mao X, Lin H, Xue C, Wei D. Cofermentation of Bacillus licheniformis and Gluconobacteroxydans for chitin extraction from shrimp waste. Biochem Eng J 2014;91:10–5.

Turning leftover to treasure 277 [85] Hajji S, Ghorbel-Belaaj O, Younes I, Jellouli K, Nasri M. Chitin extraction from crab shells by Bacillus bacteria. Biological activities of fermented crab supernatants. Int J Biol Macromol 2015;79:167–73. [86] Cheong JY. Isolation, optimization and recovery of astaxanthin from shrimp shell wastes of locally isolated Aeromonas hydrophila Steiner. [Doctoral dissertation]Serdang, Malaysia: Universiti Putra Malaysia; 2016. [87] Aytekin O, Elibol M. Cocultivation of Lactococcus lactis and Teredinobacterturnirae for biological chitin extraction from prawn waste. Bioprocess Biosyst Eng 2010;33:393–9. [88] Francisco FC, Simora RMC, Nun˜al SN. Deproteination and demineralization of shrimp waste using lactic acid bacteria for the production of crude chitin and chitosan. Aquac Aquar Conserv Legis 2015;8:107–15. [89] Harrison STL. Bacterial cell disruption: a key unit operation in the recovery of intracellular products. Biotechnol Adv 1991;9:217–40. [90] Cheong JY, Nor Azwady AA, Rusea G, Noormashela UA, Nurul Shaziera AG, Azleen AA, Muskhazli M. The availability of astaxanthin from shrimp shell wastes through microbial fermentations, Aeromonas hydrophila and cell disruptions. Int J Agric Biol 2014;16:277–84. [91] Mezzomo N, Maestri B, Dos Santos RL, Maraschin M, SRS F. Pink shrimp (P. brasiliensis and P. paulensis) processing residue: influence of extraction method on carotenoid concentration. Talanta 2011;85:1383–891. [92] Raso J, Barbosa-Canovas GV. Nonthermal preservation of foods using combined processing techniques. Crit Rev Food Sci Nutr 2003;43:265–85. [93] Hossain MS, Iqbal A. Production and characterization of chitosan from shrimp waste. J Bangladesh Agric Univ 2014;12:153–60. [94] Bonfante-Alvarez H, De Avila-Montiel G, Herrera-Barros A, Torrenegra-Alarcon M, Ginzalez-Delgado AD. Evaluation of five chitosan production routes with astaxanthin recovery from shrimp exoskeletons. Chem Eng Trans 2018;70:1969–74. [95] De Queiroz Antonino RSCM, Fook BRPL, de Oliveira Lima VA, de Farias Rached RI, Lima EPN, da Silva Lima RJ, Covas CAP, Fook MVL. Preparation and characterization of chitosan obtained from shells of shrimp (Litopenaues vannamei Boone). Mar Drugs 2017;15:141–53. [96] Antunes-Valcareggi SA, Ferreira SRS, Hence H. Enzymatic hydrolysis of blue crab (Callinectes sapidus) waste processing to obtain chitin, protein, and astaxanthin-enriched extract. Int J Environ Agric Res 2017;3:81–92. [97] Sindhu S, Sherief PM. Extraction, characterization, antioxidant and anti-inflammatory properties of carotenoids from the shell waste of arabian red shrimp Aristeus alcocki, Ramadan 1938. Open Conf Proc J 2011;2:95–103. [98] De Holanda HD, Netto FM. Recovery of components from shrimp (Xiphopenaeuskroyeri) processing waste by enzymatic hydrolysis. J Food Sc 2006;71:298–303. [99] Bhavsar SP, Singh KG, Sharma LP. Microbial production of astxanthin pigment using marine shrimp. Indian J Geomarine Sci 2016;45:352–7. [100] Khanafari A, Saberi A, Azar M, Vosooghi G, Jamili S, Sabbaghzadeh B. Extraction of astaxanthin esters from shrimp waste by chemical and microbial methods. Iranian J Environ Health Sci Eng 2007;4:93–8. [101] Pu J, Bechtel PJ, Sathivel S. Extraction of shrimp astaxanthin with flaxseed oil: effects on lipid oxidation and astaxanthin degradation rates. Biosyst Eng 2010;107:364–71. [102] Amado IR, Vazq JA, Murado MA, Gonzalez MP. Recovery of astaxanthin from shrimp cooking wastewater: optimization of astaxanthin extraction by response surface methodology and kinetic studies. Food Bioproc Tech 2015;8:371–81. [103] Felix-Valenzuela L, Higuera-Ciapara I, Goycoolea-Valencia F. Supercritical CO2/ethanol extraction of astaxanthin from blue crab (Callinectes sapidus) shell waste. J Food Process Eng 2001;24:101–12. [104] Tsiaka T, Zoumpoulakis P, Sinanoglou VJ, Markis C, Heropoulos GA, Calokerinos AC. Response surface methodology towards the optimization of high-energy carotenoid extraction from Aristeusan tennatus shrimp. Anal Chim Acta 2015;877:100–10. [105] Parjikolaei BR, El-Houri RB, Frett
e XC, Christensen KV. Influence of green solvent extraction on carotenoid yield from shrimp (Pandalus borealis) processing waste. J Food Eng 2015;155:22–8. [106] Nobre B, Marcelo F, Passos R, Beira˜o L, Palavra A, Gouveia L, Mendes R. Supercritical carbon dioxide extraction of astaxanthin and other carotenoids from the microalga Haematococcus pluvialis. Eur Food Res Technol 2006;223:787–90.

278 Chapter 13 [107] Sanchez-Camargo AP, Martinez-Correa HA, Paviani LC, Cabral FA. Supercritical CO2 extraction of lipids and astaxanthin from Brazillianredspotted shrimp waste (Farfante penaeus paulensis). J Supercrit Fluids 2011;56:164–73. [108] Radzali AA, Baharin BS, Othman R, Markom M, Rahman RA. Co-solvent selection for supercritical fluid extraction of astaxanthin and other carotenoids from Penaeus monodon waste. J Oleo Sci 2014;63:769–77. [109] Molino A, Mehariya S, Iovine A, Larocca V, Sanzo GD, Martino M, Casella P, Chianese S, Musmarra D. Extraction of astaxanthin and lutein from microalga Haematococcus pluvialis in the red phase using CO2 supercritical fluid extraction technology with ethanol as CO-solvent. Mar Drugs 2018;16:432–48. [110] Han Y, Ma Q, Wang L, Xue C. Extraction of astaxanthin from Euphausia pacific using subcritical 1,2,1,2tetraflouroethane. J Ocean Univ China 2012;11:562–8. [111] Hamdi M, Nasri R, Dridi N, Li S, Nasri M. Development of novel high-selective extraction approach of carotenoproteins from blue crab (Portunussegnis) shells, contribution to the qualitative analysis of bioactive compounds by HR-ESI-MS. Food Chem 2020;302:125334–44. [112] Du P, Jin M, Yang L, Chen G, Zhang C, Jin F, Shao H, Yang M, Yang X, She Y, Wang S, Zheng L, Wang J. Determination of astaxanthin in feeds using high performance liquid chromatography and an efficient extraction method. J Liq Chromatogr Relat Technol 2015;39:35–43. [113] Huang HW, Hsu CP, Yang BB, Wang CY. Advances in the extraction of natural ingredients by high pressure extraction technology. Trends Food Sci Technol 2013;33:54–62. [114] Gamlath S, Wakeling L. Trends in high pressure processing of foods: Food quality and bioactive components. In: Comeau M, editor. New topics in food engineering. New York: Nova Science Publisher; 2011. p. 109–10. [115] Prasad KN, Ismail A, Shi J, Jiang YM. High pressure-assisted extraction: Method, technique, and application. In: Lebovka N, Vorobiev E, Chemat F, editors. Enhancing extraction processes in the food industry. New York: CRC Press; 2011. p. 303–22. [116] Fried B, Sherma J. Thin layer chromatography revised and expanded. 4th ed. Basel: Marcel Dekker; 1999. [117] Cserhati T, Forgacs E. Separation and tentative identification of the main pigment fraction of raisins by thin-layer chromatography-fourier transform infrared and high-performance liquid chromatography-ultraviolet detection. J Chromatogr Sci 2000;38:145–50. [118] Zhang S, Sun X, Liu D. Preparation of (3R, 30 R) astaxanthin monoester and (3R, 30 ’R)-astaxanthin from Antarctic krill (Euphausia superba Dana). Eur Food Res Technol 2015;240:295–9. [119] Sila A, Ayed-Ajmi Y, Sayari N, Nasri M, Martinez-Alyarez O, Bougatel A. Antioxidant and anti-poliferative activities of astaxanthin extracted from the shell waste of deep-water pink shrimp (Parapenaeuslongirostris). J Nat Prod 2013;3:82–9. [120] Dalei J, Sahoo D. Extraction and characterization of astaxanthin from the crustacean shell waste from shrimp processing industries. Int J Pharm Sci Res 2015;6:2532–7. [121] Pongsetkul J, Benjakul S, Sampavapol P, Osako K, Faithong N. Chemical copmposition and physical properties of salted shrimp paste (Kapi) produced in Thailand. Int Aquat Res 2014;6:155–66. [122] Wang J, Guleria S, Koffas MAG, Yan Y. Microbial production of value-added nutraceuticals. Curr Opin Biotechnol 2016;37:97–104. [123] Sinthusamran S, Benjakul S, Kijroongrojana K, Prodpran T, Agustini TW. Yield and chemical composition of lipids extracted from solid residues of protein hydrolysis of pacific white shrimp cephalothorax using ultrasound-assisted extraction. Food Biosci 2018;26:169–76. [124] Lorenz RT. Thin-layer chromatography (TLC) system for naturose carotenoids. Nat Rose Technol Bull 1998;003:1–3. [125] Asker D, Awad TS, Beppu T, Ueda K. Purification and identification of astaxanthin and its novel derivative produced by radio-tolerant Sphingomonas astaxanthinfaciens. In: Barreiro C, Barredo JL, editors. Microbial carotenoids. Methods in molecular biology. vol. 1852. New York: Humana Press; 2018. p. 171–92. [126] Babu CM, Chakrabarti R, Sambasivarao KRS. Enzymatic isolation of carotenoid-protein complex from shrimp head waste and its use as a source of carotenoids. LWT-Food Sci Technol 2008;41:227–35.

Turning leftover to treasure 279 [127] Su F, Xu H, Yang N, Liu W, Liu J. Hydrolytic efficiency and isomerization during de-esterification of natural astaxanthin esters by saponification and enzymolysis. Electron J Biotechnol 2018;34:37–42. [128] Dash M, Chiellini F, Ottenbrite RM, Chiellini E. Chitosan a versatile semi- synthetic polymer in biomedical applications. Prog Polym Sci 2011;36:981–1014. [129] Gomez-Estaca J, Comunian TA, Montero P, Ferro-Furtado R, Favaro-Trindade CS. Encapsulation of an astaxanthin-containing lipid extract from shrimp waste by complex coacervation using a novel gelatin-cashew gum complex. Food Hydrocoll 2016;61:155–62. [130] Taksima T, Limpawattana M, Klaypradit W. Astaxanthin encapsulated in beads using ultrasonic atomizer and application in yogurt as evaluated by consumer sensory profile. LWT-Food Sci Technol 2015;62:431–7. [131] BBC Research. Boom in Astaxanthin supplements boosting global carotenoid market, according to BCC Research, Retrieved from: https://www.bccresearch.com/pressroom/fod/boom-in-astaxanthin-supplementsboosting-global-carotenoid-market-according-to-bcc-research; 2015. [132] Nguyen K. Astaxanthin: a comparative case of synthetic vs. natural production. Chem Biomol Eng Pub Other Works 2013;1:1–11. [133] Barredo JL, Garcı´a-Estrada C, Kosalkova K, Barreiro C. Biosynthesis of astaxanthin as a main carotenoid in the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous. J Fungi 2017;3:44–61. [134] Vaclavik VA, Christian EW. Essentials of food science. New York: Springer; 2014. [135] Ni H, Chen Q, He G, Wu G, Yang Y. Optimization of acidic extraction of astaxanthin from Phaffia rhodozyma. J Zhejiang Univ SCI B 2008;9:51–9. [136] Li Y, Zheng C, Zhao Z, Lu G, Fu X, Mei R, Li J, Wang Q. Characterization of Phomaadonidicola causing a spot blight on Adonis palaestina. Eur J Plant Pathol 2013;137:127–36. [137] Teng WL, Khor E, Tan TK, Lim LY, Tan SC. Concurrent production of chitin from shrimp shells and fungi. Carbohydr Res 2001;332:305–16. [138] Zhang L, Peng Y, Xu X, Wang S, Yu L, Hong Y, et al. Determination of other related carotenoids substances in astaxanthin crystals extracted from Adonis amurensis. J Oleo Sci 2015;64:751–9. [139] Maoka T, Etoh T, Kishimoto S, Sakata S. Carotenoids and their fatty acid esters in the petals of Adonis aestivalis. J Oleo Sci 2011;60:47–52. [140] Seybold A, Goodwin TW. Occurrence of astaxanthin in the flower petals of Adonis annua L. Nature 1959;184:1714–5. [141] Zhong Y-J, Huang J-C, Liu J, Li Y, Jiang Y, Xu Z-F, et al. Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis. J Exp Bot 2011;62:3659–69.

This page intentionally left blank

SECTION 2

Astaxanthin: Extraction, characterization, and downstream processing

This page intentionally left blank

CHAPTER 14

Industrial perspective on downstream processing of Haematococcus pluvialis Thomas O. Butlera and Ba´rbara Guimara˜esb a

Synalgae, Achterweg 65, De Kwakel, The Netherlands bPorto, Portugal

Abbreviations ADC BHA BHT BMIM CAPEX DW EMIM EPS G. settling MAP OPEX ScCO2 SFE TAG w/w

apparent digestibility coefficients butylated hydroxyanisole butylated hydroxytoluene 1-butyl-3-methylimidazolium methyl sulfate capital expenditure dry weight 1-ethyl-3-methyllimidazolium ethylsulfate extra polymeric substances gravitational settlement modified atmospheric packaging operating expenditure supercritical carbon dioxide supercritical fluid extraction triacylglycerides weight for weight

1 Downstream processing of astaxanthin-derived Haematococcus pluvialis and the challenges to overcome In 2009 91% of commercial astaxanthin (primarily synthetic) was used for animal feed pigments, and 9.1% was used for nutraceuticals [1]. But by 2016 the market share decreased to 40 % for animal feed [2]. In 2016 the astaxanthin market size was valued at US $555.4 million [3], and this year the global market for astaxanthin is expected to grow further and reach $1.5 billion; however, H. pluvialis–derived astaxanthin only represents 90%), and low energy input, leaving behind little or no toxic residues, without impairing the quality of the biomass (Table 14.1) [25]. Ideally the method employed should enable recycling of the culture medium to reduce both OPEX and total water requirement of the system for a more sustainable manufacturing process. It is anticipated that 75% of the water can be recycled providing no inhibitory molecules are found [4]. However, reusing medium has been found to result in 30% lower growth rates for H. pluvialis [26], and therefore medium recycling may actually offer a cost burden, and further investigation is required.

2.1 Batch, semicontinuous, and continuous harvesting approaches At pilot and industrial scale, it remains unconfirmed whether harvesting in batch, turbidostat (fixed cell density) or chemostat mode (fixed dilution rate) is optimal and this will depend on the strain, PBR setup, and cultivation parameters. A semicontinuous harvesting mode has been found to increase the specific growth rate and biomass productivity compared with batch production [22]. A maximum biomass productivity has been observed with a daily harvest of 20%–40% of the culture during the day (avoiding excessive dilution of the culture) [22], and semicontinuous cultivation can be maintained for several weeks to months [27] providing that biofouling and contamination are controlled. Turbidostatic operation is also a potentially viable option [22] but is difficult to implement at large scale. Future harvesting strategies should incorporate automation for harvesting, medium recycling, and culture dilution to further reduce costs.

Industrial perspective on downstream processing of H. pluvialis

287

Table 14.1: Evaluation of harvesting techniques for H. pluvialis using the criteria: harvesting efficiency, concentration factor, time efficiency, toxicity, cost, and effect on subsequent downstream processing. Criteria Harvesting efficiency

Score (1–5)

Examples (points)

Description

1–2

Electrofloculation (0)

50%–90%

1–10-fold >10–100-fold >200-fold >5 h

1–5 h 30–50 μm) and therefore can be harvested through G. settling [29,30]. In the laboratory, 95% harvesting efficiency can be attained within 40–50min [29] at a cell settling velocity of 1 cm min
1 [30]. At industrial scale, settling time is significantly longer (Fig. 14.2), attributable to industrial scale-up challenges such as flocculation tank design and higher biomass densities. G. settling has been reported to result in a concentration factor of 5.33- to 10-fold (80%–90% of the supernatant is removed), but centrifugation is required for further concentration before

Fig. 14.2 Primary downstream processing using G. settling and disk-stack centrifugation for a concentration of biomass up to 15% total solid content.

Industrial perspective on downstream processing of H. pluvialis

289

drying [4,31]. Due to the long period of G. settling, the biochemical components (including astaxanthin) can change, affecting the product quality, especially in tropical locations with high light, temperature, and humidity, and consequently this stage requires further study and optimization.

2.3 Centrifugation Subsequent to G. settling, centrifugation results in a total suspended solid content of 15% [4]. Centrifugation (conventional cream separators, disk stack, bucket, and supercentrifuges) is currently the gold standard resulting in >95% harvesting efficiency at 13,000 g [4,31]. However, centrifuges are energy intensive, and a Westfalia self-cleaning disk-stack centrifuge (Fig. 14.3A) can consume 1kWh/m3 of energy [24], translating to a consumption of 23.8–63.5 MWh annually [4], accounting for 20%–25% of the cultivation costs [13]. The Evodos spiral plate (Fig. 14.3A) and GEA Westfalia disk stack (Fig. 14.3B) are examples used in large-scale facilities producing up to 25% solids with a processing capacity of 600–800 lph with self-cleaning functions inbuilt, but residual paste still adheres to the plates and bowl requiring cleaning steps (1–2 h). Restrictions regarding opening the systems can result in some losses as high as 50%, or alternatively the centrifuges can be opened and dismantled manually to recover the biomass leading to extra operation time and cleaning steps (Fig. 14.3).

Fig. 14.3 Examples of centrifuges employed in the astaxanthin industry: (A) Evodos spiral plate and (B) GEA Westfalia disk stack. Courtesy of Synalgae, the Netherlands.

290 Chapter 14

2.4 Flocculation A two-step process has been proposed replacing G. settling with flocculation (forming a 2% dry matter slurry with up to a 40-fold concentration factor), followed by centrifugation (20% dry matter with a 10-fold further concentration) [22,32]. Alternatively, centrifugation could be bypassed altogether, with flocculation resulting in an energy reduction to 1.34 MJ kg
1 compared with centrifugation (13.8 MJ kg
1) [33]. Flocculation can be subdivided into three categories (Fig. 14.4): autoflocculation, chemical based, and bioflocculation [25]. The harvesting efficiency is influenced by cell size, cell surface charge, pH, temperature, the ionic composition of the medium, the strain, and the quantity of algogenic organic matter (AOM) [22]. 2.4.1 Chemical-based flocculation Currently, aluminum sulfate and iron chloride are widely used in industrial wastewater treatment and to harvest microalgae, but high doses are required (up to 250 and 62 mg L
1, respectively), with aluminum being known to accumulate in the biomass (>1mgg
1 on a DW basis) requiring removal through further downstream processing for incorporation in food and feed [34–36]. The limit of exposure for aluminum in humans is around 0.01 mg g
1 of body

Fig. 14.4 Flocculation can be subdivided into three categories: autoflocculation (also referred to as gravitational settling), chemical based, and bioflocculation.

Industrial perspective on downstream processing of H. pluvialis

291

weight, and higher levels can result in iron homeostasis in patients with Alzheimer’s disease [36]. Alkaline flocculation requires chemical inputs of sodium or calcium hydroxide, which creates a pH shift, inducing flocculation attributable to water hardness (increase in electrical conductivity) with coprecipitation of cells observed with Ca2+, Mg2+, carbonates, and phosphates [22]. At pH 10.5 (through the addition of sodium hydroxide), flocculation was induced in H. pluvialis within 30 min, but the harvesting efficiency and concentration factor were not reported [37]. The issue with alkaline flocculation is that it results in the formation of a white precipitate of magnesium and calcium in the biomass, which increases the mineral content above the regulatory limit of 10% [38]. A deflocculation step is required to remove the precipitated magnesium and calcium, which also adds cost to the process ($40ton
1) [38]. From an environmental and cost perspective, it is essential to transition away from conventional chemical flocculants to economical, nontoxic biobased flocculants. 2.4.2 Bioflocculation Bioflocculation is a biobased, nontoxic alternative to the methods which have been evaluated in Table 14.1 but remain to be tested in H. pluvialis. Bioflocculation includes flocculation by microorganisms and nature-derived biobased chemicals (Fig. 14.4). Bioflocculation using microorganisms includes bacteria, fungi, or yeast, which secrete biopolymers such as EPS or γ-glutamic acids aiding in floc formation [39]. Bioflocculation using nature-derived biochemicals for harvesting microalgae includes chitosan from crab shells [40], eggshells [41], Moringa oleifera seeds [42], and cationic starch [43]. Pectin from bananas could also present a viable alternative and has been found to flocculate kaolin [44] but remains to be tested in microalgae. Commercial biobased flocculants are available such as Tanfloc (Tanac, Brazil) a quaternary ammonium cationic polymer, derived from tannic acid and extracted from the bark of the invasive black wattle tree (Acacia mearnsii) [45]. Butler et al. [25] recently reported > 85% harvesting efficiency for Phaeodactylum tricornutum using Tanfloc 8025 (SL range) in only 10 min (pH 7.5–10.0 and 15–28°C) requiring only 5 mg L
1 (10.4 kg ton
1 biomass), resulting in a biomass concentration factor of 5.69 at low cost ($27.04 ton
1 microalgal biomass). Tannic acid derivatives appear to be the most commercially viable biobased flocculants, resulting in a harvesting efficiency of >90% with only 1.66-mgL
1 flocculant required for Chlorella vulgaris and Nannochloropsis oculata [45]. Guti
errez et al. [46] observed flocculation in only 15 min with 10 mg L
1of flocculant per liter of mixed microalgae consortia, but the concentration factor was not reported. Future research should investigate the effect of tannic acid derivatives for harvesting H. pluvialis and it is essential to confirm that the biomass and astaxanthin are not contaminated by the flocculant during the process and the medium can be reused without adverse effect on growth or product yield.

292 Chapter 14

2.5 Future harvesting trends Novel methods have been proposed for harvesting microalgae but have not been tested on H. pluvialis such as the use of hydrocyclones [47]; nanoparticles, for example, magnetite (Fe2O3) [32]; ultrasonic irradiation [48]; flotation [49]; and tangential flow membrane filtration (cross flow filtration) [50]. Tangential flow filtration has been reported to have a 70%–89% harvesting efficiency and a concentration factor up to 75 (15% solid content) but is constrained by a high energy consumption (0.3–2.15kWhm
3) [50]. Alternatively, utilizing attached cultivation where cells are grown on a membrane rather than in suspension culture has been proposed, and the cells are already dewatered [51,52], but this has been limited to lab-scale studies. Nevertheless, whichever harvesting method is selected, postharvest, cells must be disrupted and the astaxanthin extracted from the cells.

3 Cell disruption and extraction 3.1 The H. pluvialis aplanospore (hematocysts) challenge H. pluvialis aplanospores have a cell wall, which is three layers thick: an algaenan trilaminar sheath and secondary and tertiary cell walls (composed of mannose and cellulose) [53]. The algaenan sheath renders the cells particularly resistant to chemical [54] and mechanical (ultrasound, grinding, and freeze-thaw) [55]. There is a requirement for environmentally friendly methods of cell disruption and extraction since traditional solvent extraction techniques require large quantities of organic solvents, are labor intensive, and the labile pigments can be exposed to excessive light, heat, and oxygen [56]. New nontoxic methods should be inexpensive and utilize a green chemistry approach with generally recognized as safe (GRAS) solvents such as ethanol and acetone [57]. From an economic perspective the temperature needs to be maintained below 70°C [58].

3.2 A dry or wet process Typically the H. pluvialis wet paste is cell disrupted and then dried, but cell disruption and extraction can be conducted using a dry or a wet method. When using a wet process, the harvested biomass (5%–25% solids) must be processed rapidly (within a few hours) to avoid spoilage, especially in warm climates [24,59]. If a dry method is employed, it is also imperative that drying is initiated within a few hours postcell disruption to avoid spoilage of the biomass [4].

Industrial perspective on downstream processing of H. pluvialis

293

3.3 Cell disruption: Bead milling, the industry standard For cell disruption, high-pressure homogenization (1000–1200bar) has been suggested where cells are ejected through a tiny aperture at high speed and collisions with solid sharp objects results in shearing, crushing, and cavitation [55], but this method is constrained by high CAPEX, and often, several passes are required for complete extraction. In the past Mera Pharmaceuticals used a homogenizer at >700 bar resulting in the rupturing of >90% of the cells [30,60]. To date, bead milling has been suggested as the most effective and energy-efficient method for wet cell disruption (up to 100% lysis) with a feed of 100–200gL
1 DW equivalent described as optimal [4,61]. The sizes of the beads can range from 1 to 5mm, and the operational parameters (bead filling, bead diameter, bead density, speed of agitation, feed rate, stress number, and stress intensity) can affect the efficacy of the process [21]. It has been determined that the optimal bead diameter is 0.5 mm with a 60% bead loading volume fraction [5]. However, the two major constraints of bead milling for cell disruption are considerable heat generation, which could lead to astaxanthin degradation, in conjunction with bead fragmentation, contaminating the biomass and requiring a subsequent removal step.

3.4 Drying and cell disruption To obtain dried microalgal biomass, freeze drying, drum drying, and spray-drying have conventionally been employed using intact cells [29], but other drying methods have been explored such as solar, vacuum, and cross flow air drying [62]. The drying method used will depend on the operational scale and the use of the biomass [4]. Drum drying and spray-drying use heat, whereas freeze drying uses low temperature and pressure, which can minimize product degradation. Freeze drying is currently constrained by high CAPEX and OPEX and is typically only used for small operations with a high value product as a target [31]. Drum drying typically requires 52 kWh, but the energy consumption could be reduced if the electrically heated drying can be replaced by steam (lowering the processing cost by approximately sevenfold) in combination with utilizing a waste steam such as flue gas [63]. Drum drying has the dual advantage of partially breaking the cell walls and sterilizing the samples [63]. Spray-drying involves liquid atomization (breakup of the liquid stream into droplets), gas/ droplet mixing, and drying of liquid droplets in a hot chamber, resulting in evaporation of water resulting in a powder. The atomized droplets are sprayed in a downward movement, simultaneously with the gas phase, into a vertical chamber, with drying achieved within seconds [31]. A cyclone then separates the gas phase from the solid particles ready for storage (away from direct light, moisture, and heat) [31]. The spray-drying efficiency can be affected by the flow rate of air entering the dryer, the evaporative rate of water, and the

294 Chapter 14 residence time of the biomass [21]. Overall, spray-drying is considered the most appropriate drying method for H. pluvialis–derived astaxanthin taking into account cost and recovery rate (>95%–98%), resulting in a 5% moisture content [4,64]. Utilizing an inlet and outlet temperature of 220°C and 110°C, respectively, with storage at
21°C under nitrogen resulted in 98% during 15 days of storage at room temperature [81]. Interestingly, unstable droplet formation was observed with Zanthin® compared with AstaReal®, and this could have been due to rosemary oil being partitioned in water [81].

300 Chapter 14 4.1.5 Phase inversion temperature The phase inversion temperature method is modeled on the hydration properties of nonionic surfactant head groups when the temperature is changed, and at low temperatures the head group is hydrated, but as the temperature increases, the head group becomes progressively more dehydrated [92]. At intermediate temperatures the system exists as a bicontinuous microemulsion, and if it is rapidly cooled with stirring nanoemulsions with fine oil, droplets can be formed [92]. Few studies exist on using phase inversion temperature for astaxanthin encapsulation, but it has been reported that the thick interfacial layer formed around the oil droplets prevents oxidation, while the low water fraction results in a longer shelf life and improved gastrointestinal stability [81]. Using a concentrated emulsion termed a biliquid foam, the droplet size can be controlled by altering the emulsifier-to-oil ratio and by varying the dispersed phase concentration from 10% to 50% (droplet size 100–800 nm) with no droplet coalescence observed [10]. 4.1.6 Liposomes and nanoparticles Liposomes are spherical-shaped vesicles composed of a phospholipid bilayer structure, and astaxanthin can be encapsulated in liposomes as a drug delivery vehicle [10]. Astaxanthin has been encapsulated in liposomes with different vitamin E derivatives, and the antioxidant activity has been found to increase significantly [93]. Droplet sizes of 80nm have been observed with a retention rate of 82% after 15 days’ storage at 4°C, and this technology shows promise for effective delivery in the future [94]. Nanoparticle-encapsulated astaxanthin has droplet sizes between 100 and 300nm, and typically a solvent displacement method or a emulsion evaporation method is utilized [10]. A chitosan matrix using the solvent evaporation technique with glutaraldehyde as the cross-linking agent was resulted in a chemically stable formulation, and no significant degradation of astaxanthin was observed after 8-week storage at 25–45°C [95]. 4.1.7 Drying microencapsulation Different coating materials can be used to microencapsulate astaxanthin, including whey protein isolates, chitosan, β-cyclodextrin, soluble corn fiber, and sodium caseinate [10,96]. Freeze drying and spray-drying have both been used for microencapsulation. Shen and Quek [97] coated astaxanthin in a mixture of milk proteins and soluble corn fiber with spray-drying for the encapsulation process resulting in an encapsulation efficiency of around 95%. The freeze drying methodology was applied mostly in solvent displacement or evaporation formulations resulting in a maximum payload of 85% [98].

Industrial perspective on downstream processing of H. pluvialis

301

4.2 Challenges to address in encapsulation and formulation Currently, no optimal method has been defined for encapsulating astaxanthin, and most processes have only been described at laboratory or pilot scale. Constraints include high cost encapsulation processing, low encapsulation efficiency, large droplet size formation, astaxanthin loss, maintaining stability/functionality of the emulsion, and scalability. Most emulsification techniques incorporate heat in the emulsion system due to shear forces such as high-pressure homogenizers and microfluidizers which can lead to asatxanthin loss [81]. There is a necessity for low energy methods (membrane and microchannel emulsification) for encapsulation, but scalability is the limiting factor in the success of emulsification technologies [10]. The stability of the encapsulated astaxanthin can be affected by the matrix composition, emulsifier type, and the stabilizers used [10]. In the future, membrane and microchannel emulsification appear promising, but more focus is required on developing a vegan capsule, using environmentally friendly chemicals, ensuring compliance with food safety and regulatory bodies while making the process scalable.

5 Direct incorporation in feed and food After astaxanthin is extracted from the H. pluvialis cell for the nutraceutical sector, 90% of the biomass remains unexploited such as protein, lipid, carbohydrate, vitamin, and mineral fractions [99], and more recently, there has been an interest in using whole cell products for direct incorporation in animal/aquaculture feed and human food.

5.1 Aquaculture Most H. pluvialis–derived astaxanthin research has focused on aquaculture organisms and specifically salmonids for imparting pigmentation, improvements in growth rates, larval survival, reproductive performance, egg fecundity, feed conversion, and immune response [7,100]. Fish are unable to synthesize astaxanthin de novo, so it must be supplemented in the diet [101]. However, astaxanthin (synthetic and natural) was regulated in 1988 through the European Commission (EC) Directive 87/552 for use in salmonid feed as a color additive (E151j) at a maximum concentration of 100ppmkg
1 combined with canthaxanthin [83]. Since 2015 the European Union (EU) regulation 2015/1415 has limited synthetic petrochemically derived astaxanthin to 80

n.d.

n.d.

34

36.4

51.03

Nobre et al. [65]

Machmudah et al. [66]

Krichnavaruk et al. [83]

rate), 10% (v/v) olive oil No 500 bar, 70°C, 240 min, 3 mL/ min (CO2 flow rate) No 310 bar, 50°C, 160min, 6L/min (CO2 flow rate),9.23 mL ethanol/ g biomass (1% v/ v, ethanol as modifier) No 435 bar, 65°C, 10 L/h (CO2 flow rate), 60 min using 2.3mL ethanol/g biomass (5.52% v/v as modifier) + 150 min using 2 L/h ethanol Pretreated 200 bar, 55°C, biomass 120min, 0.06g/ supplied for the min (CO2 flow study rate), 13% (w/ w) ethanol. Ball mill 550 bar, 65°C, 80 min, 3.62 g/ min (CO2 flow rate) 550 bar, 65°C, 80 min, 3.62 g/ min (CO2 flow rate), 1 mL/min (ethanol flow rate)

10

0.5

22.8

n.d.

83.03

Thana et al. [79]

300

6.5

n.d.

n.d.

71

Pan et al. [81]

1000

240

19.75

n.d.

87.42

Wang et al. [82]

20

0.5

15.11

5.34

82.29

Reyes et al. [10]

30

1.4

18.6

10.0

93

Molino et al. [67]

18.48

6.59

92.4

n.d.: not reported. a Astaxanthin extraction yield (mg/g): mg extracted astaxanthin per g of dry biomass. b Astaxathin content (% w/w): extracted astaxanthin mass per extract mass. c Astaxanthin recovery (% w/w): extracted astaxanthin mass per initial astaxanthin mass.

364 Chapter 17 Table 17.3: Summary of studies on dried Haematococcus pluvialis in red phase using SC-CO2, gas-expanded liquids (GXLs) and pressurized liquids (PL) for antioxidant activity. Extraction conditions SC-CO2: 500bar, 70°C, 240min, 3 mL/min (CO2 flow rate) SC-CO2: 200 bar, 55°C, 120 min, 0.06 g/min (CO2 flow rate), 13% (w/w) ethanol. PLE: 103.4 bar, 100°C, 20 min, 100% hexane PLE: 103.4 bar, 100°C, 20 min, 100% ethanol GXL: 70 bar, 45°C, 120 min, 50% (w/w) ethanol

Antioxidant activity

Reference

IC50 ¼ 1.84 mg/L (ABTS method)

Thana et al. [79]

0.243 mM TE/g extract (TEAC assay)

Reyes et al. [10]

0.196 mM TE/g extract (TEAC assay) 0.265 mM TE/g extract (TEAC assay) 0.233 mM TE/g extract (TEAC assay)

Jaime et al. [51] Jaime et al. [51] Reyes et al. [10]

(80%–87%) from higher dosages (7–240g) of dried H. pluvialis without pretreatment. When the ethanol percentage is less than 5% (v/v), the extraction process is under moderate pressure (310 bar) and temperature (50°C) conditions, and a lower astaxanthin extraction efficiency (71%) was observed [81]. This value in fact was considerably lower than those (22.8 mg/g cell and 83.03%) for SC-CO2 extraction without cosolvent [79], even though the biomass dosage in the former case (6.5 g) was higher than in the latter (0.5 g). Improved astaxanthin recoveries of 92%–97%, specifically by breakage of H. pluvialis cells via mechanical pretreatment such as milling and grinding prior to ethanol-assisted SC-CO2 extraction, also have been reported [65, 67, 78]. Table 17.4 summarizes studies using GXLs and pressurized liquids (PL) for the extraction of astaxanthin from H. pluvialis. Usually, PLE turns out a faster and easier to use extraction technique with similar astaxanthin recovery than SC-CO2 (see Table 17.2). Nevertheless, the extract obtained from pure SC-CO2 contains fewer polar impurities than PLE [51]. Denery et al. [84] homogenized H. pluvialis samples and extracted astaxanthin by both acetone- and ethanol-pressurized extraction solvent technique experimental findings resulted in total astaxanthin extraction yields of 9.5mg/g dry biomass and 8.4mg/g dry biomass, respectively. Jaime et al. [51] applied mechanical disruption at freezing temperatures followed by ethanol pressurized extraction solvent (extraction conditions: 103.4bar, 100°C, 20min); experimental findings resulted in a total astaxanthin extraction yield of 20.7 mg/g dry biomass. The astaxanthin recovery is not mentioned by these authors, but if it is considered a maximum content of 3% of astaxanthin on dry biomass [39], Jaime et al. [51] would have obtained an astaxanthin recovery of 69%, similar to Denery et al. [84] and 117% in case of using hexane. Molino et al. [11] showed that by using temperature and pressure without pretreatments, very low astaxanthin recovery was achieved, since a total amount of astaxanthin close to 0.03mg/g

Table 17.4: Summary of studies on dried Haematococcus pluvialis in red phase using gas-expanded liquids (GXLs) and pressurized liquids (PL) for the extraction of astaxanthin. Extraction conditions 103.4bar, 40°C, 10 min, 100% acetone 103.4bar, 40°C, 10 min, 100% ethanol 103.4 bar, 100°C, 20 min, 100% hexane 103.4 bar, 100°C, 20 min, 100% ethanol 70 bar, 45°C, 120 min, 50% (w/w) ethanol 100 bar, 100°C, 20 min, 100% ethanol 50 bar, 100°C, 20 min, 100% hexane 100 bar, 50°C, 20 min, 100% acetone 100 bar, 40°C, 20 min, 100% acetone 100 bar, 67°C, 20 min, 100% ethanol

Pretreatment Homogenized

Haematococcus Volume of extraction vessel pluvialis loading (g) (mL) 11

Astaxanthina extraction yield (mg/g)

Astaxanthinb content (% w/w)

Astaxanthinc recovery (% w/w)

9.5

n.d.

70.4

1

8.4

Mechanical disruption at freezing temperatures

11

Pretreated biomass supply by the study

20

No

11

2

59.5

20.7

14.5

0.5

22.81

2

0.025

n.d.: not reported. a Astaxanthin extraction yield (mg/g): mg extracted astaxanthin per g of dry biomass. b Astaxathin content (% w/w): extracted astaxanthin mass per extract mass. c Astaxanthin recovery (% w/w): extracted astaxanthin mass per initial astaxanthin mass.

Denery et al. [84]

62.2

35.1

Ball mil

Reference

n.d.

Jaime et al. [51]

6.25

124.16

Reyes et al. [10]

n.d.

0.13

Molino et al. [11]

0.006

0.03

0.004

0.02

17.3

86.4

13.5

67.28

366 Chapter 17 dry biomass corresponds to a recovery close to 0.15% with respect to the initial astaxanthin in dry biomass. Therefore these authors [11] considered that the proposed mechanical pretreatment promoted the recovery of astaxanthin, which increased by three orders of magnitude for both GRAS solvents (acetone and ethanol). Besides the experimental protocols of astaxanthin extraction at 100bar by using ethanol as solvent revealed good extraction time of 20min (slightly higher than Denery et al. [84] at 40°C and 10 min) through ethanol at 67°C; it was possible to recover around 78% of the total astaxanthin extracted by acetone. Despite acetone outcomes more effective than ethanol, it is worth pointing out that without the application of mechanical pretreatment, ethanol resulted more effective than acetone. This could be due to higher extractive capacity of acetones with ester forms of astaxanthin with respect to ethanol that has greater extraction capacity with free forms of astaxanthin [11]. Reyes et al. [10] (see Table 17.4) explored a new region of pressurized liquids in which ethanol is the primarily solvent and CO2 serves as an aid in the extraction. They selected a pressure lower than the critical pressure of CO2 (73.8bar). This so-called GXL region offers several advantages compared with SC-CO2 and pressurized ethanol: it allows working at low pressures (compared with SC-CO2) and using less amount of organic solvent (compared with PLE [51]). Reyes et al. [10] and Jaime et al. [51] were capable of obtaining similar astaxanthin extraction yield. But with GXL the purity of the extract obtained is higher than PLE, and 50% of organic solvent was used. The TEAC (Trolox equivalent antioxidant capacity) assay was used by Jaime et al. [51] and Reyes et al. [10] to measure the antioxidant activity of the PLE and GXL extracts obtained at the better extraction conditions from H. pluvialis, respectively (see Table 17.3). Both authors determined that at the highest astaxanthin extraction yields achieved the antioxidant activity was the highest too. Besides, Jaime et al. [51] demonstrated that ethanol extract presented better antioxidant activity than hexane extract. In addition, they confirmed that the behavior of the antioxidant activity is a function of the extraction temperature since the higher extraction temperature, the lower the TEAC value of the extracts and therefore the lower the antioxidant activity. On the other hand, Reyes et al. [10] concluded that extracts obtained by SC-CO2 and GXL had similar antioxidant activity. Solvents used in these studies (acetone and ethanol) are listed among the so-called GRAS solvents, since toxicological and medical studies show no adverse effects on human health over their use in food over a long period [26]. Besides, European Directive 2009/32/CE [85] adds these solvents in the list of “the solvents usable in the food preparation.” Jaime et al. [51] and Molino et al. [11] used hexane as reference because it is often used during the industrial extraction processes.

5 Current global market and market players of H. pluvialis astaxanthin Synthetic astaxanthin dominates current commercial market, of the total value exceeding $200 million, corresponding to 130 metric tons of product per year [45]. In 2014 microalgae-derived

High-pressure extraction of astaxanthin from Haematococcus pluvialis

367

astaxanthin corresponds to less than 1% of the commercialized quantity due to much lower price of synthetic astaxanthin and technological problems associated with large-scale algae cultivation [46, 49]. In recent years, there has been a growing trend toward using natural ingredients in food, nutraceutical, and cosmetic markets, resulting from increasing concerns for consumer safety and regulatory issues over the introduction of synthetic chemicals into the human food chain. The demand for natural astaxanthin derived from H. pluvialis in the global market has been on the rise in recent years owing to increasing consumer awareness of its health benefits [36]. Global market for both synthetic and natural source astaxanthin in aquaculture feed, nutraceuticals, cosmetics, and food and beverages is estimated at 280 metric tons valued at $447 million in 2014. It is further projected to reach 670 metric tons valued at $1.1 billion by 2020 [86, 87]. The estimated market value of astaxanthin depending on products’ purity varies from $2500–7000/kg to about $15,000/kg pigment from H. pluvialis in some cases [48–50, 86], while the production cost is estimated at about $1000 per kg of astaxanthin for specific commercial applications (e.g., the nutraceutical market) [45]. To replace the synthetic astaxanthin, mass cultivation of H. pluvialis in industrial scale has great potential and attractive business opportunity. However, current market demand for natural astaxanthin is not met. It is expected that in the foreseeable future after the optimization of the production technology, the production costs of the natural astaxanthin from H. pluvialis should be more competitive to these of the synthetic alternative [50]. Since the mid-1990s, several leading companies are successfully producing H. pluvialis at commercial scale and marketing natural astaxanthin from H. pluvialis worldwide. Table 17.5 summarizes companies specialized in astaxanthin production conduct the SC-CO2 extraction as a part of the whole process. In addition, as demand for CO2-extracted astaxanthin is steadily increasing, also other H. pluvialis producers submit dry algae for SC-CO2 extraction, though the high-pressure facility remains expensive compared with an equipment for conventional separation methods [88]. The size of the nutraceutical astaxanthin market is growing day by day, and this market is very attractive to Haematococcus astaxanthin producers since the price of these products is significantly higher than those of feed applications. Haematococcus producers need to invest their attention for increasing astaxanthin production capacity to meet the global demand.

6 Conclusion and perspectives Supercritical carbon dioxide (SC-CO2) has attracted attention as a green solvent for the extraction of a variety of bioactive compounds [89, 90]. Supercritical CO2 features three major advantages over organic solvents: (1) It is abundant and benign to human health and the environment, (2) CO2 solvent can be removed easily by evaporation at room temperature and pressure, and (3) bioactive compounds are well preserved due to the inertness of CO2 [91, 92]. The overall extraction efficiency with SC-CO2, PLE, and GXL depends greatly on the preparation of the feedstock and the addition of any modifiers/cosolvents or the use of an unique organic solvent in case of PLE, while operating conditions (temperature, pressure, and

368 Chapter 17 Table 17.5: Leading commercial companies that produce SC-CO2-extracted astaxanthin. Company name

Country

Brand name

Product particulars

Cyanotech Corporation (www.cyanotech.com)

USA

BioAstin

Valensa International (www.valensa.com) Algatechnologies Ltd. (www.algatech.com)

USA

Zanthin

Israel

AstaPure

Atacama Bio Natural (www. atacamabionatural.com)

Chile

Supreme Asta Oil Supreme Asta powder

Fujii Chemical Industry Co. Ltd. (www. fujichemical.co.jp)

Japan, Sweden, USA

AstaREAL

AstaMAZ NZ Ltd. (www. astamaz.com)

New Zealand

Astaxanthin extract packaged in soft gel, beadlets; dietary supplement Astaxanthin extract, soft, gel, beadlets Dry algal biomass, astaxanthin beadlets, and oleoresin Oleoresin for food, nutraceutic, and cosmetic products, and powder for animal feed supplement Astaxanthin oleoresin products, water dispersible, and soluble powders Pure algae oil

Natural Astaxanthin Oil 5%, 10%, 20% Natural Astaxanthin CWS Natural astaxanthin cold water-dispersible powder 2.5%

duration) are parameters that could also be adjusted. With regard to commercial feasibility, temperatures for astaxanthin extraction are limited to a relatively low 70°C [81, 83]. Due to the rigid cell wall, direct extraction of wet microalgae biomass using SC-CO2 is generally not feasible. A common strategy is then to dehydrate and mechanically disrupt the feedstock prior to CO2 extraction, easing the downstream extraction process. However, reported astaxanthin recovery rates vary widely due to the range of disruption tools used and the ambiguity in quantifying the degree of cell wall disruption. For example, some researchers found the overall recovery rates were below 50% after cell wall disruption, even when the operating pressure was as high as 300 bar [65, 78]. Furthermore, dehydration and mechanical cell disruption add considerable time, energy, and cost to the process. Intracellular water and disrupted cell debris can also present challenges such as caking, further reducing extraction rates and/or yield over time [93]. Another strategy to aid the extraction process, as it was mentioned earlier, is to employ cosolvents [10, 83] such as ethanol and olive oil in SC-CO2. This strategy allowed for a higher recovery rate of 71% high extraction pressures (310 bar) and 160 min of extraction time [81]. The combination of mechanical cell disruption and cosolvent extraction has also been tested [10, 65, 67, 78] indicating the potential to greatly speed up extraction and enhance recovery using ethanol cosolvent.

High-pressure extraction of astaxanthin from Haematococcus pluvialis

369

Some authors as Cheng et al. [94] have proposed to minimize dry, disruption cellular and extraction steps using wet biomass to obtain high astaxanthin recoveries. They demonstrated a hydrothermal method of astaxanthin extraction from wet biomass using a high-temperature and high-pressure microfluidic platform. The hydrothermal process was performed at a pressure of 80 bar, chosen to be sufficiently high to maintain water at liquid phase at 200°C. This temperature was chosen to correspond to the approximate temperature where structural cell wall polysaccharides rapidly depolymerize without degradation of other biomass as a result of secondary reactions [95, 96]. H. pluvialis cysts were trapped within the device and visualized in situ during the cell wall disruption and astaxanthin extraction processes. The device provided a highly controlled environment and enables direct comparison of chemical versus hydrothermal processes at the cellular level. This process allowed a near-complete astaxanthin extraction recovery (98.3%) using a 4:1 CO2/ethanol flow ratio (100 and 25μL/min) at 80bar, 55°C, and 15 min of extraction time. This high recovery was attributed to two factors. First, water is miscible with ethanol phase, removing the two-phase interfacial barrier between the solvent and the water-wet surface of the cell. Secondly the solubility of astaxanthin in ethanol is five orders of magnitude higher than in SC-CO2 [97]. Here the improved performance is attribute to within the reactor, namely, that all cells had access to cosolvents (in addition to the high degree of prior cell disruption noted earlier). These results point to the possibility of extraction with significantly lower pressures. In terms of informing current and future operations, the results show that more complete cell wall disruption prior to extraction and improved transport during extraction can provide several opportunities for savings. Current operations could benefit by operating at lower pressures with existing infrastructure, the benefit being reduced pumping costs. Furthermore, future infrastructure costs would lower due to the reduced pressure requirements. These savings are likely to be very significant as cap-ex generally scales directly with pressure requirements, among other variables.

Acknowledgments I thank Casimiro Mantell, Prof. Dr. for valuable suggestions and critical reading of this manuscript. I thank Enrique Martı´nez de la Ossa, Prof. Dr. and the other members of his research group titled “Analysis and design of processes with supercritical fluids” (TEP-128) from University of Ca´diz (Spain) for their continuing support.

References [1] Rao AR, Sindhuja HN, Dharmesh SM, Sankar KU, Sarada R, Ravishankar GA. Effective inhibition of skin cancer, tyrosinase, and antioxidative properties by astaxanthin and astaxanthin esters from the green alga Haematococcus pluvialis. J Agric Food Chem 2013;61(16):3842–51. [2] Ambati R, Phang SM, Ravi S, Aswathanarayana R. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications—a review. Mar Drugs 2014;12(1):128–52. [3] Ambati RR, Gogisetty D, Aswathanarayana RG, Ravi S, Bikkina PN, Bo L, Yuepeng S. Industrial potential of carotenoid pigments from microalgae: current trends and future prospects. Crit Rev Food Sci Nutr 2019; 59(12):1880–902.

370 Chapter 17 [4] Ruen-ngam D, Shotipruk A, Pavasant P. Comparison of extraction methods for recovery of astaxanthin from Haematococcus pluvialis. Sep Sci Technol 2010;46:64–70. [5] Gil-Cha´vez GJ, Villa JA, Ayala-Zavala JF, Heredia JB, Sepulveda D, Yahia EM, Gonza´lez-Aguilar GA. Technologies for extraction and production of bioactive compounds to be used as nutraceuticals and food ingredients: an overview. Compr Rev Food Sci Food Saf 2013;12:5–23. [6] Poojary MM, Barba JF, Aliakbarian B, Donsı` F, Pataro G, Dias AD, Juliano P. Innovative alternative technologies to extract carotenoids from microalgae and seaweeds. Mar Drugs 2016;14:214. [7] Ameer K, Shahbaz HM, Kwon J-H. Green extraction methods for polyphenols from plant matrices and their byproducts: a review. Compr Rev Food Sci Food Saf 2017;16:295–315. [8] Mendes RL, Nobre BP, Cardoso MT, Pereira AP, Palavra AF. Supercritical carbon dioxide extraction of compounds with pharmaceutical importance from microalgae. Inorg Chim Acta 2003;356:328–34. [9] Crampon C, Boutin O, Badens E. Supercritical carbon dioxide extraction of molecules of interest from microalgae and seaweeds. Ind Eng Chem Res 2011;50:8941–53. [10] Reyes FA, Mendiola JA, Iban˜ez E, del Valle JM. Astaxanthin extraction from Haematococcus pluvialis using CO2-expanded ethanol. J Supercrit Fluids 2014;92:75–83. [11] Molino A, Rimauro J, Casella P, Cerbone A, Larocca V, Chianese S, Karatza D, Mehariya S, Ferraro A, Hristoforou E, Musmarra D. Extraction of astaxanthin from microalga Haematococcus pluvialis in red phase by using generally recognized as safe solvents and accelerated extraction. J Biotechnol 2018;283:51–61. [12] Temelli F. Perspectives on supercritical fluid processing of fats and oils. J Supercrit Fluids 2009;47:583–90. ´ , Cocero MJ. Carotenoid processing with supercritical fluids. J Food Eng 2009;93:255–65. [13] Mattea F, Martı´n A [14] Herrero M, Mendiola JA, Cifuentes A, Iba´n˜ez E. Supercritical fluid extraction: recent advances and applications. J Chromatogr A 2010;1217:2495–511. [15] Halim R, Danquah MK, Webley PA. Extraction of oil from microalgae for biodiesel production: a review. Biotechnol Adv 2012;30:709–32. [16] Shilpi A, Shivhare US, Basu S. Supercritical CO2 extraction of compounds with antioxidant activity from fruits and vegetables waste—a review. Focus Mod Food Ind 2013;2:43–62. [17] Brennecke JF, Eckert CA. Phase equilibria for supercritical fluid process design. AIChE J 1989;35:1409–27. [18] Randolph TW. Supercritical fluid extractions in biotechnology. Trends Biotechnol 1990;8:78–82. [19] Rozzi NL, Singh RK. Supercritical fluids and the food industry. Compr Rev Food Sci Food Saf 2002;1:33–44. [20] Han X, Poliakoff M. Continuous reactions in supercritical carbon dioxide: problems, solutions and possible ways forward. Chem Soc Rev 2012;41:1428. [21] Francis AW. Ternary systems of liquid carbon dioxide. J Phys Chem 1954;58:1099–114. [22] Reverchon E, De Marco I. Supercritical fluid extraction and fractionation of natural matter. J Supercrit Fluids 2006;38:146–66. [23] Zancan KC, Marques MOM, Petenate AJ, Meireles MAA. Extraction of ginger (Zingiber officinale roscoe) oleoresin with CO2 and CO-solvents: a study of the antioxidant action of the extracts. J Supercrit Fluids 2001;24:57–76. [24] Mendiola JA, Herrero M, Cifuentes A, Iban˜ez E. Use of compressed fluids for sample preparation: food applications. J Chromatogr A 2007;1152:234–46. [25] Kitzberger CSG, Sm^ania A, Pedrosa RC, Ferreira SRS. Antioxidant and antimicrobial activities of shiitake (Lentinula edodes) extracts obtained by organic solvents and supercritical fluids. J Food Eng 2007;80:631–8. [26] Food and Drug Administration. Substances generally recognized as safe; final rule. Fed Regist 2016;81:54959–5055. [27] Akien GR, Poliakoff M. A critical look at reactions in class I and II gas-expanded liquids using CO2 and other gases. Green Chem 2009;11:1083. [28] Sih R, Armenti M, Mammucari R, Dehghani F, Foster NR. Viscosity measurements on saturated gas-expanded liquid systems—ethanol and carbon dioxide. J Supercrit Fluids 2008;43:460–8. [29] Eckert C, Liotta C, Ragauskas A, Hallett J. Tunable solvents for fine chemicals from the biorefinery. Green Chem 2007;9:545–8. [30] Herrero M, Castro-Puyana M, Mendiola JA. Compressed fluids for the extraction of bioactive compounds. Trends Anal Chem 2013;43:67–83.

High-pressure extraction of astaxanthin from Haematococcus pluvialis

371

[31] Herrero M, Sa´nchez-Camargo AP, Cifuentes A, Iba´n˜ez E. Plants, seaweeds, microalgae and food by-products as natural sources of functional ingredients obtained using pressurized liquid extraction and supercritical fluid extraction. Trends Anal Chem 2015;71:26–38. [32] Hu J, Guo Z, Glasius M, Kristensen K, Xiao L, Xu X. Pressurized liquid extraction of ginger (Zingiber officinalis Roscoe) with bio-ethanol: an efficient and sustainable approach. J Chromatogr 2011;1218:5765–73. [33] M€aki-Arvela P, Hachemi I, Murzin DY. Comparative study of the extraction methods for recovery of carotenoids from algae: extraction kinetics and effect of different extraction parameters. J Chem Technol Biotechnol 2014;89:1607–26. [34] Yen HW, Yang SC, Chen CH, Chang JS. Supercritical fluid extraction of valuable compounds from microalgal biomass. Bioresour Technol 2015;184:291–6. [35] Catchpole O, Moreno T, Montan˜es F, Tallon S. Perspectives on processing of high value lipids using supercritical fluids. J Supercrit Fluids 2018;134:260–8. [36] Shah MMR, Liang Y, Cheng JJ, Daroch M. Astaxanthin-producing green microalga Haematococcus pluvialis: from single cell to high value commercial products. Front Plant Sci 2016;7:1–28. [37] Wayama M, Ota S, Matsuura H, Nango N, Hirata A, Kawano S. Three-dimensional ultrastructural study of oil and Astaxanthin accumulation during encystment in the green alga Haematococcus pluvialis. PLoS One 2013;8:1–9. [38] Boussiba S, Bing W, Yuan J-P, Zarka A, Chen F. Changes in pigments profile in the green alga Haeamtococcus pluvialis exposed to environmental stresses. Biotechnol Lett 1999;21:601–4. [39] Cero´n MC, Garcı´a-Malea MC, Rivas J, Acien FG, Fernandez JM, Del Rı´o E, Guerrero MG, Molina E. Antioxidant activity of Haematococcus pluvialis cells grown in continuous culture as a function of their carotenoid and fatty acid content. Appl Microbiol Biotechnol 2007;74:1112–9. [40] Sharon-Gojman R, Maimon E, Leu S, Zarka A, Boussiba S. Advanced methodsfor genetic engineering of Haematococcus pluvialis (Chlorophyceae, Volvocales). Algal Res 2015;10:8–15. [41] Hong ME, Choi HI, Kwak HS, Hwang S-W, Sung YJ, Chang WS, Sim SJ. Rapid selection of astaxanthin-hyperproducing Haematococcus mutant viaazide-based colorimetric assay combined with oil-based astaxanthin extraction. Bioresour Technol 2018;267:175–81. [42] Hagen C, Siegmund S, Braune W. Ultrastructural and chemical changes in the cell wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during aplanospore formation. Eur J Phycol 2002;37:217–26. [43] Damiani MC, Leonardi PI, Pieroni OI, Ca´ceres EJ. Ultrastructure of the cyst wall of Haematococcus pluvialis (Chlorophyceae): wall development and behaviour during cyst germination. Phycologia 2006;45:616–23. [44] Praveenkumar R, Lee K, Lee J, Oh Y-K. Breaking dormancy: an energy efficient means of recovering astaxanthin from microalgae. Green Chem 2015;17:1226–34. [45] Li J, Zhu D, Niu J, Shen S, Wang G. An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis. Biotechnol Adv 2011;29:568–74. [46] Milledge JJ. Commercial application of microalgae other than as biofuels: a brief review. Rev Environ Sci Biotechnol 2011;10:31–41. [47] Miki W. Biological functions and activities of animal carotenoids. Pure Appl Chem 1991;63:141–6. [48] Borowitzka MA. High-value products from microalgae—their development and commercialisation. J Appl Phycol 2013;25:743–56. [49] Koller M, Muhr A, Braunegg G. Microalgae as versatile cellular factories for valued products. Algal Res 2014;6:52–63. [50] Perez-Lo´pez P, Gonza´lez-Garcı´a S, Jeffryes C, Agathos SN, McHugh E, Walsh D, Murray P, Moane S, Feijoo G, Moreira MT. Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae: from lab to pilot scale. J Clean Prod 2014;64:332–44. [51] Jaime L, Rodrı´guez-Meizoso I, Cifuentes A, Santoyo S, Suarez S, Iba´n˜ez E, Sen˜orans FJ. Pressurized liquids as an alternative process to antioxidant carotenoids’ extraction from Haematococcus pluvialis microalgae. LWT-Food Sci Technol 2010;43:105–12. [52] Khoo KS, Lee SY, Ooi CW, Fu X, Miao X, Ling TC, Show PL. Recent advances in biorefinery of astaxanthin from Haematococcus pluvialis. Bioresour Technol 2019;288:1–11.

372 Chapter 17 [53] Guerin M, Huntley ME, Olaizola M. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol 2003;21:210–6. [54] Yuan J-P, Peng J, Yin K, Wang JH. Potential health-promoting effects of astaxanthin: a high-value carotenoid mostly from microalgae. Mol Nutr Food Res 2011;55:150–65. [55] Han D, Li Y, Hu Q. Astaxanthin in microalgae: pathways, functions and biotechnological implications. Algae 2013;28:131–47. [56] Han D, Li Y, Hu Q. Biology and commercial aspects of Haematococcus pluvialis. In: Handbook of microalgal culture: applied phycology and biotechnology. 2:John Wiley and Sons; 2013. p. 388–405. [57] Milledge JJ. Energy balance and techno-economic assessment of algal biofuel production systems. [Doctoral thesis] University of Southampton; 2013. [58] Panis G, Carreon JR. Commercial astaxanthin production derived by green alga Haematococcus pluvialis: a microalgae process model and a techno-economic assessment all through production line. Algal Res 2016;18:175–90. [59] Zhang W, Wang J, Wang J, Liu T. Attached cultivation of Haematococcus pluvialis for astaxanthin production. Bioresour Technol 2014;158:329–35. [60] Zou T-B, Jia Q, Li H-W, Wang C-X, Wu H-F. Response surface methodology for ultrasound-assisted extraction of astaxanthin from Haematococcus pluvialis. Mar Drugs 2013;11:1644–55. [61] Mendes-Pinto M, Raposo M, Bowen J, Young A, Morais R. Evaluation of different cell disruption processes on encysted cells of Haematococcus pluvialis: effects on astaxanthin recovery and implications for bio-availability. J Appl Phycol 2001;13:19–24. [62] Sarada R, Vidhyavathi R, Usha D, Ravishankar G. An efficient method for extraction of astaxanthin from green alga Haematococcus pluvialis. J Agric Food Chem 2006;54:7585–8. [63] Dong S, Huang Y, Zhang R, Wang S, Liu Y. Four different methods comparison for extraction of astaxanthin from green alga Haematococcus pluvialis. Sci World J 2014;2014:1–7. [64] Kang CD, Sim SJ. Direct extraction of astaxanthin from Haematococcus culture using vegetable oils. Biotechnol Lett 2008;30:441–4. [65] Nobre B, Marcelo F, Passos R, Beira˜o L, Palavra A, Gouveia L, Mendes R. Supercritical carbondioxide extraction of astaxanthin and other carotenoids from the microalga Haematococcus pluvialis. Eur Food Res Technol 2006;223:787–90. [66] Machmudah S, Shotipruk A, Goto M, Sasaki M, Hirose T. Extraction of astaxanthin from Haematococcus pluvialis using supercritical CO2 and ethanol as entrainer. Ind Eng Chem Res 2006;45:3652–7. [67] Molino A, Mehariya S, Iovine A, Larocca V, Di Sanzo G, Martino M, Casella P, Chianese S, Musmarra D. Extraction of astaxanthin and lutein from microalga Haematococcus pluvialis in the red phase using CO2 supercritical fluid extraction technology with ethanol as CO-solvent. Mar Drugs 2018;16:432. [68] Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: a review. Renew Sustain Energy Rev 2010;14:217–32. [69] Solana M, Rizza CS, Bertucco A. Exploiting microalgae as a source of essential fatty acids by supercritical fluid extraction of lipids: comparison between Scenedesmus obliquus, Chlorella protothecoides and Nannochloropsis salina. J Supercrit Fluids 2014;92:311–8. [70] Raposo MFJ, Morais AM, Morais RM. Effects of spray-drying and storage on astaxanth in content of Haematococcus pluvialis biomass. World J Microbiol Biotechnol 2012;28:1253–7. [71] Grima EM, Belarbi E-H, Ferna´ndez FA, Medina AR, Chisti Y. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol Adv 2003;20:491–515. [72] Choi S-A, Oh Y-K, Lee J, Sim SJ, Hong ME, Park J-Y, Kim M-S, Kim SW, Lee J-S. High-efficiency cell disruption and astaxanthin recovery from Haematococcus pluvialis cyst cells using room-temperature imidazolium-based ionic liquid/water mixtures. Bioresour Technol 2019;274:120–6. [73] Lee AK, Lewis DM, Ashman PJ. Disruption of microalgal cells for the extraction of lipids for biofuels: processes and specific energy requirements. Biomass Bioenergy 2012;46:89–101. [74] Subramanian B, Tchoukanova N, Djaoued Y, Pelletier C, Ferron M, Robichaud J. Investigations on the geometrical isomers of astaxanthin: Raman spectroscopy of conjugated polyenechain with electronic and mechanical confinement. J Raman Spectrosc 2014;45:299–304.

High-pressure extraction of astaxanthin from Haematococcus pluvialis

373

[75] Kaczor A, Baranska M. Structural changes of carotenoid astaxanthin in a single algal cell monitored in situ by Raman spectroscopy. Anal Chem 2011;83:7763–70. [76] Aguirre A-M, Bassi A. Investigation of high pressure steaming (HPS) as a thermal treatment for lipid extraction from Chlorella vulgaris. Bioresour Technol 2014;164:136–42. [77] Liu Z-W, Zeng X-A, Cheng J-H, Liu D-B, Aadil RM. The efficiency and comparison of novel techniques for cell wall disruption in astaxanthin extraction from Haematococcus pluvialis. Int J Food Sci Technol 2018;53:2212–9. [78] Valderrama JO, Perrut M, Majewski W. Extraction of astaxantine and phycocyanine from microalgae with supercritical carbon dioxide. J Chem Eng Data 2003;48:827–30. [79] Thana P, Machmudah S, Goto M, Sasaki M, Pavasant P, Shotipruk A. Response surface methodology to supercritical carbon dioxide extraction of astaxanthin from Haematococcus pluvialis. Bioresour Technol 2008;99:3110–5. [80] Dandge DK, Heller JP, Wilson KV. Structure solubility correlations: organic compounds and dense carbon dioxide binary systems. Ind Eng Chem Product Res Dev 1985;24:162–6. [81] Pan J-L, Wang H-M, Chen C-Y, Chang J-S. Extraction of astaxanthin from Haematococcus pluvialis by supercritical carbon dioxide fluid with ethanol modifier. Eng Life Sci 2012;12(6):638–47. [82] Wang L, Yang B, Yan B, Yao X. Supercritical fluid extraction of astaxanthin from Haematococcus pluvialis and its antioxidant potential in sunflower oil. Innov Food Sci Emerg Technol 2012;13:120–7. [83] Krichnavaruk S, Shotipruk A, Goto M, Pavasant P. Supercritical carbon dioxide extraction of astaxanthin from Haematococcus pluvialis with vegetable oils as co-solvent. Bioresour Technol 2008;99:5556–60. [84] Denery JR, Dragull K, Tang CS, Li QX. Pressurized fluid extraction of carotenoids from Haematococcus pluvialis and Dunaliella salina and kavalactones from Pipermethysticum. Anal Chim Acta 2004;501:175–81. [85] Directive 2009/32/EC of the European Parliament and of the Council. On the approximation of the laws of the Member States on extraction solvents used in the production of food stuffs and food ingredients. L141/3-L141/11. [86] Industry Experts. Global Astaxanthin market-sources, technologies and applications, Available from: http:// industry-experts.com/verticals/healthcare-and-pharma/global-astaxanthin-market-sources-technologies-andapplications; 2015. [87] Panis G. Commercial Astaxanthin production derived by green alga Haematococcus pluvialis: a microalgae process model and a techno-economic assessment all through production line [master thesis (45EC)]. Netherlands: Utrecht University; 2015. [88] Kim D-Y, Vijayan D, Praveenkumar R, Han J-I, Lee K, Park J-Y, Chang W-S, Lee J-S, Oh Y-K. Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus. Bioresour Technol 2016;199:300–10. [89] Dı´az-Reinoso B, Moure A, Domı´nguez H, Parajo´ JC. Supercritical CO2 extraction and purification of compounds with antioxidant activity. J Agric Food Chem 2006;54:2441–69. [90] Yen H-W, Yang S-C, Chen C-C, Jesisca, Chang J-S. Supercritical fluid extraction of valuable compounds from microalgal biomass. Bioresour Technol 2015;184:291–6. [91] Brunner G. Supercritical fluids: technology and application to food processing. J Food Eng 2005;67:21–33. [92] da Silva RPFF, Rocha-Santos TAP, Duarte AC. Supercritical fluid extraction of bioactive compounds. Trends Anal Chem 2016;76:40–51. [93] Reyes FA, Sielfeld CS, del Valle JM. Effect of high-pressure compaction on supercritical CO2 extraction of astaxanthin from Haematococcus pluvialis. J Food Eng 2016;189:123–34. [94] Cheng X, Qi Z, Burdyny T, Kong T, Sinton D. Low pressure supercritical CO2 extraction of astaxanthin from Haematococcus pluvialis demonstrated on a microfluidic chip. Bioresour Technol 2018;250:481–5. [95] Lorenz RT, Cysewski GR. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol 2000;18:160–7. [96] Bobleter O. Hydrothermal degradation of polymers derived from plants. Prog Polym Sci 1994;19:797–841. [97] de la Fuente JC, Oyarzu´n B, Quezada N, del Valle JM. Solubility of carotenoid pigments (lycopene and astaxanthin) in supercritical carbo´n dioxide. Fluid Phase Equilibr 2006;247:90–5.

This page intentionally left blank

CHAPTER 18

Astaxanthin extraction—Recent methods, developments and case studies Hamed Vatankhah and Hosahalli Ramasamy Department of Food Science and Agricultural Chemistry, McGill University, St-Anne-de-Bellevue, QC, Canada

Abbreviations AST HPE MAE MF SFE UAE

astaxanthin high-pressure extraction microwave-assisted extraction magnetic field-assisted extraction supercritical fluid extraction ultrasound-assisted extraction

1 Introduction Astaxanthin (AST) is extracted from different sources, the principal ones being algae (Haematococcus pluvialis [H. pluvialis]), yeast (Phaffia rhodozyma [Ph. rhodozyma]), or crustaceans (shrimp and crabs) [1]. H. pluvialis is a freshwater species of Chlorophyta from the family Haematococcaceae. This species is well known for its high content of the strong antioxidant astaxanthin (pink carotenoid), which is important in aquaculture and cosmetics. The process of extraction is involved with numerous limitations. Ph. rhodozyma is a basidiomycetous pink yeast that until recently was found exclusively in slime fluxes of certain broad-leafed trees in the northern hemisphere. The common extraction methods based on conventional solvent extraction are usually time consuming and not highly energy efficient. Thus scientists have been constantly searching for better extraction methods. Several novel extraction methods are advantageous in terms of providing a shorter time of extraction and a higher yield of recovery. In fact, using different processing approaches, the usage of solvents can be more limited, which is a positive point in terms of environmental challenges. Moreover, using most novel extraction techniques provides extraction conditions at lower temperatures. This is a very important achievement compared with the classic solvent extraction techniques where degradation of bioactive compounds—which are indeed heat susceptible—occur and result in a decrease the final obtained dosage of target materials. Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00009-X Copyright # 2021 Elsevier Inc. All rights reserved.

375

376 Chapter 18 This chapter will provide an overview on principles of existing novel extraction techniques and will present scientific data regarding extraction of AST using such approaches.

2 Solvent extraction The solvent extraction is a general method of extraction in which the target molecule is extracted based on its polarity and solubility in a liquid phase, which usually is a nonpolar solvent. Among several options available as a solvent to extract AST, methanol, isopropanol, petroleum ether hexane, and acetone are quite well known. The extraction could also be done using a mixture of selected solvents. For instant, compared with using single phase solvents, higher extraction efficiency was found when isopropyl alcohol was mixed 50%–50% with hexane. Incorporating a polar and no-polar mixture of solvents could help removing the water content using polar solvents, which facilitate extraction of nonpolar compounds such a carotenoid. In addition, the final yield of solvent extraction could be improved using multiple extractions [2]. Ruen-ngam et al. [3] compared the recovery of extraction of AST in different solvents. They found a higher recovery rate while using acetone compared with organic solvents such as methanol, ethanol, and acetonitrile. This is directly related to the high nonpolarity of acetone compared with other mentioned solvents.

3 Microwave-assisted extraction (MAE) Microwave energy could be used to improve solvent extraction (Fig. 18.1). The main advantage of using microwave is the high energy efficiency and simplicity. Microwave develops localized heated spots and intracellular pressure buildup within the sample and increases the solvent temperature that ultimately helps increasing the diffusivity and facilitates extraction of bioactive compounds from cellular structures. In this regard, extraction of AST using MAE showed that several variables such as microwave power, extraction time, and solvent type could affect the final yield. The optimal processing condition of extraction of AST from H. pluvialis was reported to be fourfold extraction at 141W power, 83s extraction time, and. 9.8mL solvent for 100mg material. In another study, approx. 75% AST was recovered form H. pluvialis using acetone at 720 W, 75°C for 5 min [3,4].

4 Ultrasound-assisted extraction (UAE) During recent years, application of ultrasound to assist solvent extraction has gained more interest [5]. The primary version of the process is based on placing the solvent containing flask inside common ultrasound baths. However, later improvements introduced the ultrasound transducer inside the solvent medium. As can be seen in Fig. 18.2, the process can be in both bath and continuous types. Compared with the common solvent extraction, UAE can reduce the

Astaxanthin extraction 377

Fig. 18.1 Schematic diagram of microwave equipment.

amount of needed solvent, extraction time, and energy. The effectiveness of ultrasound is due to its physical force imposed on the solvent and solid to develop high shear forces as a result of cavitation phenomena. By implosion of cavitation, mechanical work takes place to breakdown particles and to provide erosion in solids. Moreover, in terms of fluid dynamics, ultrasound cavitation leads to “macroturbulences and micromixing,” which is in favor of extraction improvement [6]. Vapor pressure and surface tension of solvents are important factors affecting UAE. Solvents with lower vapor pressure would intensify the mechanical effect due to the cavitation implosion. This could lead in higher extraction yield; however, it could also increase the molecular degradation of the target compound. Thus choosing the optimal solvent is essential to achieve a proper yield [3,7]. It was found that during the first 30min of extraction, amount of AST obtained during UAE did not change significantly compared with regular maceration and was at a rather low yield of approx. 40%. However, UAE for 30 min longer increased the obtained AST recovered from H. pluvialis (up to 70%). Nevertheless, compared with 60min extraction, a 90-min UAE leads to the decrease of the level of AST. The optimum condition was reported to be 60min at 45°C [3].

5 Oil stripping method In this method, different sources of edible oil alone or in combination with each other could be used to extract the nonpolar molecules of AST. The process is known to be more effective at higher temperatures. However, an optimized extraction condition regarding solvent type,

378 Chapter 18

Water out Stainless steel tank Water in

Transducers bonded to base

(A)

(B)

Transducers bonded to base

Transducer Transducer

Probe Water out

Probe

Water in

Water out

Water in

(C)

(D)

Fluid mixture

Fig. 18.2 Commonly used ultrasonic systems (A) ultrasound bath, (B) ultrasound reactor with stirring, (C) ultrasound probe, and (D) continuous sonication with ultrasound probe [6].

temperature level, and extraction time should be obtained since the AST molecule gets more fragile at higher temperature levels. Pu et al. [8] used Flaxseed oil to extract astaxanthin from shrimp waste. The extraction was done at different temperature levels between 30 and 60. The result showed that astaxanthin extraction could be improved at different time-temperature conditions up to 40 for 30min. At more intense processing condition, the final obtained dosage was affected by thermal degradation of AST as an unsaturated structure. Thus this challenge could be the important limitation associated with oil stripping extraction method of AST [8,9].

Astaxanthin extraction 379

6 Ultrafiltration AST can be separated using physical separation techniques such as ultrafiltration. The preparation step involves reaching a highly concentrated AST mixture. The source of AST could mainly be liquid wastes remained after cooking of crustacean. The yield of this solvent-free extraction method has been reported to be 10–13μg/mL cooking wastewater from shrimp using 300-kDa ultrafiltration. Overall, retention of AST is highly associated with its aggregation with other phases such as protein compounds. It is noteworthy to mention that ultrafiltered AST might need further postprocessing operations, such as enzyme hydrolysis, for liberation and purification purposes [10].

7 Supercritical fluid extraction (SFE) The SFE method is introduced because of unique physical properties of supercritical fluids due to its lower viscosity and higher diffusivity into a solid matrix (Fig. 18.3). Furthermore, this process is conducted at very low temperatures, which minimizes molecular defects of target compounds, which are usually of more heat/pressure labile molecules [12]; it could also be a very efficient method specifically for extraction of AST, which is reported to be sensitive to temperatures higher than 80°C [13]. Moreover, the most important advantage of using SFE is that the process is done using CO2, and thus, unlike other solvent extraction techniques, there is no need to remove the solvent as the final stage since CO2 is in a gaseous state under the ambient temperature. The process is considered as an environment-friendly process. This technique could be costly due to

Fig. 18.3 Schematic diagram of a pilot scale supercritical fluid extraction unit [11].

380 Chapter 18 introducing the extraction solvent in its supercritical condition. Thus there have been several studies using supercritical CO2 in combination with ethanol, and all have confirmed above 80% yield of extraction [14,15]. Wang et al. extracted ATS from H. pluvialis using SFE and ethanol [16]. They could reach a yield of 87.42% from the total available amount of 2.26% AST available in the source under 43.5 MPa pressure at 65°C. Under almost similar pressure and temperature conditions, Thana et al. found 83.78% yield of extraction (50 MPa and 70°C). Beside using pure CO2, application of cosolvents could improve the extraction yield [17]. Cheng et al. used ethanol as a cosolvent during the SFE extraction of AST [18]. The results showed that the yield reduced 1800-fold from 15h to 30s for a mixture of supercritical CO2 and cosolvent (ethanol) at 55°C. They described the improvement to be a result of ethanol removing “the interfacial barrier between the solvent and the water-wet surface and the five orders of magnitude higher solubility of AST in ethanol compared with that of CO2 according to Juan et al. [19].

8 Enzymatic hydrolysis method One of the major concerns involved with solvent extraction of AST is that the solvent removal phase may defect AST due to the high temperature involved. Thus there have been some alternative techniques to purify the AST at a lower risk of denaturation. In this regard, protease enzymes can be used for further hydrolysis of a more stable protein linked AST. The method has been studied widely, and modifications have been proposed by several researchers [20–22]. Enzymatic hydrolysis has been used as a pretreatment to extract AST from lobster. In this regard, Auerswald and G€ade used papain to disintegrate the carotenoproteinoid structures confined in the lobster tissue. Following that, methanol extraction of AST was done, and the AST was transferred to vegetable oil, and the maximum stable dosage of AST in oil was reported to be 80 mg/mL. An important technical point in the mentioned study was the utilization of papain as a protease enzyme at 0.5%–2% that showed no correlation between extraction yield and studied concentration at longer extractions times, which could be a positive cost-effective point [23].

9 Magnetic field-assisted extraction (MF) Magnetic field extraction, as a new method, is being more investigated because of its unique functionality in increasing the extraction yield. The primary concept to develop this method is based on magnetization of materials (Fig. 18.4). Under magnetic field, several physical parameters of the medium such as diffusivity, solubility and, as a result, solvent to solid equilibrium will change in a favorable way. This method has been previously tested by [25,26].

Astaxanthin extraction 381 LCD

50V

AC to DC

220V AC

Electric magnet

250V

Micro controller

400V

Sample cell

Keyboard unit

Relay

Fig. 18.4 Schematic diagram of magnetic field device [24].

Extraction of astaxanthin using MF was done by Zhao et al. [24]. The extraction efficiency of MF depends on the magnetic field intensity. It was observed that extraction yield of AST increased by more than 7% when magnetic intensity increased from 0 to 15mT. However, the extraction yield decreased at higher intensities. The mentioned behavior was reported to be due to the diamagnetic nature of the solvent phase, which indicates a decrease in the partition coefficient until reaching the yield peak and increase in the parameter at higher magnetic induction intensities [25]. Extraction time is the other important parameter during MF. It was found that ATS can be degraded if being under magnetic field for an excessive extraction time [3]. Similar trend was seen by [24] who reported to see an increase only up to 60 min of extraction. AST extraction was enhanced with increasing liquid-solid ratio. Nevertheless, at ratio of higher that 100, slight decrease in the final obtained AST was seen, which could be due to decrease in the absorbed magnetic energy by the AST source [24].

10 High-pressure extraction (HPE) During recent years the research focus has turned toward more eco-friendly processing methods in food and pharmaceutical industry. High-pressure processing has been one of the successfully introduced techniques by researchers. The main purpose of using high pressure has been to inactivate the vegetative microorganisms and enzymes for shelf-life extension purposes. This method is now widely used by the food industry to process certain foods (Fig. 18.5). During the

382 Chapter 18

Computer

Data logger

Thermocouple wire Sample thermocouple Insulator medium thermocouple

300 mm

Lid

HP medium reservoir

Vessel medium thermocouple

Water bath 85mm

Release valve

HP pump

Fig. 18.5 Schematic diagram of the high-pressure experimental system [27].

recent years, high-pressure processing has been tested for different purposes other than shelf-life extension [28,29]. One of the most recent applications of high-pressure processing is to extract bioactive molecules from vegetative, microbial, and animal tissue. This process was first introduced by Zhang and has gained considerable attention during recent years [30]. HPE is a flexible process regarding the amount of solvent to solid ratio. The technique is mainly based on applying high pressure on the target material, which is soaked in a solvent medium. The high pressure itself usually works using pressure transmitting liquids such as water and glycerin. HPE has been used to extract different bioactive compounds such as AST [31–33]. There are certain key parameters regarding HPE, which affect the yield of extraction. The pressure level involved is a crucial parameter in imposing physical pressure on the solvent and solid. The high hydrostatic pressure decreases the volume of the system, leading to induction of several molecular reactions such as protein denaturation, breakdown of salt bridges, and hydrophobic bonds. Consequently, chemical changes in macromolecules such as proteins, enzymes, lipids, and outer cell membranes could damage the cellular structure; therefore the process of solvent diffusion and extraction inside the cells is more facilitated [34].

Astaxanthin extraction 383 0.1 Mpa 100 Mpa 200 Mpa 300 Mpa 400 Mpa 500 Mpa

Astaxanthin yield (mg/g)

100

80

60

40

20 15

0

45

35

60

Liquid:solid ratio (mL/g)

Fig. 18.6 Astaxanthin (AST) yield affected by high-pressure extraction (HPE) under different liquid (solvent ethanol) to solid ratio and pressure with a 5-min pressure holding time [32].

10.1 HPE of astaxanthin: Case studies In a recent study done by [32], extraction of AST by high pressure was studied (Fig. 18.6 and Table 18.1). In this regard, shrimp waste was used as the source of astaxanthin and was mixed with different solvents such as dichloromethane, acetone, and ethanol in various solvent to solid ratios. The samples were introduced to high pressure processing and, afterward, were centrifuged, and the astaxanthin content of the supernatant was determined using spectroscopic methods. The results showed that the yield of extraction was significantly correlated to the solvent type. Ethanol, dichloromethane, and acetone showed the highest extraction yield, respectively.

Table 18.1: Astaxanthin (AST) tiled as affected by pressure holding time during high-pressure extraction (ethanol to dried shrimp waste solid ratio, 30 mL/g) [32]. Pressure holding time (min) Pressure (MPa)

0

5

0.1 100 200 300 400 500

43.62
1.76 62.98
0.59bD 55.69
2.86aC 62.65
0.94cD 50.45
0.72aB 51.64
0.39aB aA

10

42.30
1.08 62.35
0.50aB 71.14
0.94cD 71.84
0.34eD 72.94
0.84dD 69.13
1.78dC aA

15

55.60
1.49 65.60
0.62cD 66.74
0.18bD 69.42
0.64dC 57.67
0.79cB 61.33
0.59bD bA

20

55.26
2.25 63.99
1.82bcD 65.93
1.63bD 54.83
0.46bC 51.85
0.42bB 64.11
0.58cD aA

56.83
1.66bB 70.88
0.79dD 68.06
0.46bC 52.16
1.45aA 56.76
0.32cB 67.67
1.35dC

Astaxanthin (AST) yield followed by different capital letters indicate the significance analysis (a ¼ 0.05) of different pressure, while different lowercase letters indicate the significant analysis (a 0.5) of different pressure holding time.

384 Chapter 18 According to [35] the extraction yield obtained using different solvents is highly related to the ability of solvents to penetrate the cell membrane. Unlike extraction by dichloromethane and acetone, ethanol extraction showed to be responsive to the operating pressure level. In connection with the point previously mentioned, the extraction yield increased significantly from 42.3μg/g at 0.1MPa with increase in the pressure level (71.1μg/g at 200MPa). However, the extraction didn’t change between 200 and 400 MPa, and finally, the yield decreased at higher pressure levels (400–600 MPa). Irna et al. studied extraction of AST from Penaeus monodon, which is a species of shrimp. The study was done at relatively lower pressure levels (150–250MPa). However, the recovery yield was considerably higher by using a mixture of acetone and methanol as the solvent phase. The optimum point within a processing time of 10–20 min and mixture combination of 3:7 acetone was identified to be operation at approximately 239MPa for 16.29min using 6.59mL solvent/g sample resulting in 95.17 μg/g AST recovery [33]. In the available literature, it is challenging to compare different types of extraction techniques due to the fact that the yield of extraction is related to various factors such as the solvent, the solvent to solid ratio, the temperature of the samples, and most importantly the initial content of the target component in samples, which could vary based on the amount of carotenoids in the feed, other environmental and biological variations like difference in the species, and geographical origin [36]. For instance, Inra et al. compared AST extraction of six different species of shrimps using SE and HPE. Results showed higher extraction yield while using HPE. Using HPE as 210 MPa for 10 min, the highest AST was recovered from the Pe. monodon species (68.26 μg/g) [37]. Considering all mentioned variables, AST in shrimp waste has been extracted by different methods and solvents. The yield of extraction is reported to be 47–57 μg/g using enzymatic treatment. Supercritical carbon dioxide (300 bar) and ethanol mixtures at 50°C also lead to extraction of 35 μg/g AST from shrimp waste. Using pressurized ethanol extraction, 24 μg/g astaxanthin was extracted from shrimp waste. Thus comparing the data, HPE might be a promising method of extraction, which can lead to a higher extraction yield. The efficiency of high-pressure processing is associated with several pressure-related parameters including, final level of pressure, holding time, pressurization rate, and decompression process. Thus the extraction yield of HPE is also affected by the mentioned parameters. For instance, the extraction yield of astaxanthin increased as a function of pressure holding time at 100 MPa starting at 61 μg/g at zero holding time to 70.9 μg/g after 20 min. However, the trend was not similar at higher pressures. At 200–500 MPa the extraction yield reached a peak at 5th min of holding time followed by a gradual decrease in the final yield. The reason for such phenomena could be several factors being involved with the postequilibrium state. In addition, the exposure of the target compound—in this case AST—to higher pressure and longer times would lead in chemical destruction of their molecular

Astaxanthin extraction 385 structure and, as a result, a decrease in the extraction yield. Similar behavior is reported by [38] who reported a pick after 2 min of extraction of saponins under high pressure. The solvent to solid ratio is an important parameter estimating the final extraction yield and the operation costs. Extraction of astaxanthin using different solvent to solid ratio showed that the yield increases at higher ratios. However, the increasing trend reaches a peak level at a certain ratio. Similar trend has been reported by [39] during extraction of carotenoids. Irna et al. achieved a multivariable model using response surface methodology (RSM) considering the effect of t as processing time (min), P as pressure level (MPa), and V as the volume of acetone-methanol mixture (mL) (Eq. 18.1) [33]: 
AST extrction yield mg dry weight of shrimps ¼ 74:93 + 2:66P + 14:68V  1:38tP  1:31tV + 10:28PV  13:52t2  12:20P2  15:63V 2

(18.1)

Considering the effectiveness of increase in the solvent to solid ratio, technically, overuse of solvent would make the extraction and solvent removal more difficult and costly. Therefore it is important to find the proper optimum point in which an acceptable level of the target compound is extracted at the lowest possible solvent to solid ratio. In the case of HPE of ST with ethanol, a 20 mL/g ratio was seen to be the optimal point.

11 Conclusions There are more efficient ways to extract AST at a higher yield, compared with common solvent extraction methods. In fact, all novel methods mentioned in this chapter facilitated the process of extraction by increasing the heat and mass transfer parameters. Application of mentioned techniques was associated with an ascending trend as a function of processing time up to a certain level. However, overextraction of AST would lead to a decrease in the obtained dosage. This is due to the sensitivity of AST to higher pressure, temperature, and shear forces. Thus it is very important to optimize the process and consider all possible effective parameters. The current research in this area should still focus on the optimization of these novel techniques. This would also have an impact on introducing energy saving and environmentally friendly solutions to extraction of bioactive compounds.

References [1] Li J, Zhu D, Niu J. An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis. Biotechnol Adv 2011;29:568–74. https://doi.org/10.1016/j.biotechadv. 2011.04.001. [2] Sachindra NM, Bhaskar N, Mahendrakar NS. Recovery of carotenoids from shrimp waste in organic solvents. Waste Manag 2006;26:1092–8. https://doi.org/10.1016/j.wasman.2005.07.002.

386 Chapter 18 [3] Ruen-ngam D, Shotipruk A, Pavasant P. Comparison of extraction methods for recovery of astaxanthin from Haematococcus pluvialis. Sep Sci Technol 2010;46:64–70. https://doi.org/10.1080/01496395.2010.493546. [4] Zhao L, Chen G, Zhao G, Hu X. Optimization of microwave-assisted extraction of astaxanthin from Haematococcus pluvialis by response surface methodology and antioxidant activities of the extracts. Sep Sci Technol 2009;44:243–62. https://doi.org/10.1080/01496390802282321. [5] Sharayei P, Azarpazhooh E, Zomorodi S, Ramaswamy HS. Ultrasound assisted extraction of bioactive compounds from pomegranate (Punica granatum L.) peel. LWT Food Sci Technol 2019;101:342–50. https:// doi.org/10.1016/j.lwt.2018.11.031. [6] Chemat F, Rombaut N, Sicaire AG, Meullemiestre A, Fabiano-Tixier AS, Abert-Vian M. Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review. Ultrason Sonochem 2017;34:540–60. https://doi.org/10.1016/j.ultsonch.2016.06.035. [7] Zlotorzynski A. The application of microwave radiation to analytical and environmental chemistry. Crit Rev Anal Chem 1995;25:43–76. https://doi.org/10.1080/10408349508050557. [8] Pu J, Bechtel PJ, Sathivel S. Extraction of shrimp astaxanthin with flaxseed oil: effects on lipid oxidation and astaxanthin degradation rates. Biosyst Eng 2010;107:364–71. https://doi.org/10.1016/j.biosystemseng.2010.10.001. [9] Zhao T, Yan X, Sun L, Yang T, Hu X, He Z, Liu F, Liu X. Research progress on extraction, biological activities and delivery systems of natural astaxanthin. Trends Food Sci Technol 2019;91:354–61. https://doi.org/ 10.1016/j.tifs.2019.07.014. [10] Amado IR, Gonza´lez MP, Murado MA, Va´zquez JA. Shrimp wastewater as a source of astaxanthin and bioactive peptides. J Chem Technol Biotechnol 2016;91:793–805. https://doi.org/10.1002/jctb.4647. [11] Yu J, Dandekar DV, Toledo RT, Singh RK, Patil BS. Supercritical fluid extraction of limonoid glucosides from grapefruit molasses. J Agric Food Chem 2006;54:6041–5. https://doi.org/10.1021/jf060382d. [12] Routray W, Dave D, Cheema SK, Ramakrishnan VV, Pohling J. Biorefinery approach and environment-friendly extraction for sustainable production of astaxanthin from marine wastes. Crit Rev Biotechnol 2019;39:469–88. https://doi.org/10.1080/07388551.2019.1573798. [13] Ali-Nehari A, Kim SB, Lee YB, Lee HY, Chun BS. Characterization of oil including astaxanthin extracted from krill (Euphausia superba) using supercritical carbon dioxide and organic solvent as comparative method. Korean J Chem Eng 2012;29:329–36. https://doi.org/10.1007/s11814-011-0186-2. [14] Storebakken T, Sørensen M, Bjerkeng B, Harris J, Monahan P, Hiu S. Stability of astaxanthin from red yeast, Xanthophyllomyces dendrorhous, during feed processing: effects of enzymatic cell wall disruption and extrusion temperature. Aquaculture 2004;231:489–500. https://doi.org/10.1016/j.aquaculture.2003.10.034. [15] Sarada R, Vidhyavathi R, Usha D, Ravishankar GA. An efficient method for extraction of astaxanthin from green alga Haematococcus pluvialis. J Agric Food Chem 2006;54:7585–8. https://doi.org/10.1021/jf060737t. [16] Wang L, Yang B, Yan B, Yao X. Supercritical fluid extraction of astaxanthin from Haematococcus pluvialis and its antioxidant potential in sunflower oil. Innov Food Sci Emerg Technol 2012;13:120–7. https://doi.org/ 10.1016/j.ifset.2011.09.004. [17] Thana P, Machmudah S, Goto M, Sasaki M, Pavasant P, Shotipruk A. Response surface methodology to supercritical carbon dioxide extraction of astaxanthin from Haematococcus pluvialis. Bioresour Technol 2008;99:3110–5. https://doi.org/10.1016/j.biortech.2007.05.062. [18] Cheng X, Qi Z, Burdyny T, Kong T, Sinton D. Low pressure supercritical CO2 extraction of astaxanthin from Haematococcus pluvialis demonstrated on a microfluidic chip. Bioresour Technol 2018;250:481–5. https://doi. org/10.1016/j.biortech.2017.11.070. [19] Juan C, Oyarzu´n B, Quezada N, del Valle JM. Solubility of carotenoid pigments (lycopene and astaxanthin) in supercritical carbon dioxide. Fluid Phase Equilib 2006;247:90–5. https://doi.org/10.1016/j.fluid.2006.05.031. [20] Armenta-Lo´pez R, Guerrero IL, Huerta S. Astaxanthin extraction from shrimp waste by lactic fermentation and enzymatic hydrolysis of the carotenoprotein complex. J Food Sci 2002;67:1002–6. https://doi.org/10.1111/ j.1365-2621.2002.tb09443.x. [21] Chen HM, Meyers SP. Ensilage treatment of crawfish waste for improvement of astaxanthin pigment extraction. J Food Sci 1983;48:1516–20. https://doi.org/10.1111/j.1365-2621.1983.tb03528.x.

Astaxanthin extraction 387 [22] Torrissen O, Tidemann E, Hansen F, Raa J. Ensiling in acid—a method to stabilize astaxanthin in shrimp processing by-products and improve uptake of this pigment by rainbow trout (Salmo gairdneri). Aquaculture 1981;26:77–83. https://doi.org/10.1016/0044-8486(81)90111-3. [23] Auerswald L, G€ade G. Simultaneous extraction of chitin and astaxanthin from waste of lobsters Jasus lalandii, and use of astaxanthin as an aquacultural feed additive. Afr J Mar Sci 2008;30:35–44. https://doi.org/10.2989/ AJMS.2008.30.1.4.454. [24] Zhao X, Fu L, Liu D, Zhu H, Wang X, Bi Y. Magnetic-field-assisted extraction of astaxanthin from Haematococcus pluvialis. J Food Process Preserv 2016;40:463–72. https://doi.org/10.1002/elsc.201100157. [25] Sun YL, Liu Y, Wu SH, Jia SY. Effect of magnetic field on the extraction process of acetone-watertrichloroethane system. Chin J Chem Eng 2007;15:916–8. https://doi.org/10.1016/S1004-9541(08)60025-7. [26] Zielinska-Dawidziak M, Błaszak R, Piasecka-Kwiatkowska D. Influence of magnetic field on aqueous two-phase extraction of horse ferritin in the polyethylene glycol/hydroxyethyl starch system. Anal Chim Acta 2012;716:11–5. https://doi.org/10.1016/j.aca.2011.02.044. [27] Shao Y, Zhu S, Ramaswamy SH, Marcotte M. Compression heating and temperature control for high-pressure destruction of bacterial spores: an experimental method for kinetics evaluation. Food Bioprocess Technol 2010;3:71. https://doi.org/10.1007/s11947-008-0057-y. [28] Ramaswamy HS, Xu M, Vatankhah H. Investigating the influence of pH and selected heating media on thermal destruction kinetics of Geobacillus stearothermophilus (ATCC10149). J Food Meas Charact 2019;13:1310–22. https://doi.org/10.1007/s11694-019-00046-2. [29] Vatankhah H, Ramaswamy HS. High pressure impregnation of oil in water emulsions into selected fruits: a novel approach to fortify plant-based biomaterials by lipophilic compounds. LWT Food Sci Technol 2019;101:506–12. https://doi.org/10.1016/j.lwt.2018.11.080. [30] Zhang S, Zhu J, Wang C. Novel high pressure extraction technology. Int J Pharm 2004;278:471–4. https://doi. org/10.1016/j.ijpharm.2004.02.029. [31] Du J, He J, Yu Y, Zhu S, Li J. Astaxanthin extracts from shrimp (Litopenaeus vannamei) discards assisted by high pressure processing, In: 2013 Kansas City, Missouri, July 21–July 24, 2013American Society of Agricultural and Biological Engineers; 2013. p. 1. https://doi.org/10.13031/aim.20131597720. [32] Li J, Sun W, Ramaswamy HS, Yu Y, Zhu S, Wang J, Li H. High pressure extraction of astaxanthin from shrimp waste (Penaeus vannamei Boone): effect on yield and antioxidant activity. J Food Process Eng 2017;40:12353. https://doi.org/10.1111/jfpe.12353. [33] Irna C, Jaswir I, Othman R, Jimat DN. Optimization of high-pressure processing in extraction of astaxanthin from Penaeus monodon carapace using response surface methodology. J Food Process Eng 2018;41:12880. https://doi.org/10.1111/jfpe.12880. [34] Hendrickx MEG, Knorr D. Ultra high pressure treatment of foods. New York, London: Springer Science & Business Media, Kluwer Academic Press; 2012. [35] Yin C, Yang S, Liu X, Yan H. Efficient extraction of astaxanthin from Phaffia rhodozyma with polar and non-polar solvents after acid washing. Chin J Chem Eng 2013;21:776–80. https://doi.org/10.1016/S1004-9541 (13)60510-8. [36] De Holanda HD, Netto FM. Recovery of components from shrimp (Xiphopenaeus kroyeri) processing waste by enzymatic hydrolysis. J Food Sci 2006;71:298–303. https://doi.org/10.1111/j.1750-3841.2006.00040.x. [37] Irna C, Jaswir I, Othman R, Jimat DN. Comparison between high-pressure processing and chemical extraction: astaxanthin yield from six species of shrimp carapace. J Diet Suppl 2018;15:805–13. https://doi.org/ 10.1080/19390211.2017.1387885. [38] Chen RZ, Zhang SQ, Wang CZ, Dou JP, Wu H, Zhang LL. Technological process of ultrahigh-pressure extraction of saponins from Panax quinquefolius. Trans Chinese Soc Agric Eng (in Chinese) 2005;21:150–4. [39] Sachindra NM, Mahendrakar NS. Process optimization for extraction of carotenoids from shrimp waste with vegetable oils. Bioresour Technol 2005;96:1195–200. https://doi.org/10.1016/j.biortech.2004.09.018.

This page intentionally left blank

SECTION 3

Astaxanthin for food, health, pharmaceuticals—Safety and regulatory issues

This page intentionally left blank

CHAPTER 19

Efficacy of astaxanthin from different sources: Reports on the suitability for human health and nutrition Bob Capellia, Shawn Talbottb, Lixin Dingc, and Francis Capellid a

Algae Health Sciences, Irvine, CA, United States bEqqil, Draper, UT, United States cBGG, Irvine, CA, United States dFJC Design, Ocala, FL, United States

Abbreviations AMPK AX BC C. elegans EST-AX FREE-AX HEL HUVEC NAT-AX PH-AX ROS SOD SYN-AX

50 –adenosine monophosphate-activated protein kinase astaxanthin beta-carotene Caenorhabditis elegans esterified astaxanthin free astaxanthin hexanoyl lysine human umbilical vein endothelial cells natural astaxanthin from Haematococcus pluvialis microalgae astaxanthin from the genetically manipulated yeast Xanthophyllomyces dendrorhous; (former nomenclature Phaffiarhodozyma, still commonly referred to as “Phaffia”) reactive oxygen species superoxide dismutase synthetic astaxanthin from petrochemicals

1 Introduction AX is related to other members of the carotenoid family, a group of pigments that provide reactive oxygen species (ROS) scavenging activity in many plants and animals. AX is classified within the carotenoid family as a xanthophyll and is closely related to lutein, zeaxanthin, and canthaxanthin. Xanthophylls are structurally different from carotenes (the other group of carotenoids including beta-carotene [BC] and lycopene) due to hydroxyl groups and carbonyl groups at the end of the AX molecule. The hydroxyl groups and enone groups, which extend the conjugation of C]C bonds, make AX more active as an antioxidant than other carotenoids resulting in enhanced antioxidant activity and increased health benefits in humans and animals [1–3]. Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00027-1 Copyright # 2021 Elsevier Inc. All rights reserved.

391

392 Chapter 19 The wide-ranging effects of carotenoids on membranes have been hypothesized as the basis for enhanced performance of xanthophylls such as AX. BC and lycopene cause disorder of model membranes and result in lipid peroxidation; in contrast, AX preserves the structure of the model membrane [4]. In addition, while carotenes such as BC and lycopene can become prooxidants under certain conditions and hasten the proliferation of ROS, AX has never shown the capacity to create a prooxidant effect [5]. The three different forms of AX in this discussion are the result of entirely dissimilar processes: •

• •

NAT-AX is found in the highest quantities in nature in Haematococcus pluvialis, a ubiquitous unicellular microalgae, which grows in fresh water throughout the world. When these algae undergo environmental stress, they hyperaccumulate NAT-AX as a survival mechanism. SYN-AX is synthesized from petrochemicals in a highly involved, multistep process. PH-AX is produced from a species of yeast, which, in nature, produces small amounts of AX. Companies involved in the commercial production of PH-AX have genetically manipulated this species to produce exponentially more AX [6].

2 Chemical differences between forms of astaxanthin There are three significant differences chemically between NAT-AX, SYN-AX, and PH-AX: Difference #1: NAT-AX is composed of 95.7% esterified AX molecules, both monoesterified (87.0% of the total carotenoid fraction with one fatty acid molecule attached to one end of the AX molecule) and diesterified (8.7% of the total carotenoid fraction with two fatty acid molecules attached to both ends of the AX molecule) (Figs. 19.1 and 19.2). Conversely, SYN-AX and PH-AX are exclusively “free” astaxanthin (nonesterified without fatty acids attached to either end of the molecules) [7]. Difference #2: The NAT-AX, SYN-AX, and PH-AX molecules are of different shapes (Fig. 19.3). There are three distinct shapes known as diastereoisomers: diastereoisomer one (known as “S”), 3S,30 S; diastereoisomer two (known as “R”), 3R,30 R; diastereoisomer three (known as “meso”), 3R,30 S. • NAT-AX contains 100% “S” diastereoisomer3S,30 S. • SYN-AX contains a combination of three different diastereoisomers: it has 25% 3S,30 S (the same molecular shape as NAT-AX). But it contains primarily molecules shaped differently than NAT-AX: 50% is mesoastaxanthin composed of the 3R,30 S diastereoisomer and 25% is pure “R” diastereoisomer 3R,30 R. • Finally, pH-AX is exclusively “R” diastereoisomer 3R,30 R [6]. • Natural astaxanthin from algae is exclusively 3S,30 S diastereoisomer.

Efficacy of astaxanthin from different sources 393

Fig. 19.1 Natural astaxanthin carotenoid fraction: Natural astaxanthin from algae is composed primarily of esterified molecules. Approximately 95.7% of natural astaxanthin molecules have one or two fatty acid molecules attached at the ends. Approximately 1.2% is free astaxanthin (the same form as synthetic astaxanthin and Phaffia-derived astaxanthin without any fatty acid molecules attached). The remaining 3.1% is a combination of other carotenoids including canthaxanthin, BC, zeaxanthin, and lutein (in descending order).

• •

100% of Phaffia-derived astaxanthin molecules are shaped differently than natural astaxanthin with exclusively 3R,30 R diastereoisomer. 75% of synthetic astaxanthin molecules are shaped differently than the natural astaxanthin molecules. Synthetic astaxanthin contains a variety of all three diastereoisomers.

Chemical structure of di-esterifed astaxanthin O

R O

3

1

7 5

15 8

14

8⬘

1⬘

3⬘

O R⬘

7⬘

O

Fig. 19.2 Natural astaxanthin diester with two fatty acid molecules attached at either end of the astaxanthin molecule where R and R0 are either 16:0 (Palmitic acid), 18:1 (Oleic acid), or 18:2 (Linolenic acid).

394 Chapter 19

Astaxanthin enantiomers O OH 3⬘ 3 HO

Astaxanthin 3R,3ⴕR

O

O OH 3⬘

3 HO

Astaxanthin 3R,3ⴕS

O

O OH 3⬘

3 HO

Astaxanthin 3S,3ⴕS

O

(3,3⬘-dihydroxy-4,4⬘-diketo-b-carotene)

Fig. 19.3 Although all three forms of astaxanthin share the same molecular formula, they are shaped differently:

Difference #3: SYN-AX and PH-AX are exclusively AX and contain no supporting carotenoids. In contrast, NAT-AX comes naturally complexed in H. pluvialis microalgae with other carotenoids present in small quantities. When lipids are extracted from the algae, the resulting extract not only contains primarily NAT-AX but also contains four other naturally occurring carotenoids (Fig. 19.1; Table 19.1) [7].

Table 19.1 Breakdown of carotenoid fraction in natural astaxanthin from algae. 87.01% 8.70% 1.16% 0.95% 0.83% 0.71% 0.64% 100.00%

Monoesterified astaxanthin Diesterified astaxanthin Free astaxanthin Canthaxanthin Beta-carotene Zeaxanthin Lutein Total

Craft Technologies Independent Laboratory Report, November 2015.

Efficacy of astaxanthin from different sources 395

3 Differences in antioxidant activity between forms of astaxanthin Two head-to-head in vitro studies comparing NAT-AX and SYN-AX have demonstrated far superior antioxidant activity of NAT-AX ranging from 14 more activity to as much as 90 more activity. To date, we’re unaware of any antioxidant studies comparing NAT-AX with PH-AX; however, due to PH-AX being chemically more similar to SYN-AX (both are nonesterified, both have most or all R diastereoisomer, neither contains other carotenoids), we hypothesize that NAT-AX will also prove to be superior in antioxidant activity to PH-AX when tested head to head. A study published in 2013 featured antioxidant testing as both university research at Creighton University in the lab of Debasis Bagchi and independent laboratory analyses at Brunswick Laboratories. In these tests, NAT-AX was found to be a minimum of 14  stronger in antioxidant activity than SYN-AX. In the study at Creighton University, NAT-AX was tested for free radical elimination against SYN-AX and several other commonly used supplemental antioxidants. In each case, NAT-AX was far more active with free radical elimination effects ranging from 14  greater than Vitamin E to 65  greater than Vitamin C. In the case of NAT-AX versus SYN-AX, the difference in antioxidant strength was greater than 20-fold (Fig. 19.4; Table 19.2) [8].

Fig. 19.4 Natural astaxanthin from algae showed far greater free radical eliminating activity than several other common antioxidants (ranging from 14 to 65). In the case of natural astaxanthin versus synthetic astaxanthin, the difference was 21 .

396 Chapter 19 Table 19.2: NAT-AX tested for free radical elimination against several commonly used supplemental antioxidants.

Material Vitamin C Vitamin E Beta carotene Pycnogenol SYN-AX NAT-AX

mg of active material used

% free radical inhibition in study

% free radical inhibition per mg active material

100 50 100 100 100 5

19 43 23 69 59 61.7

0.19 0.86 0.23 0.69 0.59 12.34

NAT-AX relative performance N-AX N-AX N-AX N-AX N-AX

65  stronger 14  stronger 53  stronger 18  stronger 21  stronger N/A

From Capelli B, Bagchi D, Cysewski G. Synthetic astaxanthin is significantly inferior to algal-based astaxanthin as an antioxidant and may not be suitable as a human nutritional supplement. NutraFoods. 2013;12:145–52.

The independent lab testing performed at Brunswick Laboratories used Total ORACFN analyses, which tests for five free radicals commonly found in the human body. NAT-AX was many times more active in eliminating singlet oxygen, the superoxide ion, and peroxyl radicals, while SYN-AX was more active in eliminating peroxynitrite. Results for NAT-AX were not determined for elimination of hydroxyl radicals, while SYN-AX obtained a positive result. The comparison proffered by Brunswick Laboratories totaled the results for all five compounds and found that, overall, NAT-AX is 14 more active than SYN-AX. With regard to the harmful singlet oxygen radical, NAT-AX was 55 more active than SYN-AX (Table 19.3) [8]. A recent study published by French university professors and sponsored by the French National Institute of Health and Medical Research [9] again showed the superior antioxidant activity of Table 19.3: NAT-AX versus SYN-AX Total ORACFN antioxidant power test results (all numbers in micromoles Trolox equivalent per gram). Test

NAT-AX

SYN-AX

NAT-AX vs SYN-AX

Antioxidant power against singlet oxygen

12,055

220

55  stronger

Antioxidant power against superoxide ion

5377

258

21  stronger

Antioxidant power against peroxyl radicals

574

165

3.5 stronger

Antioxidant power against peroxynitrite

28

115

0.24  of SYN-AX

Antioxidant power against hydroxyl radicals

Not determined

538

Not Comparable

Total ORACFN antioxidant power

18,034

1296

14  stronger

From Capelli B, Bagchi D, Cysewski G. Synthetic astaxanthin is significantly inferior to algal-based astaxanthin as an antioxidant and may not be suitable as a human nutritional supplement. NutraFoods. 2013;12:145–52.

Efficacy of astaxanthin from different sources 397 Table 19.4: Intracellular antioxidant capacity of SYN-AX and two forms of NAT-AX.

Product SYN-AX NAT-AX (DMSO extract) NAT-AX (CO2 extract)

Cellular antioxidant activity %

Comparative increased cellular antioxidant activity (using SYN-AX as the base)

0.3 +/ 0.2 25.4 +/ 9.5 30.4 +/ 12.7

N/A 85  101 

From Regnier P, Bastias J, Rodriguez-Ruiz V, Caballero-Casero N, Caballo C, Sicilia D, et al. Astaxanthin from Haematococcus pluvialis prevents oxidative stress on human endothelial cells without toxicity. Mar Drugs. 2015;13:2857–2874.

NAT-AX compared with SYN-AX. This study was designed to examine the intracellular antioxidant capacity of these forms of AX as a means to understand potential cardiovascular benefits. The researchers tested two forms of NAT-AX against SYN-AX. The two natural forms tested were algae extracts produced by supercritical CO2 extraction and by solvent extraction using DMSO. The researchers did additional testing above and beyond a standard in vitro antioxidant comparison to investigate the potential cardiovascular benefits for the different forms of astaxanthin more deeply. They examined the intracellular antioxidant capacity of astaxanthin using a stress-based model in human umbilical vein endothelial cells (HUVEC). The HUVEC cells were subjected to stress by introducing tert-butyl hydroperoxide (a molecule that increases intracellular production of ROS by damaging mitochondrial membranes). NAT-AX extracted by supercritical CO2 performed the best (although the difference with NAT-AX extracted with the solvent DMSO was not statistically significant). Both natural forms outperformed SYN-AX by tremendous margins ranging from 85\ to 101 (Table 19.4). The researchers concluded that NAT-AX can inhibit intracellular-induced stress in human endothelial cells without toxicity. Since the intracellular antioxidant activity of NAT-AX was approximately 90 stronger than SYN-AX, they suggested that NAT-AX may have potential therapeutic or preventive properties for cardiovascular diseases [9].

4 Animal research shows superior efficacy and bioactivity of NAT-AX in comparison with SYN-AX and PH-AX In addition to the in vitro antioxidant studies cited earlier, preclinical research directly comparing the three sources of AX in different species of animals has clearly established the functional superiority of NAT-AX. In total, five animal trials have been published to date comparing AX sources; in each case, NAT-AX has demonstrated superior efficacy. In addition, an innovative study comparing esterified and nonesterified forms of AX separated from H. pluvialis microalgae and tested in rats for inhibition of skin cancer and other health parameters has further corroborated these five animal trials; this study establishes a mechanism for the superior functioning of NAT-AX and is clear evidence that esterified forms of AX are more bioactive and provide enhanced health benefits than nonesterified AX.

398 Chapter 19

5 Survival rates, stress resistance and growth rates in shrimp The first study of health differences in animals supplemented with different forms of AX was done in 1998. It focused on a species of shrimp called Penaeus monodon (known as the “giant tiger prawn”). This study was done by university researchers in Thailand in support of the large shrimp-farming industry in that country. These professors did a series of tests on three different larval and postlarval stages during the shrimp’s life cycle. They separated the shrimp into four different groups: • • • •

One treatment group was fed with a commercial diet supplemented with NAT-AX. The second treatment group was fed with a commercial diet supplemented with SYN-AX. One control group was fed with the same commercial diet without any addition of AX. A different control group was fed with a natural diet that the shrimp would normally eat in the wild.

Fifteen days after the postlarval stage, shrimp fed NAT-AX were experiencing significantly better survival rates than all three other groups (including shrimp fed the natural diet). And in each of the three life stages, shrimp fed NAT-AX survived at higher rates than shrimp fed SYN-AX. (Remarkably, in the zoea larval stage, 82.5% of the shrimp fed NAT-AX survived while only 27.8% of the shrimp fed SYN-AX survived.) The differences were statistically significant between NAT-AX and SYN-AX in both other life stages, albeit with less dramatic results (Table 19.5). In addition, tests of low water salinity were done to examine the different groups’ tolerance levels to environmental stress, and the shrimp fed the NAT-AX diet again outperformed all others. Shrimp from all three other groups died faster than the NAT-AX fed shrimp when subjected to this stressful condition (Fig. 19.5). Finally, there were differences in growth rates as well between diets. Measured at 15 days after the larval stage, shrimp fed with the NAT-AX diet were larger on average than all other groups. In each case the result was statistically significant (Table 19.6). Table 19.5: Survival rates (%) of shrimp in three different life stages, based on their diet. Diets Natural diet Commercial diet without astaxanthin added Commercial diet with algae-based astaxanthin added Commercial diet with synthetic astaxanthin added

Zoea

Mysis

Postlarvae

82.0  3.30 74.0  5.19

76.7  8.61 57.3  5.01

55.2  6.14 68.6  5.73

82.5  3.53

69.7  12.05

76.0  9.46

27.8  4.01

58.1  0.29

64.4  11.86

Aquatic Resources Research Institute, Department of Marine Science and Marine Biotechnology Research Unit, Chulalongkorn University, Bangkok 10330, Thailand, 2015.

Efficacy of astaxanthin from different sources 399

Fig. 19.5 Shrimp larvae subjected to environmental stress survived longer when fed a diet containing natural astaxanthin from algae compared with all three other diets.

It’s interesting to note that shrimp fed with SYN-AX were generally the worst performers. Most notably, in the Zoea larval stage, all three other groups outlived the shrimp fed SYN-AX by vast margins. This is particularly surprising when considering that one of the control diets was precisely the same commercial diet contained in the NAT-AX and SYN-AX diets but without any AX added. The researchers hypothesized that shrimp accept NAT-AX better than SYN-AX, which is why the shrimp supplemented with NAT-AX survive at much higher rates, grow faster, and resist environmental stress more efficiently. They cited the difference in esterification (NAT-AX

Table 19.6: Growth rate of shrimp fed different diets. Diets Natural diet Commercial diet without astaxanthin added Commercial diet with algaebased astaxanthin added Commercial diet with synthetic astaxanthin added

Length of post larval shrimp (cm) 0.9843  0.073 0.9674  0.074 1.0019  0.067 0.9652  0.075

Aquatic Resources Research Institute, Department of Marine Science and Marine Biotechnology Research Unit, Chulalongkorn University, Bangkok 10330, Thailand, 2015.

400 Chapter 19 being predominantly esterified and SYN-AX being nonesterified “free astaxanthin”) as the probable basis for NAT-AX outperforming SYN-AX [10]. This study provides substantial evidence for the superiority of NAT-AX over SYN-AX in this species due to the variety of tests carried out with consistent results. They tested differences at multiple life stages (two larval stages and 15 days into the postlarval stage), and they tested effects in both healthy environments and under environmental stress, in all cases, NAT-AX outperformed SYN-AX.

6 Gastric ulcers in rats—Study #1 A study done in 2005 at a university in Japan examined the effects of AX on gastric ulcers in rats. Results indicated significantly better potential for NAT-AX to prevent gastric ulcers than both PH-AX and SYN-AX. Rats were stressed by putting them into chest-level water for 24h after having fasted for 24h. This study tested the three forms of AX and beta-carotene. All the rats fed with carotenoids (including all three forms of AX and beta-carotene) before being stressed were appreciably protected against the formation of gastric ulcers as compared with rats in the control group. The rats given NAT-AX experienced improved health over the other groups as evidenced by smaller ulcer indexes. The researchers theorized that AX improves antitumor immune response through prevention of lipid peroxidation induced by stress [11].

7 Gastric ulcers in rats—Study #2 In 2008 a similar study was done on the effects of AX on ulcers in rats. This study tested SYN-AX against NAT-AX, but did not include PH-AX. The researchers used ethanol to induce ulcers and found that pretreatment with NAT-AX outperformed SYN-AX in inhibiting enzymes associated with the formation of ulcers. SYN-AX did not show any inhibition at all. Remarkably, NAT-AX showed inhibition of ulcers at a higher level than the ulcer drug omeprazole (sold under the brand name Prilosec). NAT-AX showed gastroprotective effects dose dependently on ethanol-induced gastric lesions. The researchers pointed out that NAT-AX showed better stability than Free AX (the form found in SYN-AX) and hypothesized that this may be the basis for its superior health benefits [12]. The better stability is likely due to esterified AX being more stable than free AX.

8 Antioxidant activity and increase of lifespan in model organism for longevity in mammals A recent study sponsored by the Chinese government’s National Natural Science Foundation tested all three forms of AX in a model organism for longevity testing in mammals to see if AX can increase lifespan. This study was published as a joint project between the Department of Food Science at University of Massachusetts and the Department of Food Science at South China Agricultural University. The model organism is a worm named Caenorhabditis elegans

Efficacy of astaxanthin from different sources 401 (C. elegans). This worm is extensively used in longevity testing for two strategic reasons: Firstly, it has 60%–80% of the human gene homologues (linking experiments with this worm to potential increase in longevity in humans) [13]. Secondly, this worm has a 3-week lifespan, allowing for rapid testing and results. The worms were separated into four groups: a control group, a group treated with NAT-AX, a group treated with PH-AX, and a group treated with SYN-AX. Similar to our goal with this review chapter, the goal of this worm study was to test functional differences between the different forms of AX to see if there is a preferred form for human supplementation. The worms underwent oxidative stress for 24 h. The researchers created oxidative stress by introducing paraquat (a toxic, fast-acting herbicide) to the worms. The worms were tracked for 5 days after exposure to paraquat. Reactive oxygen species were reduced by all forms of AX, with NAT-AX being significantly superior in quenching these free radicals, particularly versus SYN-AX in which it was over 80% more effective (Fig. 19.6). NAT-AX steadily increased survival rates of the worms at all five measurement intervals versus control. By day 3, the survival rates were 19% better than control, and by day 5, survival rates were 110% better. NAT-AX outperformed SYN-AX and PH-AX with regard to survival rates slightly; however, contrary to the profound differences in shrimp larvae cited previously, in this

Fig. 19.6 Natural astaxanthin from algae eliminated ROS much more effectively than synthetic astaxanthin (82.3% more effective) and Phaffia-derived astaxanthin (33.2% more effective).

402 Chapter 19 study the differences were not statistically significant. The researchers concluded that AX can increase oxidative resistance, decrease levels of reactive oxygen species, increase enzyme activity of superoxide dismutase (SOD) and Catalase, and enhance expression of SOD-3, all of which lead to increasing survival of the worms [14].

9 Endurance in mice This is the most recent study comparing the effects of NAT-AX to other forms in animals. The mice in this study were divided into four groups: control, mice fed with NAT-AX, mice fed with SYN-AX, and mice fed with PH-AX. The study duration was 5 weeks, during which supplementation of the various AX forms was consistently administered. At the end of 5 weeks of supplementation, the mice run until exhaustion on a treadmill. Mice fed NAT-AX ran significantly longer than mice fed both other AX forms (Fig. 19.7). Interestingly, mice fed SYN-AX and PH-AX ran slightly shorter duration than mice in the control group (although these differences were not statistically significant). Additional differences between the groups were noted: •

Plasma concentration of AX was significantly higher in the NAT-AX group than all other groups.

Fig. 19.7 NAT-AX significantly increased endurance in mice as measured by time to exhaustion on a treadmill, while SYN-AX and PH-AX decreased endurance as compared with the control group.

Efficacy of astaxanthin from different sources 403 • • •

Similarly, tissue concentration of AX was significantly higher in the NAT-AX group than all other groups. NAT-AX increased 50 –adenosine monophosphate-activated protein kinase (AMPK) levels in skeletal muscle. Although mice in the NAT-AX group ran for a longer time, hexanoyl lysine (HEL) adduct levels in skeletal muscle mitochondria were similar in the control and NAT-AX groups.

The researchers hypothesized that accumulation of AX in muscle tissue may increase endurance. They investigated energy metabolism and oxidative damage to understand the mechanisms involved in increasing endurance. AMPK has a substantial influence in regulating metabolism of both carbohydrates and lipids. Furthermore, AMPK is responsible for increasing mitochondrial biosynthesis and activity. NAT-AX increased AMPK levels, which they attributed to the increase in running endurance due to activation of energy metabolism. HEL is a marker used to measure oxidative stress, which triggers the initial phase of lipid peroxidation. NAT-AX inhibited the increase in HEL caused by endurance exercise in mice in this study. NAT-AX was successful in preventing oxidative damage in skeletal muscle mitochondria. This may be due to suppression of lactic acid levels. Previous clinical research has found NAT-AX capable of reducing lactic acid levels in athletes after running [15]. Although lactic acid levels were not measured in this study, the authors suggested a possible correlation and offer lactic acid suppression as another possible mechanism. In fact, they proffered that, due to NAT-AX’s variety of functions, its effects on exercise performance may involve various other mechanisms. Their conclusion stated that NAT-AX promotes energy production and protects tissue from oxidative damage during exercise. They attribute these results to the esterified form (NAT-AX) having superior absorption characteristics than nonesterified forms (SYN-AX and PH-AX) [16].

10 Skin cancer, antioxidant activity, retinol levels and tyrosinase enzyme levels in rats This unique study was sponsored by the government of India. It employed an alternate means to obtain the different forms of AX than the previously cited studies. In each of the five animal trials referenced earlier (as well as the antioxidant surveys), SYN-AX was obtained from petrochemicals as free astaxanthin (FREE-AX), pH-AX was obtained from genetically-manipulated yeast as FREE-AX, and NAT-AX was obtained from H. pluvialis microalgae as predominantly Esterified AX (EST-AX). In this study the researchers extracted the total carotenoid fraction from H. pluvialis and then separated the fractions into FREE-AX, monoesterified AX, and diesterified AX. This process separated the algae extract into the FREE-AX form, which is exclusively found in SYN-AX and PH-AX and two different esterified forms that are the main components of NAT-AX. They analyzed these three forms in rats along with samples of the total carotenoid fraction before separation.

404 Chapter 19 A crucial finding of this study indicated that EST-AX from H. pluvialis microalgae inhibited skin tumors in rats significantly better than FREE-AX (the form found in SYN-AX and PH-AX) (Fig. 19.8). In addition, EST-AX had far superior antioxidant activity compared with FREE-AX (Fig. 19.9), increased retinol conversion in the liver more efficiently, and augmented tyrosinase enzyme more successfully (Fig. 19.10). The conclusion stated that EST-AX from algae has better anticancer potential than FREE-AX (as found in SYN-AX and PH-AX), which may be due to better bioavailability (Rao et al., 2013). This study, while not done on SYN-AX synthesized from petrochemicals or PH-AX derived from mutated yeast, is perhaps the most significant examination of the effect of esterification of AX to date. While all three forms of AX in this study were derived from H. pluvialis microalgae, they were separated into different esterified and nonesterified forms. Results indicated that the esterified forms show far superior therapeutic and preventive health potential than the nonesterified form. Since the source of all forms was the same (H. pluvialis microalgae), other variables possible in commercial AX products were eliminated. Hence the superiority of EST-AX over FREE-AX in this study can only be attributed to the presence of esters. This is a significant factor in our conclusion that EST-AX from algae is clearly the superior choice for human supplementation.

Fig. 19.8 Both diesterified NAT-AX and monoesterified NAT-AX inhibited skin cancer in rats more effectively than Free-AX and total carotenoid fraction from H. pluvialis microalgae. In the case of diesterified NAT-AX, inhibition reached a level of 96.7%.

Efficacy of astaxanthin from different sources 405

Fig. 19.9 Esterified forms of astaxanthin from microalgae had far superior antioxidant activity compared with free astaxanthin and total carotenoids from H. pluvialis microalgae.

Fig. 19.10 Diesterified AX had 2.4–2.8 more tyrosinase enzyme inhibitory activity than free astaxanthin and total carotenoids from H. pluvialis microalgae (respectively), while monoesterified AX had 1.5–1.7 more inhibitory activity (respectively). All results were statistically significant.

406 Chapter 19

11 Summary of human clinical research While a thorough review of the human research on NAT-AX is outside of the scope of this chapter due to space constraints, there have been over 100 human clinical trials showing 10 different areas of health benefits, which are listed in the succeeding text: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

supports eye health; supports brain health; improves skin health and appearance when taken internally; has 12 different benefits for athletes and active people; supports cardiovascular health; promotes modulation of the immune system; has many antiaging properties; supports male fertility; works as an antiinflammatory to reduce pain and mitigate systemic inflammation; is the most powerful antioxidant found in nature to date, with six exceptional qualitative properties as an antioxidant that most other antioxidants do not share [17].

12 Conclusions NAT-AX is the logical choice as a consumer dietary supplement among forms of AX currently in the market as evidenced in this review of the literature. The majority of the comparative studies cited earlier were sponsored by and/or conducted by governmental agencies and universities; as such, they are impartial and free of conflicts of interest. NAT-AX has proven far more active as an antioxidant than SYN-AX by a factor 14–90. Due to the chemical similarities between PH-AX and SYN-AX, we postulate that NAT-AX will prove far more active than PH-AX, if tested in head-to-head antioxidant surveys. However, in vitro antioxidant testing does not necessarily equate to corporal actuality in humans. While human clinical trials directly comparing forms of AX for safety and health benefits would be the determining factor, no such research has been conducted to date. In fact, there is a complete dearth of research in humans for SYN-AX and PH-AX demonstrating efficacy or even safety. This is a huge concern for SYN-AX and PH-AX—we don’t currently know if they have health benefits for humans or if they are completely safe for human consumption on a long-term basis. Due to the lack of human clinical research for SYN-AX and PH-AX, we turn to animal research directly comparing the three sources to better understand their potential as human dietary supplements for preventive or therapeutic effects. Five animal trials in different animal species directly comparing different forms of AX have undisputedly demonstrated that NAT-AX from

Efficacy of astaxanthin from different sources 407 algae is the most effective form for a variety of health benefits. This research has established that NAT-AX is superior in the following: • • • • • •

increasing survival rates improving resistance to environmental stress improving exercise endurance preventing gastric ulcers expanding antioxidant activity increasing growth rates

In addition, a unique study in rats comparing esterified and nonesterified forms provides weighty evidence that NAT-AX is superior to SYN-AX and PH-AX—and this study gives a basis for why this is so. By separating esterified and nonesterified AX from a single source— H. pluvialis microalgae—the researchers conducting this study eliminated other potential complicating factors that could surface by using AX from different sources. They definitively demonstrated the superiority of esterified astaxanthin (the form present in NAT-AX) over free astaxanthin (the form present in SYN-AX and PH-AX) by demonstrating that esterified AX is superior to free astaxanthin at: • • • •

preventing the formation of skin cancer amplifying antioxidant activity augmenting retinol conversion in the liver increasing tyrosinase enzyme levels.

This innovative study establishes one mechanism by which NAT-AX consistently outperforms SYN-AX and PH-AX in the studies cited in this review—esterification. The two additional chemical differences between AX forms (stereochemistry and presence of other carotenoids) may also play a role in the functional superiority of NAT-AX, but at present, these factors have not been isolated and tested. In addition, possible explanations for the functional superiority of NAT-AX proffered in three of the animal trials cited previously (improved bioavailability of NAT-AX and better stability of NAT-AX) may also be found true; we hope to see research in this area in the future. Due to the nonexistence of clinical trials showing health benefits and the complete absence of direct safety research in humans for SYN-AX and PH-AX, we highly recommend that consumers supplement with NAT-AX as the sole clinically validated form. NAT-AX has proven safe and effective over many years of consumer use. NAT-AX has amassed an abundance of safety data and has demonstrated extensive health benefits in more than 100 human clinical studies. These factors have led regulatory bodies throughout the world to accept NAT-AX for direct human consumption in supplement form. Owing to the lack of such research, SYN-AX and PH-AX have only been accepted in a few countries to date.

408 Chapter 19 As a result of the preponderance of evidence previously, we recommend that consumers supplementing with AX ensure that the brand of AX they consume sources raw materials from H. pluvialis algae.

Competing interests Bob Capelli and Lixin Ding, PhD, work for companies involved in the production of natural astaxanthin from microalgae. Shawn Talbott, PhD, is the owner of an independent clinical research organization and is not involved in the production of astaxanthin. Francis Capelli is an independent consultant and is not involved in production of astaxanthin.

Authors’ contributions All authors contributed equally to this manuscript.

Acknowledgments and funding While Bob Capelli and Lixin Ding work for companies involved in the production of astaxanthin from microalgae, this manuscript was written independently of their responsibilities at those companies. No funding for this review was given by any company or other entity.

Description of additional file The authors felt that it is important to bring to the readers’ attention the extensive human clinical research on astaxanthin derived from algae; however, the scope of this manuscript did not allow for an extensive review of this literature. For this reason, we directed attention to a hyperlink for a document that contains this clinical research: www.algaehealthsciences.com/medicalresearch/abstractlist

References [1] Astaxanthin KP. Cell membrane nutrient with diverse clinical benefits and anti-aging potential. Altern Med Rev 2011;16:355–64. [2] Fassett R, Coombes J. Astaxanthin, oxidative stress, inflammation and cardiovascular disease. Futur Cardiol 2009;5:333–42. [3] Guerin M, Huntley M, Olaizola M. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol 2003;21:210–6. [4] McNulty H, Jacob R, Mason R. Biologic activity of carotenoids related to distinct membrane physicochemical interaction. Am J Cardiol 2008;101:20D–29D. [5] Beutner S, Bloedorn B, Frixel S, Blanco I, Hoffmann T, Martin H, Mayer B, Noack P, Ruck C, Schmidt M, Schulke I, Sell S, Ernst H, Haremza S, Seybold G, Sies H, Stahl W, Walsh R. Quantitative assessment of antioxidant properties of natural colorants and phytochemicals: carotenoids, flavonoids, phenols and indigoids. The role of B-carotene in antioxidant functions. J Sci Food Agric 2001;81:559–68. [6] Visioli F, Artaria C. Astaxanthin in cardiovascular health and disease: mechanisms of action, therapeutic merits, and knowledge gaps. Food Funct 2017;8:39–63. [7] Craft Technologies Independent Laboratory Report. On file at BGG North America. Irvine, CA.

Efficacy of astaxanthin from different sources 409 [8] Capelli B, Bagchi D, Cysewski G. Synthetic astaxanthin is significantly inferior to algal-based astaxanthin as an antioxidant and may not be suitable as a human nutritional supplement. Nutrafoods 2013;12:145–52. [9] Regnier P, Bastias J, Rodriguez-Ruiz V, Caballero-Casero N, Caballo C, Sicilia D, Fuentes A, Maire M, Crepin M, Letourneur D, Gueguen V, Rubio S, Pavon-Djavid G. Astaxanthin from Haematococcus pluvialis prevents oxidative stress on human endothelial cells without toxicity. Mar Drugs 2015;13:2857–74. [10] Darachai J, Piyatiratitivorakul S, Kittakoop P, Nitihamyong C, Menasveta P. Effects of astaxanthin on larval growth and survival of the giant tiger prawn, Penaeus monodon. In: Flegel TW, editor. Advances in shrimp biotechnology. Bangkok: National Center for Genetic Engineering and Biotechnologoy; 1998. p. 117–22. [11] Nishikawa Y, Minenaka Y, Ichimura M, Tatsumi K, Nadamoto T, Urabe K. Effects of astaxanthin and vitamin C on the prevention of gastric ulcerations in stressed rats. J Nutr Sci Vitaminol 2005;51:135–41. [12] Kamath B, Srikanta B, Dharmesh S, Sarada R, Ravishankar GA. Ulcer preventive and antioxidative properties of astaxanthin from Haematococcus pluvialis. Eur J Pharmacol 2008;590:387–95. [13] Kaletta T, Hengartner M. Finding function in novel targets: C. elegans as a model organism. Nat Rev Drug Discov 2008;5:387–99. [14] Liu X, Luo Q, Cao Y, Goulette T, Liu X, Xiao H. Mechanism of different stereoisometric astaxanthin in resistance to oxidative stress in Caenorhabditis elegans. J Food Sci 2016;81:H2280–7. [15] Sawaki K, Yoshigi H, Aoki K, Koikawa N, Azumane A, Kaneko K, Yamaguchi M. Sports performance benefits from taking natural astaxanthin characterized by visual acuity and muscle fatigue improvement in humans. J Clin Ther Med 2002;18:1085–100. [16] Aoi W, Maoka T, Abe R, Fujishita M, Tominaga K. Comparison of the effect of non-esterified and esterified astaxanthins on endurance performance in mice. J Clin Biochem Nutr 2018;62:161–6. [17] Capelli B, Ding L. Natural astaxanthin: the supplement you can feel. ISBN-13:978-0-9992223-0-0.

This page intentionally left blank

CHAPTER 20

Oxidation products of astaxanthin: An overview Takashi Maoka Research Institute for Production Development, Kyoto, Japan

Abbreviations MS HPLC LC/PDA ESI-MS NMR ESR

mass spectrometry high performance liquid chromatography high performance liquid chromatography/photodiode array detection electro spray ionization mass spectrometry nuclear magnetic resonance spectrometry electro spin resonance spectrometry

1 Introduction Several apocarotenoids such as apocarotenones and apocarotenals have been reported in nature as oxidative degradation products of C40-carotenoids in plants [1] and animals [2]. Furthermore, these apocarotenoids play several important biological roles in plants and animals [1,2]. However, there were a few reports on apoastaxanthins in nature. Only 80 -apoastxanthinal (3-hydroxy-4-oxo-80 -apo-β-caroten-80 -al) was reported as a natural occurring apoastaxanthin. In 1992 Tanaka et al. isolated 80 -apoastacenal (3-hydroxy-4-oxo-2,3-didehydro-80 -apoβ-caroten-80 -al) from a saponified extract of eggs of nudibranch belonging to Hexabranchus [3]. It was well known that carotenoid with 3-hydroxy-4-keto-β-end group such as astaxanthin was oxidized to 2,3-didehydro-3-hydroxy-4-keto-β-end group such as astacene during saponification procedure [4]. Therefore, they assumed that the original form of 80 -apoastacenal in the eggs of nudibranch was 80 -apoastaxanthinal [3]. 80 -Apoastaxanthinal was also isolated from Rhodococcus sp. and Gordonia sp. with astaxanthin dirhamnoside and 3,4dihydroxyretinolin by Cabello et al. [5] as shown in Fig. 20.1. 80 -Apoastaxanthinal found in these organisms was assumed to be oxidative metabolites of astaxanthin. 9-Apoastaxanthinone was reported in the metabolites of astaxanthin in primarily cultures of rat hepatocytes [6]. Furthermore, 9-apoastaxanthinone (3-hydroxy-4-oxo-β-ionone) and 9-apoastaxanthinol (3-hydroxy-4-oxo-β-iononol) were identified as metabolites of astaxanthin in human plasma and primary cultured human hepatocytes [7]. Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00013-1 Copyright # 2021 Elsevier Inc. All rights reserved.

411

412 Chapter 20

O HO O

8ⴕ-Apoastaxanthinal (Original form) Saponification O

HO O

8ⴕ-Apoastacenal

8ⴕ-Apoastacenal from a saponified extract of eggs of nudibranch OH HO

OH

O O

HO

O

O

OH

Astaxanthin dirhamnoside

O HO

O

OH OH O HO O

8ⴕ-Apoastaxanthinal OH

HO OH 3,4-Dihydroxyretinol

Found only in Gordonia sp.

Carotenoids in Rhodococcus sp. and Gordonia sp. O OH

HO

Astaxanthin

O

in human hepatocytes O HO O

9-Apoastaxanthinone

OH HO O

9-Apoastaxanthinole

Fig. 20.1 Naturally occurring apoastaxanthins: 80 -apoastaxanthinal from nudibranch egg, Rhodococcus sp. and Gordonia sp. 9-apoastaxanthinone and 9-apoastaxanthinole as oxidative metabolites of astaxanthin in human plasma and primary cultured human hepatocytes.

Oxidation products of astaxanthin: An overview 413 Recently, several oxidation products of astaxanthin were found as reaction products of astaxanthin with reactive oxygen and nitrogen species. In this chapter, I review these oxidation products of astaxanthins from chemical point of view.

2 Apoastaxanthins as autooxidation products of astaxanthin In 2012 Etoh et al. [8] reported that a series of apoastaxanthins were identified as autooxidation products of astaxanthin. In this experiment, astaxanthin extracted from Haematococcus pluvialis was placed in petri dish and allowed to react with atmospheric oxygen at 55°C in the dark for 35days. Then, reaction products were analyzed by HPLC, UV-vis, MS, and 1H NMR. Nine apoastaxanthins were identified as autooxidation products of astaxanthin as shown in Fig. 20.2. Among them, 13-apoastaxanthinone was obtained as a major product (56.5% of the total aposataxanthins formed by auto-oxidation), followed by 11-apoastaxanthinal (9.3%), 9-apoastaxanthinone (8.4%), and 100 -apoastaxanthinal (8.2%), whereas 7-apoastaxanthinal (0.5%), 15-apoastaxanthinal (0.5%), 140 -apoastaxanthinal (0.5%), 120 -apoastaxanthinal (0.5%), and 80 -apoastaxanthinal (0.5%) were detected as minor components. These results indicated that the double bond at C13dC14 in astaxanthin was more easily oxidized than other double bonds followed by double bonds at C9dC10 and C11dC12. These results were in agreement with the results of molecular orbital calculations. The relative cleavage energy of the double bonds at C7dC8, C9dC10, C11dC12, and C13dC14 in astaxanthin, when the cleavage energy of the double bond at C15dC150 was 0 kJ, were 6.72, 29.53, 12.24, and 30.53 kJ, respectively. Therefore the double bond at C13dC14 in astaxanthin was most easily cleaved by oxidation as shown in Fig. 20.2. These results indicated that astaxanthin could take up oxygen by its polyene chain by the formation of apoastaxanthinal and apoastxanthinone. Further drastic condition, astaxanthin was polymerized and formed insoluble materials or decomposed to form colorless materials. These degradation compounds were also formed by reaction of oxygen atom with astaxanthin.

3 Astaxanthin epoxides and endperoxides as reaction products of astaxanthin with active oxygen species Astaxanthin is an excellent antioxidant not only for quenching singlet oxygen (1O2) but also for inhibition of lipid peroxidation. Furthermore, it was revealed that astaxanthin could scavenge hydroxyl radical (•OH) and superoxide anion radical (•O 2 ) [9,10]. Recently Nishino et al. investigated the reaction products of astaxanthin with hydroxyl radical, superoxide anion radical, and singlet oxygen by LC/PDA ESI-MS and ESR spectrometry [11]. The ESR study revealed that astaxanthin could quench not only singlet oxygen but also superoxide anion radical and hydroxyl radical. The LC/PDA ESI-MS study revealed that

414 Chapter 20

Fig. 20.2 Autooxidation products of astaxanthin. Molecular orbital calculations were performed by using the Gaussian 03 program. Details were described in literature [8].

astaxanthin epoxides were major reaction products of astaxanthin with superoxide anion radical and hydroxyl radical. Astaxanthin endoperoxides were identified as major reaction products of astaxanthin with singlet oxygen (Fig. 20.3). Similar results were also obtained in the case of β-carotene, zeaxanthin [12], capsanthin, and capsorubin [13]. These results suggest that carotenoids could take up singlet oxygen, superoxide anion radical, and hydroxyl radical by the formation of endoperoxide or epoxide in its molecule.

Oxidation products of astaxanthin: An overview 415 O OH

HO

Astaxanthin

O -.

O2

1

.

OH

O2

O

O

OH OH

O

O O

HO

HO

O

O

O

O

OH

OH O

HO O

Astaxanthin epoxides

HO

O

O

O

Astaxanthin endoperoxides

Fig. 20.3 Reaction products of astaxanthin with singlet oxygen, superoxide anion radical, and hydroxyl radical.

Details of reaction of reactive oxygen species with astaxanthin were as follows: Hydroxyl radical, superoxide anion radical, and singlet oxygen were generated by UV-A irradiation of hematoporphyrin, riboflavin, and hydrogen peroxide (H2O2) solution, respectively. An acetonitrile solution of astaxanthin or its acetate was added to these reactive oxygen species-generation systems. At regular intervals of UV-A irradiation, reaction products were analyzed by LC/PDA ESI-MS and ESR spin trapping. In the case of the reaction of astaxanthin acetate with hydroxyl radical, reaction products were increased with UV-A irradiation time dependently. The amount of reaction products reached a maximum with 6 min of UV-A irradiation. Then, astaxanthin acetate and oxidation products were markedly decreased, and the reaction solution was bleached. Fig. 20.4 A shows the HPLC profiles of the reaction product of astaxanthin acetate with hydroxyl radical with 6 min of UV-A irradiation. Three reaction products (Peaks 1 to 3) were detected on HPLC. They were identified to be astaxanthin acetate triepoxide (Peak 1), astaxanthin acetate 5,8-epoxide (or 7,8-epoxide) (Peak 2), and astaxanthin acetate 5,6-epoxide (Peak 3) based on the MS, MS/MS, and UV-vis spectral data. Similar results were obtained in the case of reaction products of astaxanthin acetate with superoxide anion radical. In the case of the reaction of astaxanthin acetate with 1O2, astaxanthin acetate 5,6endperoxide was obtained as major reaction product (Peak 3) along with astaxanthin acetate 5,6-endperoxide (Peak 2) as shown in Fig. 20.4B. Similar results as those for astaxanthin acetate were obtained in the case of free-form astaxanthin with these reactive oxygen species. Namely, astaxanthin epoxides were obtained by the reaction of hydroxyl radical and superoxide anion radical, and astaxanthin endperoxides

416 Chapter 20 Abs 450 nm

Peak 4

Peak 1 Peak 2

(A)

0

1

Peak 5

Peak 3

2

3

Abs 450 nm

4

6

7

8

9

10 min

7

8

9

10 min

Peak 4

Peak 1

Peak 5

Peak 2 Peak 3

(B)

5

Peak 6

0

1

2

3

4

5

Peak 6

6

Fig. 20.4 (A) HPLC of reaction products of astaxanthin acetate with hydroxyl radical for 6 min. Peak 1, astaxanthin acetate triepoxide; Peak 2, astaxanthin acetate 5,8-epoxide (or 7,8-epoxide); Peak 3, astaxanthin acetate 5,6-epoxide; Peak 4, astaxanthin acetate; Peaks 5 and 6: astaxanthin acetate cisisomers. (B) HPLC of reaction products of astaxanthin acetate with singlet oxygen for 6min. Peak 1, hematoporphyrin; Peak 2, astaxanthin acetate 5,6-endperoxide; Peak 3, astaxanthin acetate 5,8endperoxide; Peak 4, astaxanthin acetate; Peaks 5 and 6, astaxanthin acetate cis-isomers. HPLC condition; column, BEH Shield RP18 (1.7μm, 2.1100mm, waters); mobile phase, MeOH; column temperature, 40°C; flow rate, 0.2 mL/min; detection, 450 nm.

were produced by the reaction with singlet oxygen. It was found that oxidation products of astaxanthin acetate were more than those of free astaxanthin. It was assumed that acetylation might protect the oxidation of hydroxyl groups of astaxanthin from reactive oxygen species and inhibit the rapid degradation of the astaxanthin molecule. Structures of astaxanthin and its acetate and their major reaction products with reactive oxygen species are shown in Fig. 20.5 [11]. To reveal the reaction mechanism of astaxanthin and its acetate with these reactive oxygen species, these reactions were monitored with the ESR spin-trapping method using 5,5dimethyl-1-pyrroline-N-oxide (DMPO) [11]. Astaxanthin acetate peroxide radical (AsxOO•) and astaxanthin acetate oxide radical (AsxO•) were detected during the reaction of astaxanthin

Oxidation products of astaxanthin: An overview 417

Fig. 20.5 Structures of astaxanthin and its acetate and their major reaction products with reactive oxygen species.

acetate with superoxide anion radical (•O 2 ) by the ESR spin-trapping method, as shown in Fig. 20.6. Namely, a DMPO-OOAsx signal was observed in ESR for the 0–2.5 min reaction of astaxanthin acetate with •O 2 . The intensity of the DMPO-OOAsx signal reached it maximum with a 5 min reaction. Subsequently the DMPO-OOAsx signal was decreased, and DMPO-OAsx signal was observed for the 12.5–15.0 min reaction. These results indicate that astaxanthin acetate takes up •O 2 together with the formation of corresponding peroxy radical. Subsequently, astaxanthin acetate alkoxy radical (AsxO•) was formed with the production of •OH from peroxy radical. Eventually, epoxide was formed from alkoxy radical, as shown in Fig. 20.7. The ESR spectrum showed that astaxanthin (acetate) scavenged hydroxyl radical (•OH) directly. Although signals of alkoxy and/or peroxy radicals were not observed in ESR spin trapping during the reaction of astaxanthin (acetate) with •OH, the formation of epoxide by this reaction could be considered as shown in Fig. 20.7. Namely, an •OH radical was attached at C-5 of astaxanthin (acetate). Then an epoxide ring was formed by the intramolecular homolytic substitution reaction.

418 Chapter 20

Fig. 20.6 ESR spectra due to 8 μg/mL of astxanthin acetate in acetonitrile (100 μL), 0.025 mM of riboflavin solution (100μL), and 250mM of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (10μL). After 2.5min of UV-A irradiation (upper), after 7.5 min of UV-A irradiation (middle), after 15 min of UV-A irradiation (lower). ESR spectra due to both the DMPO-OOAsx (astaxanthin acetate-OO•) and DMPO-OAsx (astaxanthin acetate-O•) were observed. Initially, ESR signal of DMPO-OOAsx was dominant, and time-dependently ESR signal of DMPO-OAsx was increased. On the other hand, only ESR spectra due to both the DMPO-OOH and DMPO-OH were observed without astxanthin acetate, indicating the formation of •O2 followed by •OH in the control riboflavin/UV-A system.

Oxidation products of astaxanthin: An overview 419

Fig. 20.7 Reaction schemes of astaxanthin and its acetate with reactive oxygen species.

Peroxide radical (AsxOO•) and astaxanthin acetate oxide radical (AsxO•) were detected during the reaction of astaxanthin acetate with superoxide anion radical (•O2) by the ESR spin-trapping method, as shown in Fig. 20.6. Namely, a DMPO-OOAsx signal was observed in ESR for the 0–2.5 min reaction of astaxanthin acetate with •O2. The intensity of the DMPO-OOAsx signal reached it maximum with a 5-min reaction. Subsequently the DMPO-OOAsx signal was decreased and DMPO-OAsx signal was observed for the 12.5–15.0 min reaction. These results indicate that astaxanthin acetate takes up •O2 together with the formation of corresponding peroxy radical. Subsequently, astaxanthin acetate alkoxy radical (AsxO•) was formed with the production of •OH from peroxy radical. Eventually, epoxide was formed from alkoxy radical, as shown in Fig. 20.7. The ESR spectrum showed that astaxanthin (acetate) scavenged hydroxyl radical (•OH) directly. Although signals of alkoxy and/or peroxy radicals were not observed in ESR spin trapping during the reaction of astaxanthin (acetate) with •OH, the formation of epoxide by this reaction could be considered as shown in Fig. 20.7. Namely, an •OH radical was attached at C-5 of astaxanthin (acetate). Then an epoxide ring was formed by the intramolecular homolytic substitution reaction.

420 Chapter 20

4 Reaction products of astaxanthin with hypochlorous acid/hypochlorite (HOCl/OCl2) Weesepoel et al. [14] reported that palmitoyl epoxy apo-9-astaxanthinone and palmitoyl epoxy apo-13-astaxanthinone were formed from palmitoyl astaxanthin with reaction of hypochlorous acid/hypochlorite (HOCl/OCl). They reacted free astaxanthin and mono- and dipalmitate astaxanthin esters with hypochlorous acid/hypochlorite (HOCl/OCl) in a methanolic model system and analyzed degradation products by LC/PDA-ESI-MS system. They reported that apoastaxanthinals and apoastaxanthinones containing 3–10 conjugated double bonds were found upon autooxidation for all three types of astaxanthin (except free apo-8-astaxanthinal). Astaxanthin monopalmitate degradation resulted in a mixture of free and palmitate apoastaxanthins. HOCl/OCl rapidly converted the astaxanthins into a mixture of apo-9astaxanthinone epoxides and apo-13-astaxanthinone epoxides as shown in Fig. 20.8. The palmitate ester bond was hardly affected by autooxidation, whereas for HOCl/OCl the ester bond of the apoastaxanthin palmitoyl esters was degraded.

Fig. 20.8 Reaction products of astaxanthin monopalmitate with hypochlorous acid/hypochlorite.

Oxidation products of astaxanthin: An overview 421

5 Reaction products of astaxanthin with active nitrogen reactive nitrogen species Peroxynitrite (ONOO), which is one of the reactive nitrogen species, is formed from superoxide (O 2 ) and nitric oxide (NO•) and is a highly reactive oxidant that causes nitration of the aromatic ring of free tyrosine and protein tyrosine residues. Furthermore, peroxynitrite was found to induce various forms of oxidative damage such as LDL oxidation, lipid peroxidation, and DNA strand breakage [15]. In 1997, Kikugawa et al. reported that β-carotene was an effective scavenger of nitrogen dioxide (NO2), peroxynitrous acid (ONOOH), and peroxynitrite and that the nitrogen atoms derived from nitrogen dioxide were tightly bound to the β-carotene molecule [16]. Furthermore, Maoka et al. reported that paprika carotenoids such as capsanthin inhibited carcinogenesis initiated by peroxynitrite and nitric oxide [17]. These results suggested that carotenoids might be scavenged reactive nitrogen species. Therefore, Yoshikawa et al. [18] and Hayakawa et al. [19] investigated reaction of carotenoids with peroxynitrite by chemical model. Namely, astaxanthin was reacted with peroxynitrite, and the reaction products were analyzed by HPLC, as shown in Fig. 20.9. Apoastaxanthins (13-apoastaxanthinone, 120 -apoastaxanthinal, and 100 -apoastaxanthinal), nitroastaxanthins, and cis-astaxanthins were produced by this reaction. Novel-nitrated astaxanthins, named nitroastaxanthins, were obtained from this reaction as major products. They were determined to be 140 -s-cis-150 -nitroastaxanthin (1), 100 -s-cis110 -cis-110 -nitroastaxanthin (2), 9-cis-150 -s-cis-150 -nitroastaxanthin (3), 13-cis-14’s-cis150 -nitroastaxanthin (4), and 13, 15, 130 -tri-cis-150 -nitroastaxanthin (5) (Fig. 20.8) based on MS and NMR spectral data. Nitroastaxanthins obtained by this reaction showed cis configurations in the polyene chain. Nitro group is bulky functional group. To react with polyene chain, cis isomerization might be formed during reaction process of nitrogen group.

Fig. 20.9 HPLC of peroxynitrite reaction products of astaxanthin (HPLC conditions were shown in [7]).

422 Chapter 20 Furthermore, aponitroastaxanthins were also obtained as minor components. Their structures were determined as 120 -apo-150 -nitroastaxanthin (6) and 13-apo-8-nitroastaxanthinone (7) [19] (Fig. 20.9). They were assumed to be further oxidative cleavage product of nitroastaxanthins or nitration products of apoastaxanthins (Figs. 20.10 and 20.11). It was reported that phenolic compounds such as p-coumaric acid and pelargonidin could scavenge peroxynitrite by the formation of nitro-p-coumaric acid and nitropelargonidin, respectively, and protect against the nitration of tyrosine [20,21]. Our investigations also indicated that carotenoids could take up peroxynitrite through the formation of nitrocarotenoids. These results suggest that carotenoids might contribute to the prevention of cytotoxicity and genotoxicity by peroxynitrite in vivo. In fact, paprika carotenoid was shown to inhibit the carcinogenesis initiated by peroxynitrite and nitric oxide using a two-stage mouse carcinogenesis model [17]. Furthermore, it was reported that some carotenoids could inhibit the generation of superoxide or nitric oxide in cells [22]. Therefore, carotenoids might have the potential to reduce the risk of disease induced by reactive nitrogen species.

OH

NO2

O

HO O NO2

HO O

10'-s-11'-cis-11'-nitroastxanthin (2)

OH O

O OH

14'-s-cis-15'-nitroastxanthin (1)

OH

HO O

O

NO2

13-cis-14'-s-cis-15'-nitroastxanthin (4)

HO

O

O

NO2

HO O

NO2

9-cis-14'-s-cis-15'-nitroastxanthin (3)

13, 15,13'-tri-cis-15'-nitroastxanthin (5)

Fig. 20.10 Structures of nitroastaxanthins (1–5).

OH

Oxidation products of astaxanthin: An overview 423

Fig. 20.11 Structures of aponitroastaxanthins (6, 7a, 7b).

6 Apoastaxanthins as impurity of synthetic astaxanthin Rao et al. analyzed impurity of synthetic samples of astaxanthin by HPLC [23]. They found a series of aposataxanthins. They were also assumed to be an oxidative degradation products of C40 astaxanthin.

7 Conclusion It was reported that the mechanism whereby carotenoids such as astaxanthin scavenge singlet oxygen was a physical reaction [24]. Namely, carotenoids take up thermal energy from singlet oxygen and release this energy by polyene vibration. Furthermore, resent studies revealed that carotenoids also scavenged reactive oxygen species by chemical reactions [25–28]. In this chapter, I described oxidation products of astaxanthins as reaction products of astaxanthin with reactive oxygen and nitrogen species. A series of apoastaxanthin was obtained as out-oxidation products of astaxanthin. Astaxanthin endperoxides and epoxides were identified as reaction products of astaxanthin with singlet oxygen and superoxide anion and hydroxyl radicals. Nitro-astaxanthins were found by reaction products of astaxanthin peroxynitrite. These results indicate that astaxanthin is able to capture reactive oxygen and nitrogen species its polyene chain.

424 Chapter 20

References [1] Wahlberg I, Eklund A-M. Degraded carotenoids. In: Britton G, Liaaen-Jensen S, Pfander H, editors. Carotenoids: biosynthesis and metabolism. vol. 3. Basel, Switzerland: Birkh€auser; 1998. p. 195–216. [2] Schiedt K. Absorption and metabolism of carotenoids in birds, fish and crustaceans. In: Britton G, Liaaen-Jensen S, Pfander H, editors. Carotenoids: biosynthesis and metabolism. vol. 3. Basel, Switzerland: Birkh€auser; 1998. p. 285–358. [3] Tanaka Y, Yamada S, Sameshima M. Novel apocarotenoid apoastacenal isolated from nudibranch eggmasses. Nippon Suisan Gakkaishi 1992;58:1549. [4] Britton G, Liaaen-Jensen S, Pfander S. Carotenoids: isolation and analysis. vol. 1. Basel, Switzerland: Birkh€auser; 1995. [5] Cabelloa LO, Mendezb HIP, Alvarezb NM, Manjarrezc LAM, Herna´ndezb JC, Cano ETQ, Lunab AL. Characterization and antioxidant activity of carotenoid mixtures present in Rhodococcus sp. and Gordonia sp. J Chem Pharm Res 2016;8:879–88. [6] Wolz E, Lichti H, Notter B, Oesterhet G, Kister A. Characterization of metabolites of astaxanthin in primarily cultures of rat hepatocytes. Drug Metab Dispos 1999;27:456–62. [7] Kistler A, Liecht H, Pichard L, Wolz E, Oosterhelt G, Hayes A, Maurel P. Metanolism and CYP-induce properties of astaxanthin in man and primary human hepatocytes. Arch Toxicol 2002;75:665–75. [8] Etoh H, Suhara M, Tokuyama S, Kato H, Nakahigashi R, Maejima Y, Ishikura M, Terada Y, Maoka T. Auto-oxidation products of astaxanthin. J Oleo Sci 2012;61:17–21. [9] Hama S, Uenishi S, Yamada A, Ohgita T, Tsuchiya H, Yamashita E, Kogure K. Scavenging of hydroxyl radicals in aqueous solution by astaxanthin encapsulated in liposomes. Biol Pharm Bull 2012;35:2238–42. [10] Murata K, Oyagi A, Takahira D, Tsuruma K, Shimazawa M, Ishibashi T, Hara H. Protective effects of astaxanthin from Paracoccus carotinifaciens on murine gastric ulcer models. Phytother Res 2012;26:1126–32. [11] Nishino A, Maoka T, Yasui H. Analysis of reaction products of astaxanthin and its acetate with reactive oxygen species using LC/PDA ESI-MS and ESR spectrometry. Tetrahedron Lett 2016;57:1967–70. [12] Nishino A, Yasui H, Maok T. Reaction and scavenging mechanism of β-carotene and zeaxanthin with reactive oxygen species. J Oleo Sci 2017;66:77–84. [13] Nishino A, Yasui H, Maoka T. Reaction of paprika carotenoids capsanthin and capsorubin with reactive oxygen species. J Agric Food Chem 2016;64:4786–92. [14] Weesepoel Y, Gruppen H, de Bruijn W, Vincken J-P. Analysis of palmitoyl Apo-astaxanthinals, Apo-astaxanthinones, and their epoxides by UHPLC-PDA-ESI-MS. J Agric Food Chem 2014;62:10254–63. [15] Maoka T, Etoh H. Shi J, editor. Biological antioxidation mechanism: quenching of peroxynitrite, in Japanese food, in functional food ingredicents and nutraceuticals processing technologies. 2nd ed. Boca Raton: CRC Press; 2016. p. 589–607. [16] Kikugawa K, Hiramoto K, Tomiyama S, Asano Y. ß-Carotene effectively scavenges toxic nitrogen oxides: nitrogen dioxide and peroxynitrous acid. FEBS Lett 1997;404:175–8. [17] Maoka T, Mochida K, Kozuka M, Enjo F, Kuchide M, Nobukuni Y, Tokuda H, Nishino H. Chemopreventive activity of paprika extract and capasnthin on nitric oxide or peroxynitrite induced carcinogenesis (in Japanese). Food Clin Nutr 2006;1:7–14. [18] Yoshioka R, Hayakawa T, Ishizuka K, Kulkarni A, Terada Y, Moaka T, Etoh H. Nitration reaction of astxanthin and β-carotene by peroxynitrite. Tetrahedron Lett 2006;47:3637–40. [19] Hyayakawa T, Kulkarni A, Terada Y, Maoka T, Etoh H. Reaction of astaxanthin with peroxynitrite. Biosci Biotechnol Biochem 2008;72:2716–22. [20] Goaa SP, Hogg N, Kalyanaraman B. The effect of α-tocopherol on the nitration of γ-tocopherol by peroxynitrite. Arch Biochem Biophys 1990;363:333–40. [21] Mortion LW, Ward NC, Crof KD, Puddey IB. Evidence for the nitroration of γ-tocopherol in vitro; 5-nitroγ-tocopherol is elevated in the plasma of subjects with coronary heart disease. Biochem J 2002;364:625–8. [22] Halliwell B. Free radicals, antioxidants, and human disease: curiosity, cause or consequence? Lancet 1994;344:721–4.

Oxidation products of astaxanthin: An overview 425 [23] Rao RN, Alvi SN, Rao BN. Preparative isolation and characterization of some minor impurities of astaxanthin by high-performance liquid chromatography. J Chromatogr A 2005;1076:189–92. [24] Foote CS, Denny RW. Chemistry of singlet oxygen. VII quenching by β-carotene. J Am Chem Soc 1968;90:6233–5. [25] Yamauchi R, Tsuchihashi K, Kato K. Oxidation product of β-carotene during the peroxidation of methyl linolate in the bulk phase. Biosci Biotechnol Biochem 1998;62:1301–6. [26] Martin HD, Ruck C, Schmidt M, Sell S, Beutner S, Mayer B, Walsh R. Chemistry of carotenoid oxidation and free radical reactions. Pure Appl Chem 1999;71:2253–62. [27] Fiedor J, Fiedor L, Haeβner R, Scheer H. Cyclic endoperoxides of β-carotene, potential pro-oxidants, as products of chemical quenching of singlet oxygen. Biochim Biophys Acta Bioenerg 2005;1709:1–4. [28] Mordi RC, Walton JC, Burton GW, Hughes L, Keith IU, David LA, Douglas MJ. Oxidative degradation of beta-carotene and brta-apo-80 -carotenal. Tetrahedron 1993;49:911–28.

This page intentionally left blank

CHAPTER 21

Anticancer properties of astaxanthin: A molecule of great promise Pinar Buket Demirela and Bilge Guvenc Tunab a

Department of Medical Biology and Genetics, Faculty of Medicine, Maltepe University, Istanbul, Turkey Department of Biophysics, Faculty of Medicine, Yeditepe University, Istanbul, Turkey

b

Abbreviations 8-OHdG 8-oxoG Akt APCs ARE ATX CAT ECM EMT eNOS Erk GPx GR IL-1α IL-2 INF-γ JAK/STAT-3 MAPK MMPs ncRNAs NF-κB NK NOXs NRF2 PI3K Prxs ROS SOD TAA TNFα USFDA

8-hydroxy-20 -deoxyguanosine 7,8-dihydro-8-oxoguanine protein kinase B antigen presenting cells antioxidant response element astaxanthin catalase extracellular matrix epithelial mesenchymal transition nitric oxide synthase extracellular-signal-regulated kinase glutathione peroxidase glutathione reductase interlukin-1α interleukin-2 interferon-γ signal transducers and activators of transcription 3 mitogen-activated protein kinases matrix metalloproteinases noncoding RNAs nuclear factor kappaB natural killer NADPH oxidases nuclear factor erythroid 2-related factor 2 phosphoinositide 3-kinase peroxiredoxins reactive oxygen species superoxide dismutase tumor-associated antigen tumor-necrosis factor-α US Food and Drug Administration

Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00003-9 Copyright # 2021 Elsevier Inc. All rights reserved.

427

428 Chapter 21

1 Introduction Astaxanthin (ATX) is a fat-soluble xanthophyll carotenoid, which naturally occurs in a variety of microorganisms (i.e., marine bacteria, yeast, and microalgae), marine animals (i.e., shrimp, salmon, lobster, and clamp), and birds (i.e., flamingos and quail) [1]. ATX cannot be biosynthesized by mammals including humans and it must be obtained from the diet. It is approved by the US Food and Drug Administration (USFDA) and European Commission as a color additive for animal and fish feed and a dietary supplement [2]. Carotenoids are among the most powerful natural antioxidants. Although ATX exhibits very similar metabolic and physiological functions associated with carotenoids such as lutein, lycopene, zeaxanthin, canthaxantin, and α- and β-carotene, it has a significantly higher antioxidant property relative to other carotenoids [3]. ATX possesses a significant capacity for singlet oxygen quenching and scavenging of free radicals such as superoxide anion and hydrogen peroxide [4,5]. A strong indirect antioxidant activity of ATX through inducing the transcription of nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor that regulates the expression of hundreds of antioxidant genes containing an enhancer sequence called the antioxidant response element (ARE) has also been reported [6,7]. The strong antioxidant activity of ATX is mainly attributed to its unique molecular structure. Specifically, the presence of both keto and hydroxyl groups in each terminal ion rings provides a higher antioxidant capacity and a more polar structure to ATX compared with other carotenoids [8]. Its polar structure enables ATX to span the membrane bilayer: Its polar terminal rings extend toward the bilayer, while the polyene chain is embedded in the interior of the membrane. Therefore free radical quenching and scavenging effects of ATX occur in both layers of the cell membrane leading to a significantly higher antioxidant property relative to carotenoids that function either only in the inner or the outer cell membrane [9]. There is a well-established link between dietary intake and overall cancer risk. Diet ranks the highest as a risk factor for cancer-related death among other known risk factors including smoking, pollution, occupation, and infection [10]. On the other hand, regular consumption of vegetables and fruits is correlated with a reduced overall cancer risk in humans [11]. Since many fruits and vegetables are rich in carotenoids, many natural carotenoids have been extensively evaluated for their anticancer activities and reported to exert anticancer effects in vivo and in vitro [12]. Accumulating evidence suggests that, as a dietary carotenoid, ATX possesses an effective antitumoral activity in a variety of animal carcinogenesis models and human cancer cell lines. Dietary ATX exposure was reported to protect against chemically induced oral [13], skin [14], and colon [15] cancer in rat models. Colitis-associated [16] and chemically induced colon [17] and urinary bladder [18] carcinogenesis were also inhibited in ATX fed mice. Additionally, intragastric administration of ATX significantly suppressed tumor growth in PC-3 xenograft nude mice [19]. In a hamster oral squamous cell carcinoma model, dietary supplementation of ATX inhibited tumor growth [20]. Cancer preventive effects of

Anticancer properties of astaxanthin: A molecule of great promise

429

ATX have also been noted in a variety of cancer cell lines including breast [21], colon [22], gastric [23], colorectal [24], hepatic [25], and melanoma [26] (Table 21.1). This chapter reviews the potential mechanisms of cancer preventive effects of ATX, outlines its inhibitory effects on the processes involved in malignant transformation, and highlights its use in combination with anticancer drugs during chemotherapy.

2 Cancer preventive mechanisms of ATX Preventive effects of ATX on cancer may be mediated by its antioxidant, immunomodulatory, and antiinflammatory activities (Fig. 21.1).

2.1 Antioxidant activity Antioxidants protect cells from the damaging effects of reactive oxygen species (ROS) via preventing oxidation either by directly scavenging ROS, inhibiting ROS generation or removing/repairing the ROS-induced damage [46]. Antioxidant defense mechanisms are composed of both enzymatic and nonenzymatic antioxidants. Enzymatic antioxidants break down and remove free radicals. For example, superoxide dismutase (SOD) has an important role of converting superoxide anion to hydrogen peroxide and molecular oxygen in the first line of defense. Hydrogen peroxide is neutralized by multiple other enzymatic antioxidants such as catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and peroxiredoxins (Prxs). Carotenoids are among nonenzymatic antioxidants, and they impede free radical chain reactions by interacting with ROS [47]. ROS are oxygen containing small, reactive chemical molecule species and include free oxygen radicals and nonradical ROS. Free oxygen radicals, such as superoxide anion, hydroxyl radical, and nitric oxide, contain one or more unpaired electron(s) in their outer shell; therefore they are highly reactive. Nonradical ROS, including singlet oxygen and hydrogen peroxide, do not contain any unpaired electrons [48]. ROS are endogenously generated as natural by-products of aerobic metabolic reactions carried out mainly in mitochondria, peroxisomes, and endoplasmic reticulum. Since approximately 2% of the oxygen consumed by the mitochondria is converted to superoxide anion, mitochondria are considered to be the major intrinsic source of ROS production [49]. Aside from the nonenzymatic sources, several enzymatic reactions involving membrane-bound NADPH oxidases (NOXs), cytochrome P450 enzymes, and nitric oxide synthase (eNOS) also generate ROS constantly [50]. Accumulated evidence suggests that at low to moderate concentrations, ROS function as important second messengers participating in a variety of signaling cascades associated with cell growth, proliferation, differentiation, and survival [51,52]. However, intracellular ROS levels higher than the physiological concentration disrupt the redox homeostasis and cause oxidative damage to important cellular components such as DNA, proteins, and lipids.

430 Chapter 21 Table 21.1 Anticancer activity of ATX. In vivo/ in vitro

Intervention

Colorectal cancer

LS-180

50–150 μM

Colorectal cancer and colon cancer

HCT116, CT26 Nude mice

50 μM

Oral cancer

SCC131, SCC4 Hamster

400 μM, 15 mg/kg

Ovarian cancer

Drugresistant SKOV3

Lung cancer

A549, H1975

Esophageal cancer

Rat (male F344)

Breast cancer

MCF-7, MCF10A, MDA-MB231 A375, A2058 Xenograft model

Cancer type

Malignant melanoma

Molecular targets

PBK/Akt, NF-κB, STAT3

NF-κB, MAPK

20 μM + erlotinib

MAPK

NFkB, COX2

10–50 μM

5–125 μg/mL; 25 mg/kg

Outcomes

References

Decreased proliferation; increased apoptosis; upregulation of Bax, caspase-3; downregulation of Bcl-2; decreased MDA; increased SOD, catalase and glutathione peroxidase Decreased EMT; upregulation of miR-29a3p, miR-200a; downregulation of MMP2, ZEB1 Decreased proliferation invasion and angiogenesis; increased apoptosis; downregulation of cyclin D1, p21, Bcl-2; upregulation of Bax, caspase-3, -9, MMP2, MMP9, VEGF Decreased proliferation and migration; increased apoptosis; upregulation of p53, caspase-3; increased Bcl-2/Bax ratio Decreased proliferation; upregulation of p53, p-MKK3/6 and p-p38 MAPK Suppressed oxidative stress; decreased MDA; increased SOD and glutathione peroxidase Decreased proliferation; suppressed cell migration; non-toxic to normal breast cancer cells Decreased proliferation; decreased tumor size; increased apoptosis; suppressed metastasis; decreased ROS; downregulation of MMP 1, 2, 9; non-toxic to normal human epidermal and dermal cells

[24]

[27]

[20]

[28]

[29]

[30]

[21]

[26]

Anticancer properties of astaxanthin: A molecule of great promise Table 21.1 Cancer type

In vivo/ in vitro

431

Anticancer activity of ATX—cont’d

Intervention

Prostate cancer

PC-3 prostate xenograft nude mice

100 mg/kg

Squamous carcinoma

VX2, RAW264.7 Rabbit

10–300 μg/mL

Esophageal squamous cell carcinoma

ECA109, TE13

1–500 μmol/L + irradiation

Breast cancer

MCF-7

0–50 μM

Pancreas cancer

GR-HPCCs Xenograph mice

0–300 μM + gemcitabine 500 mg/kg + gemcitabine

Human nonsmall cell lung cancer

A549, H1703

20 μM + mitomycin C

Molecular targets

Outcomes

References

Inhibited tumor growth; increased apoptosis; decreased Ki67, PCNA; upregulation of cleaved caspase-3, miR-375 and miR-487b; increased amount of Lactobacillus sp. and Lachnospiraceae in mice stools Cytotoxic on squamous carcinoma cells, non-toxic to nonmalignant macrophages Decreased proliferation; increased apoptosis; enhanced radiosensitivity of ESCC cells; cell cycle arrest at G2/M; upregulation of Bax; downregulation of Bcl2, CyclinB1, Cdc2 Decreased proliferation; increased apoptosis; cell cycle arrest at G0/G1; upregulation of Bax, p53; downregulation of Bcl-2, Cyclin D1; decreased MDA, increased glutathione peroxidase Decreased proliferation of gemcitabine-resistant cells; inhibited tumor growth; increased gemcitabine sensitivity; increased EMT; downregulation of ribonucleoside diphosphate reductase (RRM) 1, RRM2, TWIST1, ZEB1; upregulation of human equilibrative nucleoside transporter 1 Decreased proliferation; inactivation of Akt; downregulation of Rad51, phospho-Akt

[19]

[31]

[32]

[33]

[34]

[35]

Continued

432 Chapter 21 Table 21.1 Cancer type

In vivo/ in vitro

Anticancer activity of ATX—cont’d

Intervention

Lung cancer

A549

Hepatocellular carcinoma

LM3, SMMC7721

Oral cancer

Hamster

Oral cancer

Hamster

15 mg/kg

Skin cancer Hepatocellular carcinoma

Rat CBRH7919

200 μg/kg

Esophageal cancer

TE-1, TE-4

6–10 μg/mL, 6–10 μg/mL + α-tocopherol

Hepatocellular carcinoma

CBRH7919, SHZ-88, HL-7702

39 μM

Breast cancer

WAZ-2T (-SA) inoculated mice

100–300 μM

Molecular targets

Outcomes

References

JAK/STAT

Decreased proliferation; increased apoptosis; cell cycle arrest at G0/G1; upregulation of Bax; downregulation of Bcl-2

[36]

PI3K/Akt, MAPK/ERK NF-κB, Wnt/ β-catenin

Decreased proliferation; increased apoptosis; upregulation of Bax/Bcl-2

[37]

Decreased proliferation invasion and angiogenesis Decreased proliferation; increased apoptosis; downregulation of Bcl-2, p-Bad, surviving; upregulation of Bax, caspase, Bad; release of Smac/Diablo, cytochromec into; induced cleavage of poly (ADP-ribose) polymerase (PARP). Reduced tumor incidence Increased apoptosis; upregulation of Bax, nm231; downregulation of Bcl-2, Bcl-xl, c-myc; downregulation of JAK1 and STAT3 ATX alone decreased proliferation; increased apoptosis; upregulation of p27, cleaved caspase-3, p-p38; downregulation of p-Akt ATX+ α-tocopherol decreased cyclin D1 but did not enhanced induction of apoptosis Decreased proliferation; increased apoptosis; nontoxic to normal human hepatocytes; upregulation of Bax; downregulation of Bcl-2 Delayed tumor growth; reduced plasma IL-1α

[38]

PI3K/Akt, NF-κB, Wnt/βcatenin, Erk/ MAPK

JAK/STAT

PI3K/Akt, MAPK

[39]

[14] [40]

[41]

[42]

[43]

Anticancer properties of astaxanthin: A molecule of great promise Table 21.1 Cancer type

In vivo/ in vitro

Anticancer activity of ATX—cont’d

Intervention

Hepatocellular carcinoma

Rat

25 mg/kg + cyclophospha mide

Colon cancer

HCT-116

15–25 μ/mL

Fibrosarcoma

Meth-A tumor cell inoculated mice, mice TDLN and spleen cells Rat (male F344)

40 mg/kg

Oral cancer

433

Molecular targets

JNK, Akt, ERK1/2

Outcomes

References

Decreased cyclophosphamideinduced oxidative stress and DNA damage; decreased p53, p38; increased Nrf2, phase-II enzymes (NQO-1, HO-1)

[44]

Decreased proliferation; increased apoptosis; upregulation of p-JNK, p-38, p-Akt, p-ERK1/2, p53, p21, p27, Bax/Bcl-2 ratio and Bcl-XL; downregulation of cyclin D1 Inhibited tumor growth; upregulation of cytotoxic T lymphocyte activity and IFN-γ

[22]

Decreased proliferation; decreased preneoplastic lesions and neoplasms in the oral cavity

[13]

[45]

Therefore modulating intracellular ROS levels by maintaining the balance between ROS generation and detoxification by the antioxidant defense mechanisms is critical for preserving cellular homeostasis. Elevated intracellular ROS levels are a hallmark of cancer cells and are closely related to malignant transformation by contributing to proliferation and metastasis of cancer cells and angiogenesis [53]. Oxidative damage to DNA is considered as an important trigger for carcinogenesis as many types of oxidative DNA lesions have been indicated to have intrinsic potential for mutagenesis. For instance, one of the most abundant oxidative DNA lesions, 7,8-dihydro-8-oxoguanine (8-oxoG) [54], leads to misinsertion of adenine residues opposite 8-oxoG resulting in aG/C to T/A transversion if not corrected [55]. ATX may reduce carcinogenesis through suppression of the ROS-mediated oxidative damage. Consistently, dietary ATX significantly reduces a predominant form of oxidative DNA lesion 8-hydroxy-20 -deoxyguanosine (8-OHdG) in young healthy adult female human subjects [56]. UVA-mediated DNA damage in rat trachea epithelial cells is also significantly reduced by ATX treatment in a dose-dependent manner [57]. Moreover, ATX exposure significantly decreases the frequency of chromosomal

434 Chapter 21

Fig. 21.1 Mechanisms of cancer prevention and inhibition of tumor progression by ATX.

aberrations induced by gamma radiation in human peripheral blood lymphocytes regardless of the cell cycle phase at which gamma radiation was applied [58].

2.2 Immunomodulatory activity Another important common biological function of carotenoids is modulation of the immune response. Immune system protects the host from infectious agents such as viruses and bacteria via cell-mediated and humoral immune responses. Cell-mediated immune response is mediated by several types of immune system cells, such as antigen-presenting cells (APCs), helper T cells, cytotoxic T cells, and natural killer (NK) cells, while antibodies are the mediators of the humoral immune response. Immune system recognizes infectious agents by the “nonself” antigens on their cell surfaces, which mark them as “foreign.” APCs of the immune system process and deliver the foreign antigen to certain lymphocytes for recognition. Several tumor-associated antigens (TAA) that are present on the surface of tumor cells, but not present at all or present at very low levels on the surface of normal cells, have been identified. Dendritic cells, a major group of migratory APCs, monitor the environment, take up and process TAAs, and present them to T cells [59] providing tumor immunity [60]. There is a line of evidence suggesting that the proper immune function is critical in providing protection against cancer cells. For instance, primary [61] and HIV-induced [62] immunedeficiencies and immunosuppression in organ transplant patients [63] have been reported to be associated with an increased risk of cancer development. Immune system recognition of the cancer cell-specific mutated proteins has also been revealed [63]. Increasing evidence reveals that carotenoids augment immune activity, which is critical in fighting against cancer cells. Immunomodulatory effects of ATX are reported to be higher when compared with the effect of β-carotene [64]. Enhancing effects of ATX on cell-mediated

Anticancer properties of astaxanthin: A molecule of great promise

435

immune response has been demonstrated both in vitro and in vivo. Activity and proliferation of different cell types involved in cellular immune response are stimulated by ATX treatment. Activity of cytotoxic T cells is increased upon dietary ATX intake in mice inoculated with Meth-A tumor cells [45]. Besides, stress-induced inhibition of the antitumor activity of NK cells is suppressed in ATX-fed mice [65]. Consistently, the cytotoxic activity of NK cells is enhanced by ATX treatment in cell culture [66]. Proliferative effects of ATX on mouse thymocytes [66] and lymphocytes [67] as well as murine spleen cells have also been reported [68]. In addition to its positive influence on the proliferation and activity of the immune system cells, ATX treatment has also been shown to boost the production of cytokines in these cells both in vitro and in vivo. Cytokines are small proteins essential for the immune function due to their critical function of mediating the communication between different immune responses. Oral administration of ATX leads to increased interferon-γ (INF-γ) production in mice inoculated with Meth-A tumor cells in vivo [45] and lymphocytes of mice ex vivo [67]. In line with these findings, increased plasma levels of INF-γ have been detected in humans upon ATX supplementation [56]. In addition to INF-γ, ATX treatment significantly enhanced the release of tumor-necrosis factor-α (TNFα) and interlukin-1α (IL-1 α) in murine peritoneal adherent cells [68] and production of interleukin-2 (IL-2) in mice lymphocytes ex vivo [67]. ATX exhibits significant immunomodulating activity in humoral immune response as well. Humoral immune response is mediated by antibodies or immunoglobulins, which recognize, bind, and inactive foreign antigens including TAA. ATX exposure significantly enhances in vitro polyclonal antibody production (immunoglobulin M and G) in murine spleen cells [68]. In another in vitro culture system, Jyonouchi et al. [64] showed that ATX treatment significantly increases antibody production to antigenic stimulation by sheep red blood cells without stimulating lymphocyte proliferation or IL-2/IL-4 production. The same group later reported the ability of ATX to preferentially stimulate production of specific antibodies against T cell-dependent antigen in vitro [69] and in vivo [70]. Moreover, the enhancing effect of ATX supplementation on in vivo antibody production against T cell-dependent antigens partially restored humoral immune response in old mice [70].

2.3 Antiinflammatory activity Inflammation is a normal immune response to injuries or infections. Inflammatory response stimulates the immune system to repair the damaged tissue at the site of the injury or resolve the infection and to initiate the healing process. Failure in the resolution of the acute inflammation due to the persistence of the cause may lead to chronic inflammation, which is associated with a number of diseases including cancer [71]. About 20% of all cancers have been reported to be linked to chronic inflammation [72], which is a well-established stimulatory factor for tumor initiation, progression, and metastasis [73]. ATX has been demonstrated to turn off the expression of proinflammatory genes, which may directly contribute to cancer progression [71]

436 Chapter 21 by inhibiting the activation of the nuclear factor kappa B (NF-κB) in a lipopolysaccharide-induced murine macrophage cell (RAW264.7) and in primary culture of mouse peritoneal macrophages [10]. In line with these data, ATX exhibits antiinflammatory effects by suppressing the production of nitric oxide, prostaglandin E2, and tumor necrosis factor-alpha in endotoxin-induced uveitis in rats in vivo and in lipopolysaccharide-induced RAW264.7 cells in vitro [74]. ATX feeding also suppresses inflammation-associated colon carcinogenesis by reducing NF-κB and inflammatory cytokine expression in mice [16].

3 Effects of ATX on tumor progression Tumor progression is characterized by increased tumor aggressiveness associated with invasion and migration. Various signaling cascades controlling cell proliferation and apoptosis as well as cross talk between these cascades are deregulated during tumor progression. A line of evidence reveal that carotenoids inhibit tumor progression depending on the cell type, concentration of the carotenoid applied, and the redox level of the target cell [75]. Several recent studies demonstrated the inhibitory effect of ATX treatment on tumor progression in various cancer types including oral [20,39], ovarian [28], colon [24,27], lung [29], breast [33], pancreatic [34], prostate [19], and esophagal [30]. To explain the inhibitory effect of ATX treatment on tumor progression, pathways that are modulated by ATX and molecular targets of ATX related to cell proliferation, apoptosis, invasion, and metastasis were investigated. The inhibitory effect of ATX treatment on tumor progression has been shown to be controlled by the inhibition of NF-κB, Wnt/β-catenin, signal transducers, and activators of transcription 3 (JAK/STAT-3) and phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) or mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (Erk) signaling pathways (Fig. 21.1). Signaling pathways modulated by ATX are interlinked and activated commonly in cancer cells. For example, the cross talk between PI3K/Akt, NF-κB, and STAT-3 is critical for regulating cancer cell proliferation, and inhibition of any of these proteins results in inhibition of cell proliferation. Additionally, NF-κB has also been shown to be interlinked with Wnt/β-catenin pathway in suppressing proliferation of hepatocarcinoma cells. Information transfer among these signaling pathways is also crucial for the regulation of cancer cell apoptosis. For instance, PI3K/Akt and MAPK/Erk signaling pathways both regulate apoptosis through Bcl-2 family proteins [75].

3.1 Proliferation ATX treatment inhibits proliferation of oral cancer cell lines (SCC131 and SCC4) though inhibition of PI3K/Akt signaling, which is associated with NF-κB and JAK/STAT signaling pathways. PI3K/Akt pathway regulates STAT-3 phosphorylation leading to downregulation of cyclin D1 and proliferating cell nuclear antigen (PCNA) (p21) and nuclear translocation of

Anticancer properties of astaxanthin: A molecule of great promise

437

NF-κBp65. Additionally, noncoding RNAs (ncRNAs) influencing PI3K/Akt signaling such as miR-21 and HOTAIR were shown to be downregulated as a result of ATX treatment [20]. Treatment of ATX in combination with human serum albumin inhibits proliferation of drug-resistant human ovarian cancer cells (SKOV3) via inactivation of NF-κB, p53, and MAPK signaling pathways [28]. Chen et al. showed that ATX treatment also inhibits proliferation of human lung adenocarcinoma cell lines (A549, H1975) through inhibition of p38 MAPK, which negatively regulates p53 [29].

3.2 Apoptosis Apoptosis, an active and genetically regulated mode of cell death, is critical in maintaining tissue homeostasis and disruption of tissue homeostasis is closely related to tumorigenesis. Apoptotic cell death balances cell proliferation and thus prevents abnormal accumulation of cells, which defines cancer. Apoptosis also eliminates damaged/mutated cells with a potential of becoming cancerous. Therefore, it is not surprising that evasion of apoptosis is a main driving force for malignant transformation [76]. Owing to the fact that apoptosis evasion is a hallmark of cancer cells independent of the cause or the origin of the cancer and it does not induce inflammation, targeting apoptosis provides a successful treatment of cancer that is effective in all cancer types while causing a minimal damage to the tissue. Major mediators of apoptosis are a family of cysteine proteases called caspases. Two main pathways of apoptosis that activate caspases are the intrinsic (mitochondrial) and extrinsic (death receptor) pathways. Cell death in mammals is mainly mediated by the intrinsic pathway of apoptosis in response to a plethora of signals including DNA damage and oxidative stress [77]. Major regulators of the intrinsic pathway of apoptosis is the Bcl-2 protein family composed of both proapoptotic (e.g., Bax) and antiapoptotic (e.g., Bcl-2) proteins, all of which are located on the outer membrane of the mitochondria, where they mainly function [78]. Stimulation of the intrinsic pathway of apoptosis due to ATX treatment has been shown in various cancer cell types by analyzing the expression of pro- and antiapoptotic members of the Bcl2 family, caspases, and cytochrome c release. ATX was found to induce apoptosis by downregulation of Bcl-2 and upregulation of Bax in rat hepatocarcinoma (CBRH-7919) [42], human breast cancer (MCF-7) [33], esophageal squamous cell carcinoma (ECA109 and TE13) [32], colorectal cancer (LS-180) [24], and malignant melanoma (A375, A2058) [26] cells. Apoptosis-inducing effect of ATX in oral cancer cells was accompanied by downregulation of the PI3K/Akt activity [20]. In line with these data, ATX increases apoptosis via PI3K/Akt downregulation in human leukemic monocyte lymphoma (U937) [79] and esophageal cancer (TE-1, TE-4) [41] as well. Apoptosis of drug-resistant ovarian carcinoma (SKOV3) cells is induced upon ATX treatment through modulation of NF-κB and MAPK pathways [28]. ATX induces apoptosis of human lung cancer (A549) [36] and rat

438 Chapter 21 hepatocarcinoma (CBRH-7919) cells, as revealed by upregulation of Bax and downregulation of Bcl-2, via inactivation of JAK/STAT [40]. Apoptosis-inducing effect of ATX on cancer cells has also been reported in various rodent carcinogenesis models in vivo. Kavitha et al. showed that dietary intake of ATX induced intrinsic apoptotic pathway, revealed by inactivation of Bcl-2, activation of Bax, and cytochrome c release, through suppression of MAPK/Erk and PI3K/Akt signaling in a hamster oral carcinoma model [39]. Apoptosis of colon cancer cells is also stimulated by ATX, as the number of apoptotic cells and caspase-3 expression was increased in ATX fed in rats with DMH-induced colon carcinogenesis [15]. Similarly, dietary ATX treatment has been found to increase the ratio of apoptotic cells and caspase-3 expression in a PC-3 prostate xenograft nude mice model [19].

3.3 Invasion and metastasis Invasion and metastasis are two key processes involved in tumor progression [80]. Metastasis, a major cause of cancer lethality, is the process during which primary tumor cells invade adjacent tissues and migrate to distant sites where they are colonized into secondary tumors [81]. Invasion and metastasis of cancer cells require them to undergo several changes in cell structure to become motile and cross several physical barriers including extracellular matrix (ECM). Degradation of the ECM is mainly mediated by a family of zinc-binding endopeptidases known as the matrix metalloproteinases (MMPs) [82]. Additionally, cancer cells undergo several morphological alterations losing epithelial features and achieving a motile mesenchymal phenotype via a process called epithelial-mesenchymal transition (EMT) enabling their migration from the primary site to distant secondary sites. It is now well established that elevated ROS levels in cancer cells play a pivotal role in their malignant transformation by actively participating in both EMT and degradation of the ECM [83]. Consistently, ROS can regulate the activity and expression of MMPs directly or indirectly by modulating the suppression of the endogenous inhibitors of MPPs. In this context, ATX treatment has been reported to inhibit expressions of MMP-2 and MMP-9 by inhibiting PI3K, NF-κB, and STAT-3 in oral cancer cells (SCC131 and SCC4) as well as in a hamster oral carcinogenesis model, indicating an antiinvasive activity for ATX both in vitro and in vivo [20,38]. ATX was also found to reduce invasion and migration of human malignant melanoma cells (A2058 and A375), which was accompanied by decreased expressions of MMP-1, MMP2, and MMP-9 [26]. Consistently, Su et al. [28] reported that ATX treatment inhibited migration of drug-resistant ovarian carcinoma cells (SKOV3) through NF-κB inactivation. Antimigratory and antiinvasive effects of ATX have also been noted in in vitro and in vivo models of metastatic colon cancer. ATX exposure suppressed the activity of MPP-2 through upregulation of miR-29a-3p and inhibited the expression of ZEB1, a critical activator of EMT, through enhancement of miR-200a in metastatic colon cancer cells (CT26, HCT116) and in a mouse model of metastatic colon carcinoma [27]. A significant decrease in the expressions of MMP-2

Anticancer properties of astaxanthin: A molecule of great promise

439

and MMP-9 upon dietary ATX intake has also been reported in a rat colon carcinogenesis model [15]. Consistent with these data, ATX is found to reduce migration of human breast cancer cells (MCF7 and MDA-MB-231) compared with normal breast epithelial cells (MCF10A) [21].

4 Combination cancer treatment with ATX ROS is an important second messenger at physiological concentrations modulating signaling cascades associated with cell growth, proliferation, differentiation, and cell death [51,52]. Intracellular ROS levels higher than the physiological concentration cause DNA damage, which is a key step involved in mutagenesis leading to cancer. Elevated ROS levels, a hallmark of cancer cells, are known to promote malignant transformation. There is a positive feedback between the processes involved in malignant transformation and further increase of intracellular ROS levels, which increase aggressiveness of cancer cells. On the other hand, excessively higher levels of ROS can activate cell cycle checkpoints and stimulate apoptosis [49]. Many anticancer drugs are known to induce apoptosis in cancer cells by increasing intracellular ROS production. For instance, erlotinib [84], mitomycin C [85], and paclitaxel [86], which are successfully used in the treatment of a variety of cancers including pancreas, prostate, nonsmall lung, cervical, and breast, induce apoptosis in cancer cells by increasing intracellular ROS levels. Antioxidant supplementation during chemotherapy is controversial. There are studies suggesting that antioxidants reduce chemotherapy-related cytotoxicity by selectively protecting normal cells and not compromising the anticancer activity of the chemotherapeutic agent. On the other hand, a number of studies claim that antioxidants protect cancer cells along with normal cells and thus reduce the oncologic efficacy of the chemotherapeutic agent. There are also studies reporting that antioxidant supplementation during chemotherapy increases the effectiveness of the anticancer drug by enhancing its cytotoxic effect, whereas in some studies, no significant enhancing effect of antioxidants on the oncologic effectiveness of the anticancer drug was found [87,88]. ATX treatment has been shown to decrease proliferation of human breast cancer (MCF-7) [21] and malign melanoma (A375) cells while being nontoxic to normal breast (MCF-10A) and epidermal cells (A2058), suggesting that ATX treatment alone selectively protects normal cells [26]. However, the effect of combination treatment with ATX and anticancer drugs on the cytotoxicity and efficacy of the anticancer drugs has been investigated only in a limited number of in vitro studies. The synergistic cytotoxic effect of combination treatments with ATX and erlotinib [29] as well as ATX and mitomycin C [35] has been demonstrated in nonsmall cell lung cancer cells (A549, H1703). Concomitant treatments with both ATX and erlotinib [29] as well as ATX and mitomycin C [35] resulted in a greater loss of cell viability in nonsmall A549 and H1703 cells. There is only one study examining the effect combination treatment with ATX and an anticancer drug on cancer cell proliferation as well as on intracellular ROS levels. In this study, Atalay et al. reported that cotreatment of breast cancer

440 Chapter 21 cells (MCF-7) with ATX and carbendazim leads to a significant decrease in cell proliferation while reducing intracellular ROS levels [89]. Data from the only in vivo study examining the effect of combination treatment with ATX and an anticancer drug (gemcitabine) on tumor growth in a gemcitabine-resistant pancreatic carcinogenesis xenograft mouse model revealed that ATX selectively decreases the proliferation of gemcitabine-resistant pancreatic cells and inhibits tumor growth by increasing gemcitabine sensitivity [34].

5 Conclusions There is a well-established link between dietary habits and the risk of developing cancer. Earlier small-scale epidemiological studies indicate that individuals consuming higher amounts of fruits and vegetables that are rich in carotenoids have a lower risk of developing certain types of cancer [90]. However, in more recent large-scale prospective studies, only a few significant associations have been observed between fruit and vegetable consumption and risk for cancers associated with consumption of smoking and/or alcohol, suggesting that the associations observed might be due to potentially confounding factors, smoking and alcohol [91]. Therefore, further large-scale prospective cohort studies should be conducted and the data must be interpreted cautiously considering potential confounding factors to elucidate the association between fruit and vegetable consumption and risk for cancer development. ATX is a promising anticancer agent due to its potent cancer preventing effects, which may be mediated by its strong antioxidant, immunomodulatory, and antiinflammatory activities. Additionally, ATX has an inhibitory effect on the key processes involved in tumor progression such as proliferation, apoptosis, invasion, and metastasis through modulation of various tumor progression-associated signaling pathways, including PI3K/Akt, NF-κB, Wnt/β-catenin, MAPK/Erk, and JAK/STAT. The effect of combination treatment with ATX and anticancer drugs has been investigated only in a very limited number of in vitro and in vivo studies. Data obtained from these studies are not sufficient to draw conclusions regarding the impact of ATX treatment on the anticancer activity and cytotoxicity of anticancer drugs. Therefore, further studies are needed to be able to comprehensively evaluate the safety/efficacy of combination treatment with ATX and anticancer drugs during chemotherapy.

References [1] Guerin M, Huntley ME, Olaizola M. Haematococcusastaxanthin: applications for human health and nutrition. Trends Biotechnol 2003;21(5):210–6. [2] Ambati RR, Phang SM, Ravi S, Aswathanarayana RG. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications – a review. Mar Drugs 2014;12(1):128–52. https://doi.org/10.3390/ md12010128. [3] Higuera-Ciapara I, Felix-Valenzuela L, Goycoolea FM. Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr 2006;46(2):185–96.

Anticancer properties of astaxanthin: A molecule of great promise

441

[4] Chintong S, Phatvej W, Rerk-Am U, Waiprib Y, Klaypradit W. In vitro antioxidant, antityrosinase, and cytotoxic activities of astaxanthin from shrimp waste. Antioxidants (Basel) 2019;8(5):128. https://doi.org/ 10.3390/antiox8050128. [5] Dose J, Matsugo S, Yokokawa H, Koshida Y, Okazaki S, Seidel U, Eggersdorfer M, Rimbach G, Esatbeyoglu T. Free radical scavenging and cellular antioxidant properties of astaxanthin. Int J Mol Sci 2016;17(1):103. https://doi.org/10.3390/ijms17010103. [6] Saw CL, Yang AY, Guo Y, Kong AN. Astaxanthin and omega-3 fatty acids individually and in combination protect against oxidative stress via the Nrf2-ARE pathway. Food ChemToxicol 2013;62:869–75. https://doi. org/10.1016/j.fct.2013.10.023. [7] Xie X, Chen Q, Tao J. Astaxanthin promotes Nrf2/ARE signaling to inhibit HG-induced renal fibrosis in GMCs. Mar Drugs 2018;16(4):117. https://doi.org/10.3390/md16040117. [8] Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H. Astaxanthin, a carotenoid with potential in human health and nutrition. J Nat Prod 2006;69(3):443–9. [9] Davinelli S, Nielsen ME, Scapagnini G. Astaxanthin in skin health, repair, and disease: a comprehensive review. Nutrients 2018;10(4):522. https://doi.org/10.3390/nu10040522. [10] Lee S, Bai S, Lee K, Namkoong S, Na H, Ha K, Han J, Yim S, Chang K, Kwon Y, Lee S, Kim Y. Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing IkB kinase-dependent NFR-kB activation. Mol Cells 2003;16(1):97–105. [11] Weisburger JH. Nutritional approach to cancer prevention with emphasis on vitamins, antioxidants, and carotenoids. Am J Clin Nutr 1991;53(1 Suppl):226S–237S. https://doi.org/10.1093/ajcn/53.1.226S. [12] Tanaka T, Shnimizu M, Moriwaki H. Cancer chemoprevention by carotenoids. Molecules 2012;17 (3):3202–42. https://doi.org/10.3390/molecules17033202. [13] Tanaka T, Makita H, Ohnishi M, Mori H, Satoh K, Hara A. Chemoprevention of rat oral carcinogenesis by naturally occurring xanthophylls, astaxanthin and canthaxanthin. Cancer Res 1995;55(18):4059–64. [14] Rao AR, Sindhuja HN, Dharmesh SM, Sankar KU, Sarada R, Ravishankar GA. Effective inhibition of skin cancer, tyrosinase, and antioxidative properties by astaxanthin and astaxanthin esters from the green alga Haematococcuspluvialis. J Agric Food Chem 2013;61(16):3842–51. https://doi.org/10.1021/jf304609j. [15] Nagendraprabhu P, Sudhandiran G. Astaxanthin inhibits tumor invasion by decreasing extracellular matrix production and induces apoptosis in experimental rat colon carcinogenesis by modulating the expressions of ERK-2, NFkB and COX-2. Invest New Drugs 2011;29(2):207–24. [16] Yasui Y, Hosokawa M, Mikami N, Miyashita K, Tanaka T. Dietary astaxanthin inhibits colitis and colitis-associated colon carcinogenesis in mice via modulation of the inflammatory cytokines. Chem Biol Interact 2011;193(1):79–87. [17] Kochi T, Shimizu M, Sumi T, Kubota M, Shirakami Y, Tanaka T, Moriwaki H. Inhibitory effects of astaxanthin on azoxymethane-induced colonic preneoplastic lesions in C57/BL/KsJ-db/db mice. BMC Gastroenterol 2014;14:212. https://doi.org/10.1186/s12876-014-0212-z. [18] Tanaka T, Morishita Y, Suzui M, Kojima T, Okumura A, Mori H. Chemoprevention of mouse urinary bladder carcinogenesis by the naturally occurring carotenoid astaxanthin. Carcinogenesis 1994;15(1):15–9. [19] Ni X, Yu H, Wang S, Zhang C, Shen S. Astaxanthin inhibits PC-3 xenograft prostate tumor growth in nude mice. Mar Drugs 2017;15(3)https://doi.org/10.3390/md15030066. [20] Kowshik J, Nivetha R, Ranjani S, Venkatesan P, Selvamuthukumar S, Veeravarmal V, Nagini S. Astaxanthin inhibits hallmarks of cancer by targeting the PI3K/NF-κΒ/STAT3 signalling axis in oral squamous cell carcinoma models. IUBMB Life 2019;71(10):1595–610. https://doi.org/10.1002/iub.2104. [21] McCall B, McPartland CK, Moore R, Frank-Kamenetskii A, Booth BW. Effects of astaxanthin on the proliferation and migration of breast cancer cells in vitro. Antioxidants (Basel) 2018;7(10):135. https://doi.org/ 10.3390/antiox7100135. [22] Palozza P, Torelli C, Boninsegna A, et al. Growth-inhibitory effects of the astaxanthin-rich alga Haematococcus pluvialis in human colon cancer cells. Cancer Lett 2009;283:108–17. [23] Kim JH, Park JJ, Lee BJ, Joo MK, Chun HJ, Lee SW, Bak YT. Astaxanthin inhibits proliferation of human gastric cancer cell lines by interrupting cell cycle progression. Gut Liver 2016;10(3):369–74. https://doi.org/ 10.5009/gnl15208.

442 Chapter 21 [24] Hormozi M, Ghoreishi S, Baharvand P. Astaxanthin induces apoptosis and increases activity of antioxidant enzymes in LS-180 cells. Artif Cells Nanomed Biotechnol 2019;47(1):891–5. https://doi.org/ 10.1080/21691401.2019.1580286. [25] Nagaraj S, Rajaram M, Arulmurugan P, Baskaraboopathy A, Karuppasamy K, Jayappriyan K, Sundararaj R, Rengasamy R. Antiproliferative potential of astaxanthin-rich alga HaematococcuspluvialisFlotow on human hepatic cancer (HepG2) cell line. Biomed Prev Nutr 2012;2(3):149–53. [26] Chen YT, Kao CJ, Huang HY, Huang SY, Chen CY, Lin YS, Wen ZH, Wang H-MD. Astaxanthin reduces MMP expressions, suppresses cancer cell migrations, and triggers apoptotic caspases of in vitro and in vivo models in melanoma. J Funct Foods 2017;31:20–31. [27] Kim HY, Kim YM, Hong S. Astaxanthin suppresses the metastasis of colon cancer by inhibiting the MYC-mediated downregulation of microRNA-29a-3p and microRNA-200a. Sci Rep 2019;9(1):9457. https:// doi.org/10.1038/s41598-019-45924-3. [28] Su XZ, Chen R, Wang CB, Ouyang XL, Jiang Y, Zhu MY. Astaxanthin combine with human serum albumin to abrogate cell proliferation, migration, and drug-resistant in human ovarian carcinoma SKOV3 cells. Anticancer Agents Med Chem 2019;19(6):792–801. https://doi.org/10.2174/1871520619666190225123003. [29] Chen JC, Wu CH, Peng YS, Zheng HY, Lin YC, Ma PF, Yen TC, Chen TY, Lin YW. Astaxanthin enhances erlotinib-induced cytotoxicity by p38 MAPK mediated xeroderma pigmentosum complementation group C (XPC) down-regulation in human lung cancer cells. Toxicol Res (Camb) 2018;7(6):1247–56. https://doi.org/ 10.1039/c7tx00292k. [30] Cui L, Xu F, Wang M, Li L, Qiao T, Cui H, Li Z, Sun C. Dietary natural astaxanthin at an early stage inhibits N-nitrosomethylbenzylamine-induced esophageal cancer oxidative stress and inflammation via downregulation of NFκB and COX2 in F344 rats. Onco Targets Ther 2019;12:5087–96. https://doi.org/10.2147/OTT.S197044. [31] Nguyen VP, Kim SW, Kim H, Kim H, Seok KH, Jung MJ, Ahn YC, Kang HW. Biocompatible astaxanthin as a novel marine-oriented agent for dual chemo-photothermal therapy. PLoS One 2017;12(4)e0174687. https:// doi.org/10.1371/journal.pone.0174687. [32] Qian X, Tan C, Yang B, Wang F, Ge Y, Guan Z, Cai J. Astaxanthin increases radiosensitivity in esophageal squamous cell carcinoma through inducing apoptosis and G2/M arrest. Dis Esophagus 2017;30(6):1–7. https:// doi.org/10.1093/dote/dox027. [33] Sowmya PR, Arathi BP, Vijay K, Baskaran V, Lakshminarayana R. Astaxanthin from shrimp efficiently modulates oxidative stress and allied cell death progression in MCF-7 cells treated synergistically with β-carotene and lutein from greens. Food Chem Toxicol 2017;106(Pt A):58–69. https://doi.org/10.1016/j. fct.2017.05.024. [34] Yan T, Li HY, Wu JS, Niu Q, Duan WH, Han QZ, Ji WM, Zhang T, Lv W. Astaxanthin inhibits gemcitabine-resistant human pancreatic cancer progression through EMT inhibition and gemcitabine resensitization. Oncol Lett 2017;14(5):5400–8. https://doi.org/10.3892/ol.2017.6836. [35] Ko JC, Chen JC, Wang TJ, Zheng HY, Chen WC, Chang PY, Lin YW. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells. Biochem Pharmacol 2016;105:91–100. https://doi.org/10.1016/j.bcp.2016.02.016. [36] Wu C, Zhang J, Liu T, Jiao G, Li C, Hu B. Astaxanthin inhibits proliferation and promotes apoptosis of A549 lung cancer cells via blocking JAK1/STAT3 pathway. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2016;32(6):784–8. [37] Li J, Dai W, Xia Y, Chen K, Li S, Liu T, Zhang R, Wang J, Lu W, Zhou Y, et al. Astaxanthin inhibits proliferation and induces apoptosis of human hepatocellular carcinoma cells via inhibition of NF-κB P65 and Wnt/Β-catenin in vitro. Mar Drug 2015;13:6064–81. [38] Kowshik J, Baba AB, Giri H, Deepak Reddy G, Dixit M, Nagini S. Astaxanthin inhibits JAK/STAT-3 signaling to abrogate cell proliferation, invasion and angiogenesis in a hamster model of oral cancer. PLoS One 2014;9 (10):e109114. https://doi.org/10.1371/journal.pone.0109114. [39] Kavitha K, Kowshik J, Kishore TK, Baba AB, Nagini S. Astaxanthin inhibits NF-κB and Wnt/β-catenin signaling pathways via inactivation of Erk/MAPK and PI3K/Akt to induce intrinsic apoptosis in a hamster

Anticancer properties of astaxanthin: A molecule of great promise

[40]

[41]

[42] [43] [44]

[45] [46] [47] [48] [49] [50] [51] [52] [53]

[54] [55]

[56] [57] [58]

[59] [60]

443

model of oral cancer. Biochim Biophys Acta 2013;1830(10):4433–44. https://doi.org/10.1016/j. bbagen.2013.05.032. Song X, Wang M, Zhang L, Zhang J, Wang X, Liu W, Gu X, Lv C. Changes in cell ultrastructure and inhibition of JAK1/STAT3 signaling pathway in CBRH-7919 cells with astaxanthin. Toxicol Mech Methods 2012;22 (9):679–86. https://doi.org/10.3109/15376516.2012.717119. Lim S, Lee JY, Jung WH, Lim EH, Joo MK, Lee BJ, Park JJ, Kim JS, Bak YT, Jung SW, Lee SW. Anticancer effects of astaxanthin and α-tocopherol in esophageal cancer cell lines. Korean J Helicobacter Up Gastrointest Res 2011;11:170–5. Song XD, Zhang JJ, Wang MR, Liu WB, Gu XB, Lv CJ. Astaxanthin induces mitochondria-mediated apoptosis in rat hepatocellular carcinoma CBRH-7919 cells. Biol Pharm Bull 2011;34(6):839–44. Nakao R, Nelson OL, Park JS, Mathison BD, Thompson PA, Chew BP. Effect of astaxanthin supplementation on inflammation and cardiac function in BALB/c mice. Anticancer Res 2010;30(7):2721–5. Tripathi DN, Jena GB. Astaxanthin intervention ameliorates cyclophosphamide-induced oxidative stress, DNA damage and early hepatocarcinogenesis in rat: role of Nrf2, p53, p38 and phase-II enzymes. Mutat Res 2010;696(1):69–80. https://doi.org/10.1016/j.mrgentox.2009.12.014. Jyonouchi H, Sun S, Iijima K, Gross MD. Antitumor activity of astaxanthin and its mode of action. Nutr Cancer 2000;36(1):59–65. Li R, Jia Z, Trush MA. Defining ROS in biology and medicine. React Oxyg Species (Apex) 2016;1(1):9–21. https://doi.org/10.20455/ros.2016.803. Nimse SB, Dilipkumar P. Free radicals, natural antioxidants, and their reaction mechanisms. RSC Adv 2015;5:27986–8006. Liou GY, Storz P. Reactive oxygen species in cancer. Free Radic Res 2010;44(5):479–96. https://doi.org/ 10.3109/10715761003667554. Qian Q, Chen W, Cao Y, Cao Q, Cui Y, Li Y, Wu J. Targeting reactive oxygen species in cancer via Chinese herbal medicine. Oxid Med Cell Longev 2019;2019:9240426. https://doi.org/10.1155/2019/9240426. Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as an anticancer strategy. Nat Rev Drug Discov 2013;12(12):931–47. Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 2001;11(4):173–86. Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, Dong W. ROS and ROS-mediated cellular signaling. Oxid Med Cell Longev 2016;2016:4350965. https://doi.org/10.1155/2016/4350965. Aggarwal V, Tuli HS, Varol A, Thakral F, Yerer MB, Sak K, Varol M, Jain A, Khan MA, Sethi G. Role of reactive oxygen species in cancer progression: molecular mechanisms and recent advancements. Biomolecules 2019;9(11):735. Laval J, Jurado J, Saparbaev M, Sidorkina O. Antimutagenic role of base-excision repair enzymes upon free radical-induced DNA damage. Mutat Res 1998;402:93–102. Michaels ML, Cruz C, Grollman AP, Miller JH. Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc Natl Acad Sci U S A 1992;89(15):7022–5. https:// doi.org/10.1073/pnas.89.15.7022. Park JS, Chyun JH, Kim YK, Line LL, Chew BP. Astaxanthin decreased oxidative stress and inflammation and enhanced immune response in humans. NutrMetab (Lond) 2010;7:18. https://doi.org/10.1186/1743-7075-7-18. Santocono M, Zurria M, Berrettini M, Fedeli D, Falcioni G. Influence of astaxanthin, zeaxanthin and lutein on DNA damage and repair in UVA-irradiated cells. J Photochem Photobiol B 2006;85(3):205–15. Kurinnyi D, Rushkovsky S, Demchenko O, Pilinska M. Astaxanthin as a modifier of genome instability after γ-radiation. In: Zepka LQ, Jacob-Lopes E, De Rosso VV, editors. Progress in carotenoid research. IntechOpen; 2018. https://doi.org/10.5772/intechopen.79341. Steinman RM, Banchereau J. Taking dendritic cells into medicine. Nature 2007;449(7161):419–26. Diamond MS, Kinder M, Matsushita H, Mashayekhi M, Dunn GP, Archambault JM, Lee H, Arthur CD, White JM, Kalinke U, Murphy KM, Schreiber RD. Type I interferon is selectively required by dendritic cells for immune rejection of tumors. J Exp Med 2011;208(10):1989–2003.

444 Chapter 21 [61] Salavoura K, Kolialexi A, Tsangaris G, Mavrou A. Development of cancer in patients with primary immunodeficiencies. Anticancer Res 2008;28:1263–9. [62] Shiels MS, Cole SR, Kirk GD, Poole C. A meta-analysis of the incidence of non-AIDS cancers in HIV-infected individuals. J Acquir Immune Defic Syndr 2009;52:611–22. https://doi.org/10.1097/QAI.0b013e3181b327ca. [63] Corthay A. Does the immune system naturally protect against cancer? Front Immunol 2014;5:197. https://doi. org/10.3389/fimmu.2014.00197. [64] Jyonouchi H, Hill RJ, Tomita Y, Good RA. Studies of immunomodulating actions of carotenoids. I. Effects of beta-carotene and astaxanthin on murine lymphocyte functions and cell surface marker expression in in vitro culture system. Nutr Cancer 1991;16(2):93–105. [65] Kurihara H, Koda H, Asami S, Kiso Y, Tanaka T. Contribution of the antioxidative property of astaxanthin to its protective effect on the promotion of cancer metastasis in mice treated with restraint stress. Life Sci 2002;70 (21):2509–20. [66] Weihong S, Lihong X, Hong L, Kailiang L, Yuxiu Z, Xiaofang L. Assessment and comparison of in vitro immunoregulatory activity of three astaxanthin stereoisomers. J Ocean Univ China (Oceanic and Coastal Sea Research) 2016;15:283–7. [67] Lin KH, Lin KC, Lu WJ, Thomas PA, Jayakumar T, Sheu JR. Astaxanthin, a carotenoid, stimulates immune responses by enhancing IFN-γ and IL-2 secretion in primary cultured lymphocytes in vitro and ex vivo. Int J Mol Sci 2015;17(1):44. https://doi.org/10.3390/ijms17010044. [68] Okai Y, Higashi-Okai K. Possible immunomodulating activities of carotenoids in in vitro cell culture experiments. Int J Immunopharmacol 1996;18(12):753–8. [69] Jyonouchi H, Zhang L, Tomita Y. Studies of immunomodulating actions of carotenoids. II. Astaxanthin enhances in vitro antibody production to T-dependent antigens without facilitating polyclonal B-cell activation. Nutr Cancer 1993;19(3):269–80. [70] Jyonouchi H, Zhang L, Gross M, Tomita Y. Immunomodulating actions of carotenoids: enhancement of in vivo and in vitro antibody production to T-dependent antigens. Nutr Cancer 1994;21(1):47–58. [71] Singh N, Baby D, Rajguru JP, Patil PB, Thakkannavar SS, Pujari VB. Inflammation and cancer. Ann Afr Med 2019;18(3):121–6. https://doi.org/10.4103/aam.aam_56_18. [72] Aggarwal BB, Vijayalekshmi RV, Sung B. Targeting inflammatory pathways for prevention and therapy of cancer: short-term friend, long-term foe. Clin Cancer Res 2009;15(2):425–30. [73] Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010;140(6):883–99. https:// doi.org/10.1016/j.cell.2010.01.025. [74] Ohgami K, Shiratori K, Kotake S, Nishida T, Mizuki N, Yazawa K, Ohno S. Effects of astaxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo. Invest Ophthalmol Vis Sci 2003;44 (6):2694–701. [75] Palozza P, Serini S, Di Nicuolo F, Calviello G. Modulation of apoptotic signalling by carotenoids in cancer cells. Arch Biochem Biophys 2004;430(1):104–9. [76] Letai A. Apoptosis and cancer. Annu Rev Cancer Biol 2017;1:275–94. [77] Labi V, Erlacher M. How cell death shapes cancer. Cell Death Dis 2015;6(3):e1675. https://doi.org/10.1038/ cddis.2015.20. [78] Wong RS. Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res 2011;30(1):87. https:// doi.org/10.1186/1756-9966-30-87. [79] Lordan S, O’Neill C, O’Brien NM. Effects of apigenin, lycopene and astaxanthin on 7 beta-hydroxycholesterol-induced apoptosis and Akt phosphorylation in U937 cells. Br J Nutr 2008;100 (2):287–96. https://doi.org/10.1017/S0007114507898643. [80] Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 2003;3:362–74. [81] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100(1):57–70. [82] Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 2010;141(1):52–67. https://doi.org/10.1016/j.cell.2010.03.015.

Anticancer properties of astaxanthin: A molecule of great promise

445

[83] Lee DJ, Kang SW. Reactive oxygen species and tumor metastasis. Mol Cells 2013;35(2):93–8. https://doi.org/ 10.1007/s10059-013-0034-9. [84] Qian X, Li J, Ding J, Wang Z, Zhang W, Hu G. Erlotinib activates mitochondrial death pathways related to the production of reactive oxygen species in the human non-small cell lung cancer cell line A549. Clin Exp Pharmacol Physiol 2009;36(5–6):487–94. https://doi.org/10.1111/j.1440-1681. [85] Yan C, Kong D, Ge D, Zhang Y, Zhang X, Su C, Cao X. Mitomycin C induces apoptosis in rheumatoid arthritis fibroblast-like synoviocytes via a mitochondrial-mediated pathway. Cell Physiol Biochem 2015;35 (3):1125–36. https://doi.org/10.1159/000373938. [86] Ren X, Zhao B, Chang H, Xiao M, Wu Y, Liu Y. Paclitaxel suppresses proliferation and induces apoptosis through regulation of ROS and the AKT/MAPK signaling pathway in canine mammary gland tumor cells. Mol Med Rep 2018;17(6):8289–99. https://doi.org/10.3892/mmr.2018.8868. [87] D’Andrea GM. Use of antioxidants during chemotherapy and radiotherapy should be avoided. CA Cancer J Clin 2005;55(5):319–21. [88] Singh K, Bhori M, Kasu YA, Bhat G, Marar T. Antioxidants as precision weapons in war against cancer chemotherapy induced toxicity – exploring the armoury of obscurity. Saudi Pharm J 2018;26(2):177–90. https://doi.org/10.1016/j.jsps.2017.12.013. [89] Atalay PB, Kuku G, Tuna BG. Effects of carbendazim and astaxanthin co-treatment on the proliferation of MCF-7 breast cancer cells. In Vitro Cell Dev Biol Anim 2019;55(2):113–9. https://doi.org/10.1007/s11626018-0312-0. [90] Krinsky NI, Johnson EJ. Carotenoid actions and their relation to health and disease. Mol Aspects Med 2005;26 (6):459–516. [91] Key TJ. Fruit and vegetables and cancer risk. Br J Cancer 2011;104(1):6–11. https://doi.org/10.1038/sj. bjc.6606032.

This page intentionally left blank

CHAPTER 22

Astaxanthin for improved muscle function and enhanced physical performance Karen A. Hecht, Ph.D.a, Joerg Schnackenberg, Ph.D.b, Arun Nair, Ph.D.a, and ˚ ke Lignell, Ph.D.c A a

AstaReal Inc., Moses Lake, WA, United States bAstaReal Co. Ltd., Tokyo, Japan cAstaReal AB, Nacka, Sweden

Abbreviations 1RM 4-HNE 8-OHdG AST ATP BAP CK CPT1 CRP CSA d-ROM HDL LDL MDF MVC Nrf-2 PGC-1α PON1 ROS SOD TA

one-repetition maximum 4-hydroxynonenal 8-hydroxydeoxyguanosine aspartate aminotransferase adenosine triphosphate blood prooxidant-antioxidant balance creatine kinase carnitine palmitoyltransferase 1 C-reactive protein muscle cross-sectional area derivatives of reactive oxygen metabolites high-density lipoprotein low-density lipoprotein mean dynamic force maximum voluntary contraction nuclear factor erythroid 2-related factor 2 proliferator-activated receptor gamma coactivator 1-alpha paraoxonase 1 reactive oxygen species superoxide dismutase tibialis anterior

1 Introduction Antioxidants have a recognized role in healthy aging because they help to offset the chronic oxidative stress that is caused by age-related impairment in redox homeostasis [1, 2]. Notably, aging is not the only physiological condition known to produce oxidative stress. Physical exercise produces acute oxidative stress, the extent of which depends on exercise type, intensity, duration, frequency, the individual’s age, and level of training [3, 4]. Excess free Global Perspectives on Astaxanthin. https://doi.org/10.1016/B978-0-12-823304-7.00033-7 Copyright # 2021 Elsevier Inc. All rights reserved.

447

448 Chapter 22 radical accumulation during exhaustive exercise has been shown to produce oxidative damage, resulting in deleterious effects on energy production and muscle recovery [5, 6]. However, there is also evidence that free radicals play a beneficial role in exercise adaptation when present at physiological levels [7, 8]. While the benefit of an antioxidant-rich diet for athletes has been identified, the role of antioxidant supplementation in supporting athletic performance is still subject to debate [3, 4, 9, 10]. It is important to note that antioxidants may interact differently with metabolic signaling pathways, depending on the antioxidant’s molecular structure, cellular localization, antioxidant capacity, and affinity for various types of free radicals. An antioxidant regimen tailored to the athlete’s individual redox status has been suggested to achieve the greatest benefit [9, 11–13]. It is important to understand the mechanism of action and properties of antioxidants in order to inform experimental design, and leverage each antioxidant nutrient for optimal gains. With these goals in mind, this chapter examines available evidence pertaining to the dietary antioxidant, natural astaxanthin, on exercise performance and adaption. Natural astaxanthin (3,30 -dihydroxy-β, β0 -carotene-4,40 -dione) is a red-orange, lipid-soluble xanthophyll carotenoid produced by various microorganisms, including algae [14]. The first widely used commercial application for this nutrient was as a feed additive for farmed salmon [15, 16]. It was found that wild salmon acquired natural astaxanthin in their diet. Astaxanthin has been shown to bind actomyosin in salmon muscle tissue, giving salmon their characteristic color and supporting their growth [17–20]. Further study revealed the potential of natural astaxanthin as an antioxidant for human health by demonstrating its superior inhibition of lipid peroxidation [21, 22]. For human applications, the microalga Haematococcus pluvialis has been the preferred source of natural astaxanthin, based on numerous safety and efficacy studies [14, 16]. Natural astaxanthin from H. pluvialis has been approved as a dietary supplement for human consumption globally [23]. Natural astaxanthin was first launched as a commercial dietary ingredient for supplementation in Sweden in 1995 [24]. The astaxanthin industry has grown significantly, and is expected to reach a global market value of $1.3 billion USD by 2030 [25]. Astaxanthin functions in mitochondria-rich muscles during exercise, where it modulates redox homeostasis and exercise-induced inflammation [26–29]. In view of the many biological activities of astaxanthin, the focus of this chapter is on evidence demonstrating that astaxanthin boosted endogenous antioxidant systems, localized in muscles and mitochondria, and has been coined a mitochondrial nutrient. Clinical studies examining the impact of natural astaxanthin on aerobic exercise, anaerobic exercise, and training effect are also discussed. Finally, the role of natural astaxanthin in maintaining redox homeostasis is examined in relation to age, training level, and exercise protocol throughout the review of clinical studies in this chapter. This chapter may be a valuable resource to many readers, and to members of the scientific community who are researching astaxanthin health benefits in the context of sport nutrition and muscle health.

Astaxanthin for improved muscle function 449

2 The impact of oxidative stress in skeletal muscle during exercise The observation that free radicals can cause tissue damage during exercise dates back to 1978. Since then, it has been widely accepted that exercise induces the production of free radicals (Table 22.1). However, the impact of free radicals on muscle performance and exercise adaptation is still subject to debate. Exercise-induced free radicals can have both beneficial and deleterious effects, depending on their quantity, the duration of exposure, level of exercise adaptation, and metabolic status of skeletal muscle as affected by age or disease [30,31]. Physiological levels of reactive oxygen species (ROS) are involved in force generation and in signaling pathways associated with exercise adaptation, such as mitochondrial biogenesis, increased aerobic capacity, insulin sensitivity, and antioxidant capacity. However, high levels of ROS and chronic exposure to ROS has been linked to reduced force generation, muscle fatigue, muscle atrophy, and muscular dystrophies. Therefore endogenous antioxidant mechanisms are in place to maintain redox homeostasis during exercise [31]. Endogenous antioxidants include antioxidant enzymes, such as superoxide dismutase (SOD) and catalase; nonenzymatic antioxidants, like glutathione; and dietary antioxidants, including vitamin E and astaxanthin [30,41]. Endogenous and dietary antioxidants work together to reduce free radical levels, not to eliminate them. Different antioxidants target different parts of the cell, and exert their action on different types of free radicals. Therefore, a suite of both endogenous and dietary antioxidants can work in concert to maintain redox homeostasis [42,43]. Dietary supplementation with natural astaxanthin has been shown to boost endogenous antioxidant capacity by increasing levels of SOD, catalase, and glutathione [44]. This suggests that rather than competing with the endogenous antioxidant system, astaxanthin may provide additive antioxidant benefits. One mechanism by which astaxanthin is thought to increase endogenous antioxidant levels is through binding and activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) [45,46]. Nrf2 is an important regulator of exercise adaptation that is activated by free radicals produced during exercise, and which acts as a

Table 22.1 Sources of free radical production during exercise. Free radical producers

Originating in

Reference

Mitochondria (complexes I and III) NADPH oxidases Phospholipase A2 Xanthine oxidase Lipoxygenases Neutrophils (oxidative burst) Macrophages (oxidative burst) Catecholamine autooxidation Endothelial cells

Muscle Muscle Muscle Muscle Muscle Immune cell Immune cell Adrenal gland Blood vessels

[32] [33] [34] [35] [36] [37] [38] [39] [40]

450 Chapter 22 master switch to induce endogenous antioxidant expression that helps to restore redox homeostasis [47]. As a dietary antioxidant, astaxanthin has been shown in human clinical studies to reduce total hydroperoxides, increase biological antioxidant potential (BAP) in serum, decrease oxidation of polyunsaturated fatty acids in serum, reduce protein oxidation, and reduce derivatives of reactive oxygen metabolites (d-ROM) [26,27,48–55]. Astaxanthin is thought to play a significant role in modulating redox status during exercise in both young and in aging muscles. In cases where free radical production overwhelms endogenous antioxidant capacity, astaxanthin has been shown to support mitochondrial function, muscle endurance, and recovery from exercise [56].

3 The role of astaxanthin on mitochondrial function and oxidative stress during exercise Orally administered astaxanthin was found to accumulate in all organs of the body, as shown in animal models [28]. Within cells, astaxanthin is able to span lipid bilayers that form the cell and mitochondrial membranes. Its protective role in membranes is attributed to astaxanthin’s unique molecular structure, which allows astaxanthin to reach across the full thickness of the lipid bilayer, and quench free radicals both outside and inside the membrane [57–59]. Astaxanthin has been shown to accumulate in skeletal and cardiac muscle of both sedentary and exercising mice (Fig. 22.1A) [60,61]. Subcellular localization of natural astaxanthin has been examined in canine and feline leukocytes, where it was found that as much as 50% of membrane-bound astaxanthin partitioned to mitochondrial membranes (Fig. 22.1B) [59]. Astaxanthin-enriched mitochondria were reported in Park et al. [62] to increase ATP production in leukocytes of supplemented dogs [62]. Therefore, it has been proposed that astaxanthin is enriched in the mitochondria of skeletal muscles, where it can protect mitochondria from oxidative damage caused by exercise-induced ROS accumulation (Fig. 22.1C). Astaxanthin has been shown to protect mitochondrial integrity in HeLa cells challenged with antimycin A, which inhibits complex III of the mitochondrial electron transport chain, and causes overproduction of superoxide anions [63,64]. Astaxanthin’s position in the mitochondrial membrane, and its strong singlet oxygen and superoxide anion quenching capacity explains this protective effect of astaxanthin on the mitochondria of HeLa cells in vitro [65]. In addition to this direct evidence of mitochondrial protection, astaxanthin lowered oxidation levels in both skeletal and cardiac muscles of exercising rodents, as measured by 4-HNE or HEL modified proteins; indicators of lipid peroxidation, and 8-OHdG; a marker of DNA oxidation [29,60,66]. Published studies have tested the hypothesis that reduced oxidative damage to mitochondria and muscle tissue would increase exercise endurance. Mice supplementing with H. pluvialis

Astaxanthin for improved muscle function 451 Sedentary

Running

1379.2

1033.3

294.5

(A)

277.5

Plasma (ng/ml)

268.2

318.0

Skeletal muscle (ng/g)

Heart (ng/g)

Fig. 22.1 Natural astaxanthin distribution in muscle and mitochondria. (A) Natural astaxanthin concentration (ng/mL) in serum, skeletal muscle (ng/g), and heart (ng/g) of sedentary (n¼8) or running mice (n¼8) after 5weeks of supplementation. No significant difference in astaxanthin levels was observed between sedentary and running mice. (Continued)

Fig. 22.1, cont’d (B) Subcellular distribution of membrane-bound natural astaxanthin in leukocytes of dogs supplemented with 40mg/day for 8days (left). Subcellular distribution of membrane-bound natural astaxanthin in leukocytes of cats supplemented with 10 mg/day for 6 days (right). (Continued)

452 Chapter 22

Fig. 22.1, cont’d (C) Schematic of natural astaxanthin localization in mitochondrial membranes. Natural astaxanthin is thought to span the lipid bilayer, and has been shown to provide antioxidant protection both inside and outside the membrane. Its preferential localization in mitochondrial membranes suggests that it is a targeted mitochondrial antioxidant effective in quenching exerciseinduced reactive oxygen species produced by mitochondria. (A) From Aoi W, Maoka T, Abe R, Fujishita M, Tominaga K. Comparison of the effect of non-esterified and esterified astaxanthins on endurance performance in mice. J Clin Biochem Nutr 2018;62(2):161–6. (B) From Park JS, Kim HW, Mathison BD, Hayek MG, Massimino S, Reinhart GA, et al. Astaxanthin uptake in domestic dogs and cats. Nutr Metab (Lond) 2010;7:52. (C) From Pashkow FJ, Watumull DG, Campbell CL. Astaxanthin: a novel potential treatment for oxidative stress and inflammation in cardiovascular disease. Am J Cardiol 2008;101(Suppl.):58D–68D.

astaxanthin for 5weeks exhibited greater endurance in a swimming test compared with control (P