Renewable Resources and Biotechnology for Material Applications 9781619422575

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MATERIALS SCIENCE AND TECHNOLOGIES

RENEWABLE RESOURCES AND BIOTECHNOLOGY FOR MATERIAL APPLICATIONS

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MATERIALS SCIENCE AND TECHNOLOGIES

RENEWABLE RESOURCES AND BIOTECHNOLOGY FOR MATERIAL APPLICATIONS

G.E. ZAIKOV D.P. PUDEL AND

GRZEGORZ SPYCHALSKI EDITORS

Nova Science Publishers, Inc. New York

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Renewable resources and biotechnology for material applications / editors, G.E. Zaikov, Frank Pudel, Grzegorz Spychalski. p. cm. -- (Materials science and technologies) Includes bibliographical references and index. ISBN 978-1-61942-257-5 (EBook) 1. Plant biotechnology. 2. Renewable natural resources. 3. Materials--Biotechnology. 4. Biomass energy. I. Zaikov, G. E. (Gennadii Efremovich), 1935- II. Pudel, Frank. III. Spychalski, Grzegorz. TP248.27.P55R458 2011 630--dc22 2011003559

Published by Nova Science Publishers, Inc. † New York

This volume is dedicated to the memory of Frank Columbus On December 1st 2010, Frank H. Columbus Jr. (President and Editor-in-Chief of Nova Science Publishers, New York) passed away suddenly at his home in New York. We lost our colleague, our good friend, a nearly perfect person who helped scientists from all over the world. Particularly Frank did much for the popularization of Russian and Georgian scientific research, publishing a few thousand books based on the research of Soviet (Russian, Georgian, Ukranian etc.) scientists. Frank was born on February 26th 1941 in Pennsylvania. He joined the army upon graduation of high school and went on to complete his education at the University of Maryland and at George Washington University. In 1969, he became the Vice-President of Cambridge Scientific. In 1975, he was invited to work for Plenum Publishing where he was the Vice-President until 1985, when he founded Nova Science Publishers, Inc. Frank Columbus did a lot for the prosperity of many Soviet (Russian, Georgian, Ukranian, Armenian, Kazakh, Kyrgiz, etc.) scientists. He did the same for scientists from East Europe – Poland, Hungary, Czeckoslovakia, Romania and Bulgaria. He was a unique person who enjoyed studying throughout the course of his life, who felt at home in his country which he loved and was proud of, as well as in Russia and Georgia. There is a famous Russian proverb: "The man is alive if people remember him.‖ In this case, Frank is alive and will always be in our memories while we are living. He will be remembered for his talent, professionalism, brilliant ideas and above all – for his heart. Professor Gennady Efremovich Zaikov Honored Member of Russian Science Head of Polymer Division, IBCP D. P. Pudel An Enterprise of OHMI Consulting Germany Grzegorz Spychalski Institute of Material Fibres and Medical Plants Poland

CONTENTS Preface

xi

Chapter 1

Sorghum as Biogas Crop Anja Hartmann, Karen Zeise and Maendy Fritz

Chapter 2

Enzyme Modified Grain by-Product Reinforced Polypropylene Composites Abdullah Al Mamun, Andrzej K. Bledzki and Jürgen Volk

Chapter 3

Biomass as Combustion Fuel – Experiences and Findings Grete Bach, Stefanie Bachmann, Daniel Kolb, Peter Kosel and Wendy Franke

Chapter 4

Efficiency of Hemp Essentials Oil Depending on Sowing Density and Time of Inflorescence Harvest H. Burczyk, R. Kaniewski, W. Konczewicz, N. Kryszak and J. Turowski

Chapter 5

Chapter 6

IWN-11 the New Biostymulator for Increasing Crops Resistance to Drought Stress Krzysztof Heller, Małgorzata Byczyńska and Zenon Woźnica Yellow Natural Dyestuffs and their Light Fastness Properties and UV Protection on Natural Fabrics Katarzyna Schmidt-Przewoźna and Jakub Kowalinski

Chapter 7

Multipurpose White Mulberry (Morus Alba L.) Malgorzata Lochynska and Grzegorz Oleszak

Chapter 8

Agricultural Residues as a Renewable Source of Bio-Energy with Special Focus on Cereal Straws Narra S., C. Glaser, H.-J. Gusovius, C. Stollberg and P. Ay

1

11 21

31

41

49 59

67

viii Chapter 9

Contents Prevention of Disposal of Greenhouse Gas from Digested Residues by Optimal Use of the Nitrogen Fertilizer Potential Ute Bauermeister

81

Chapter 10

Vetiveria Zizanioides Grass a Useful Tool G. Bach and R. Hommel

89

Chapter 11

State of the Art of the Renewable Resources in Poland R. Kozłowski, K. Seidler-Lozykowska, M. Mackiewicz-Talarczyk, P. Baraniecki, J. Mankowski, Cz. Ogurkowski and I. Pniewska

97

Chapter 12

Energy Efficiency of Four Crop Species Jerzy Pudelko, Jerzy Mankowski and Jacek Kolodziej

123

Chapter 13

Enzymatic Biomass Hydrolysis Viktor Antonov, Josef Marousek, Jan Marek, Stanislav Kuzel and Tomas Rosenberg

131

Chapter 14

Sustainable Logistics Centers Hartmut Zadek and Robert Schulz

141

Chapter 15

Relation between the Cell-Free DNA Content and the Lipid Peroxidation in the Blood Plasma of Mice under Damaging Action Lyudmila N. Shishkina, Mikhail A. Klimovich, Mikhail V. Kozlov and Margarita A. Smotryaeva

Chapter 16

Chapter 17

Chapter 18

Chapter 19

Chapter 20

Mechanism of Stable Radical Generation in Lignin under the Action of Nitrogen Dioxide E.Ya.Davydov, I.S. Gaponova, S.M. Lomakin, G.B. Pariiskii, T.V. Pokholok and G.E. Zaikov Spray Formation of Alternative Diesel Fuels under Engine-Like Conditions Dennis Backofen, Michael Könnig, Helmut Tschöke and Jürgen Schmidt DNA Fingerprinting and Characterisation of Genetic Variation of Different Clones of Urtica Dioica L. via RAPD and RAPD-Derived SCAR Markers Bettina Biskupek-Korell, Sabrina Becker, Jasmin Dufrenne, Patricia Rauscher and Carolin Schneider IFT Performance of MES Surfactant from Palm Olein for EOR Application Erliza Hambali, Mira Rivai, Putu Suarsana, Sugiharjo, Edi Zulchaidir and Hermansyah Handoko The Development Process of Jatropha Methyl Ester Sulfonic Acid (MESA) to Enhance Oil Recovery Siti Mujdalipah, Mira Rivai, Erliza Hambali, Ani Suryani, Hermansyah Handoko and Edi Zulchaidir

147

157

167

181

191

201

Contents Chapter 21

Rapeseed Proteins – Recent Results on Extraction and Application Frank Pudel

Chapter 22

PCR-Based Detection of Different Endophytic Bacteria Appearing in In Vitro Cultures of Different Clones of Urtica Dioica L. Using Specific PCR-Primers, Derived from Bacterial 16S rDNA Sequences Carolin Schneider, Sven Wartenberg, Jasmin Dufrenne and Bettina Biskupek-Korell

Chapter 23

Chapter 24

Chapter 25

Chapter 26

Chapter 27

Chapter 28

Chapter 29

Survey of Jatropha Curcas Energy Potential for an Efficient Production of Biodiesel as Renewable Energy Sékou Traoré, Amadou Diarra, Macki Traoré and Sékou Magassouba

209

223

229

Genetic Resources of Cannabis Sativa L. in the Collection of the Gene Bank at Infandmp in Poznan Magda lena Chudy and Grażyna Mańkowska

241

Production of Biodegradable Carrier Materials for the Immobilization of Microorganisms for the Treatment of Water Alvaro E. Gonzalias, A. Werner and Hans-Joachim Radusch

251

The Changes in the Protein Profile in Response to Cadmium Stress in Flax (Linum Usitatissimum L.) Milena Szalata, Szalata Marlena and Wielgus Karolina

257

Regional Value Added of Bioenergy Activities A Material Flow Approach Ruth Offermann, Thilo Seidenberger and Daniela Thrän

263

Simple Kinetics of Methane Formation Using Model Substrates: A Short Discussion Robert Reinhard Pätz and Jan-Henryk Richter-Listewnik

271

Biosafety of Transgenic Potatoes Producing the Biopolymer Cyanophycin Christoph Unger, Maja Hühns and Inge Broer

279

Chapter 30

Provision Pathways for Biomethane Diana Weigl, Katja Oehmichen, Michael Seiffert, Franziska Müller-Langer and Frank Scholwin

Chapter 31

Flammability of Polymers Reinforced with Lignocelullosic Raw Materials Maria Władyka-Przybylak and Krzysztof Bujnowicz

Chapter 32

ix

Feedstocks and (Bio) Technologies for Biorefineries Joachim Venus

283

293 299

x

Contents

Chapter 33

Non-Food Crops as a Feed Stock for Modern Bio-Based Industry Maria Mackiewicz-Talarczyk, Krzysztof Heller, Przemyslaw Baraniecki and Irena Pniewska

Chapter 34

Multiscale Metabolic Modeling of Cereals: An Integrated Systems Biology Approach for Research Biomass Mohammad R. Hajirezaei, Mohammad R. Ghaffari, Björn H. Junker, Johannes Müller, Björn Usadel Michael Leps, Rainer Lemke and Falk Schreiber

311

325

Chapter 35

Bioactive Substances from the Balsam Poplar S. Ludewig, S. Gille, D. Orzessek and C. Griehl

333

Chapter 36

The Potential of Microalgae to Produce Lipids for Biofuels C.Griehl, H. Polhardt, D. Müller and S. Bieler

347

Chapter 37

Biomaterials for Consumer Products Peter Gerth, Thomas Bagusch, Melanie Poschke and Johann Zimprich

357

Chapter 38

Use of Rice Spelt as Component in Building Material Stefanie Bachmann, Grete Bach and Rainer Loth

363

Chapter 39

Criteria System of Sustainable Agriculture (CSSA - KSNL) Gerhard Breitschuh, Hand Eckert, Ines Matthes and Jürfgen Strümpfel, In cooperation with: Günter Bachmann, Landwirt Martin Herold, Thorsten Breitschuh and Ulrich Gernand

371

Index

395

PREFACE Gennady Zaikov, Frank Pudel1 and Grzegorz Spychalski2 N.M. Emanuel Institute of Biochemical Physics Russian Academy of Sciences, 4 Kosygin str., Moscow 119334, Russia 1 Managing Director Pilot Pflanzenöltechnologie Magdeburg e.V., Berliner Chaussee 66, D-39114 Magdeburg, Germany, 2 Institute of Material Fibres and Medical Plants, 71b, Wojska Polskiego str., 60-630 Poznan, Poland. This volume is a proceeding of the 16th International Conference for Renewable Resources and Plant Biotechnology (NAROSSA® 2010). NAROSSA® 2010 conference was held in Maritim Hotel (Magdeburg, Germany) in the period June 7 – 8, 2010. The organizers of conference were the association Pilot Pflanzenöltechnologie Magdeburg e. V. (PPM) and the sponsor of this conference was OHMI Consulting GmbH. Prof. Dr. Melvyn F. Askew (Census, Wolverhampton, United Kingdom), Prof. Dr. Grete Bach (ifn Forschungs- und Technologiezentrum GmbH, Elsteraue/OT Tröglitz, Germany), Dr. Ute Bauermeister (Gesellschaft für Nachhaltige Stoffnutzung mbH, Halle/Saale, Germany), Dr. Peter Bloß (Kunststoff-Zentrum in Leipzig gGmbH, Leipzig, Germany), Dieter Bockey (UFOP e.V., Berlin, Germany), Dr. Susanne Brandt (Landesbauernverband Sachsen-Anhalt e.V., Magdeburg, Germany), Thorsten Breitschuh (NAROSSA e.V., Magdeburg, Germany), Dr. Waldemar Buchwald (Institute for Natural Fibres and Medicinal Plants, Poznan, Poland), Dr. Matthias Gohla (Fraunhofer IFF - Institut für Fabrikbetrieb und automatisierung, Magdeburg, Germany), Torsten Graf (Thüringer Landesanstalt für Landwirtschaft, Dornburg, Germany), PhD Paul P. Kolodziejczyk (Biolink Consultancy, New Denver, Canada), Prof. Gotthard Kunze (Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung IPK, Gatersleben, Germany), Prof. Dr. Lothar Mörl (Otto-vonGuericke-Universität, Magdeburg, Germany), Prof. Dr. Ingo Schellenberg (Hochschule Anhalt (FH), Kompetenzzentrum Life Sciences, Bernburg, Germany), Dr. Heike Schimpf (Landesanstalt für Landwirtschaft, Forsten und Gartenbau / KoNaRo, Bernburg, Germany), Prof. Grzegorz Spychalski (Institute for Natural Fibres and Medicinal Plants, Poznan, Poland), Dr. Ralph Thomann (Institut für Getreideverarbeitung GmbH, Bergholz-Rehbrücke, Germany), Dr. Maria Wladyka-Przybylak (Institute for Natural Fibres and Medicinal Plants,

xii

Gennady Zaikov, Frank Prudel and Grzegorz Spychalski

Poznan, Poland), Prof. Dr. Gennady E. Zaikov (N. M. Emanuel Institute of Biochemical Physics, Moscow, Russia) were members of Scientific Committee of this conference. The program of the conference included follow main topics: 1.

Plant Breeding for non-food Application   

Renewable Resources for Material Applications

2.    3.

Biotechnology In vitro culture Smart breeding

Biobased composites (Natural fibres, Nano particles etc.) Biobased polymers Biobased additives

Processing, Characterisation and Application of Secondary Plant Metabolites   

Biomass conversion technologies Biorefineries Cosmetics and pharmaceuticals Biomass and Efficient Energy Generation

4.   

Energy crops and organic residues Thermal, thermochemical and biochemical conversion technologies Gas cleaning and product upgrading

5. Sustainable Development in Environment, Economy and Sociology   

Acceptance Sustainability in production and logistics Regional material, nutrient and energetic cycles

About 100 participants from 27 research centers from Germany, France, Russia, Poland, Switzerland, Czech Republic, Guinea, Indonesia took part in this conference. Scientific program of the conference included 3 plenary lectures and 33 oral presentations which were divided on two sessions. The topic of first plenary lecture was ―Greeting of the patron‖ (H. O. Aeikens, Minister for Agriculture and the Environment of Saxony-Anhalt, Magdeburg, Germany). The second plenary lecture was about feedstocks and (bio) technologies for biorefineries (J. Venus, Leibniz-Institut für Agrartechnik Potsdam-Bornim e.V., Germany). Socially sustainable development of the rural areas was discussed in the third lecture (G. Spychalski, Director of the Institute of Natural Fibres and Medicinal Plants, Poland). The first session of conference had 18 oral presentations. P. Gerth, T. Bagusch, M. Poschke, J. Zimprich (Centre of Competence Engineering Sciences/Renewable Materials, University of Applied Sciences, Magdeburg, Germany) spoke about biomaterials for consumer products. A. Breier, K. Gliesche, P. Govindarajulu, C. Rentsch, B. Rentsch

Preface

xiii

(Leibniz-Institut für Polymerforschung Dresden e.V., Germany; Universitätsklinikum CarlGustav-Carus, TU Dresden, Germany; Fa. Catgut GmbH, Markneukirchen, Germany) gave presentation about tissue engineering executed on textile scaffolds embroidered with degradable surgical thread materials. The lecture of M.I. Artsis, A.P.Bonartsev,, A.L.Iordanskii, G.A.Bonartseva, G. E.Zaikov (General Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia; A.N. Bachs Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia; Faculty of Biology, Moscow State University, Moscow, Russia) was devoted to biodegradation and medical application of microbial poly(3-hydroxybutyrate). The results about flammability of polymeres reinforced with lignocellulosis raw materials was included in the talk of M. Wladyka-Przybylak, K. Bujnowicz (Institute of Natural Fibres and Medicinal Plants, Poznan, Poland). J.-P. Krause (Institute of Nutrition Science, University of Potsdam, Germany) spoke about correlations between interfacial rheology and film forming behaviour of plant proteins. The lecture of P. L. Mikolajczak, A. Bogacz, M. Karasiewicz, P. M. Mrozikiewicz (Dep. of Pharmacology and Biotechnology, Institute of Natural Fibres and Medicinal Plants, Poznan, Poland; Dep. of Pharmacology; Laboratory of Experimental Pharmacogenetics, Dep. of Clinical Pharmacy and Biopharmacy Poznan University of Medical Science, Poznan, Poland) was devoted to role of puerarin and daidzin as secondary plant metabolites - study on alcoholism. The information about PCR-based detection endophytic bacteria appearing in in vitro cultures of different clones of Urtica dioica L. using specific PCR-primers, derived from bacterial 16S rDNA sequences was included in the lecture of C. Schneider, B.Eng. Sven Wartenberg, B.Eng. Jasmin Dufrenne, B. Biskupek-Korell (Institut für Pflanzenkultur e.K., Schnega, Germany; FH Hannover, Fakultät 2, Abt. Bioverfahrenstechnik, Germany). M. Chudy, G. Mankowska (Dep. of Hemp Breeding, Agronomy and Seed Production; Dep. of Biotechnology both: Institute of Natural Fibres and Medicinal Plants, Poznan, Poland) spoke about genetic resources of Cannabis sativa L. in the collection of the gene bank at INF&MP in Poznan. The lecture of B. Biskupek-Korell, S. Becker, J. Dufrenne, P. Rauscher, C. Schneider (FH Hannover, Fakultät 2, Abt. Bioverfahrenstechnik, Hannover, Germany; Institut für Pflanzenkultur e.K., Schnega, Germany) was devoted DNA fingerprinting and charakterisation of genetic variation of different clones of Urtica dioica L. via RAPD and RAPD-derived SCAR markers. The problems of detection of phytopathogenic RNA-viruses were discussed by K. Florschütz, A. Schröter, M. Körner, G. Kunze (Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany). M. Strybe, N. Kryszak (Dep. of Hemp Breeding, Agronomy and Seed Production; Harvesting Technology and Fibre Plants Evaluation Dep.; Institute of Natural Fibres and Medicinal Plants, Poznan, Poland) spoke about the effect of harvesting time on cellulose content in fibrous hemp. The application of biotechnology in flax and hemp breeding was discussed by K. Wielgus, A. Luwanska, G. Mankowska, J. Przewozna (Department of Biotechnology, both: Institute of Natural Fibres and Medicinal Plants, Poznan, Poland). Five last oral lectures were devoted to the next problems: public acceptance of biomass power plants – results of recent environmental psychological surveys (J. Zoellner, P. Schweizer-Ries, I. Rau, Institut für Psychologie, FG Umweltpsychologie, Otto-von-GuerickeUniversität Magdeburg, Germany); regional value added of bioenergy activities: a material flow approach (R. Offermann, T. Seidenberger, D. Thrän, DBFZ German Biomass Research Centre, Leipzig, Germany); sustainable intra-logistics with renewable energies and fuel cells

xiv

Gennady Zaikov, Frank Prudel and Grzegorz Spychalski

(H. Zadek, R. Schulz, Institute of Logistics and Material Handling Systems, University "Ottovon-Guericke", Magdeburg, Germany); prevention of disposal of greenhouse gases from digested residues by optimal use of the nitrogen fertilizer potential (U. Bauermeister, GNS Gesellschaft für Nachhaltige Stoffnutzung mbH und BMVerfahrenstechnologie, Halle/Saale, Germany); criteria system of sustainable agriculture (CSSA-KSNL) (G. Breitschuh, H. Eckert, I. Matthes, J. Strümpfel, G. Bachmann, T. Breitschuh, Thuringian Ministry for agriculture and environment, Erfurt, Germany; Thuringian state institute for agriculture, Jena, Germany; VAVB e.V., Jena, Germany). The second session of conference had 15 oral presentations. R. Thomann, C. Luft, S. Icking, H. Hiob, R. Schneeweiß (Institut für Lebensmittel und Umweltforschung e.V., Nuthetal, Germany; Görlitzer Kornbrennerei und Spirituosenfabrik, Seyda, Germany; Institut für Getreideverarbeitung GmbH (IGV), Nuthetal, Germany) gave presentation about economical and ecological aspects in the production of ethanol from grain with the use of roller mills. M. Seiffert, F. Langer, F. Scholwin (German Biomass Research Centre, Leipzig, Germany) spoke about provision pathway for biomethane. The title of presentation of R. Pätz (Hochschule Anhalt, Germany) was ―Simple kinetics of methane formation using model substrates‖. The lecture of D. Backofen, M. Adam, H. Tschöke, M. Könnig, J. Schmidt (Institute of Mobile Systems; Institute of Fluid Dynamics and Thermodynamics; Otto-vonGuericke-University, Magdeburg, Germany) was devoted to spray formation of alternative Diesel fuels under engine-like conditions. The information about energetic efficiency of four arable crop species was presented by J. Kolodziej, J. Pudelko, J. Mankowski (Institute of Natural Fibres and Medicinal Plants, Poznan, Poland) and the information about the potential of Sorghum varieties as bioenergy crops was included in the presentation of A. Hartmann, K. Zeise, M. Fritz (Technologie- und Förderzenturm Straubing / Sachgebiet Rohstoffpflanzen und Stoffflüsse, Straubing, Germany). Multiscale metabolic modeling: an integrative systems biology approach for biomass research was discussed in the lecture of M.-R. Hajirezaei, M. R. Ghaffari, R. Lemke, B. H. Junker, J. Müller, B. Usadel, R. Wünschiers, F. Schreiber (Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany; SunGene GmbH, a BASF Plant Science Company, Gatersleben, Germany; Martin-Luther-Universität Halle-Wittenberg, Yield and Ecophysiology, Halle/Saale, Germany; Max-Planck-Institute for Molecular Plant Physiology, Integrative Carbon Biology, Golm, Germany). M. Ernst, M. Ouzonova, T. Presterl, P. Westhoff, R. Meyer, A. Melchinger, J. M. Montes, C. Riedelsheimer, C. Grieder, J. Selbig, A. Larhlimi, M. Stitt, R. Sulpice, A. Czedik-Eysenberg, L. Willmitzer, T. Altmann (Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany; KWS Saat AG, Einbeck, Germany; Heinrich-Heine-Universität Düsseldorf, Germany; Universität Hohenheim, Germany; Universität Potsdam, Germany; Max-Planck-Institut für molekulare Pflanzenphysiologie, Golm, Germany) spoke about structural genomic research for energy maize. The problems of agricultural residues as a renewable source of bio-energy with special focus on cereal straws were discussed by S. Narra, C. Glaser, P. Ay, H.-J. Gusovius, C. Stollberg (Chair of Mineral Processing, Brandenburg University of Technology, Cottbus; Leibniz Institute for Agricultural Engineering Potsdam-Bornim, Germany; Process Technology of Biogenous Resources, Faculty of Engineering, University of Applied Sciences Wismar, Poel, Germany). M. Lochynska, G. Oleszak (Dep. of Silkworms Breeding and Mulberry Cultivation; Experimental Farm in Petkow; Institute of Natural Fibres and

Preface

xv

Medicinal Plants, Poznan, Poland) discussed the problems of multi-use of the white mulberry (Morus alba L.). G. Fleck, N. Stadermann (Pilot Pflanzenöltechnologie Magdeburg e.V., Germany; Maschinenfabrik Reinartz, Neuss, Germany) gave presentation under the title ―Technology development of decentralized processing of Jatropha-seeds‖. The lecture of S. Traoré, A. Diarra, M. Traoré, S. Magassouba (Polytechnic Institute, University of Conakry, Guinea; FSE, Dep. of Economics, University of Sonfonia, Conakry, Guinea; Institute of medical plants, Dubreka, Guinea) was devoted to survey of Jatropha curcas energy potential for an efficient production of biodiesel as renewable resource. The information about non-food crops as a feedstock for modern bio-based industry was included in the lecture of K. Heller, M. Mackiewicz-Talarczyk, P. Baraniecki (Institute of Natural Fibres and Medicinal Plants, Poznan, Poland). The last oral presentations were devoted to the next problems: oilseed Bbio-refinery – a new concept of value-added processing (F. Pudel, Pilot Pflanzenöltechnologie Magdeburg e.V., Germany); evaluation of different fractionation methods for the extraction of pure fatty acids (M. Rohrbeck, Pilot Pflanzenöltechnologie Magdeburg e.V., Germany); IFT Performance of MES surfactant from Palm Olein for EOR application (E. Hambali Surfactant and Bioenergy Research Center, Bogor Agricultural University, Indonesia); the development of Palm Methyl Ester Sulfonic Acid (MESA) to increase oil well recovery (S. Mujdalipah, E. Hambali, E. Zulchaidir (Surfactant and Bioenergy Research Center, Bogor Agricultural University, Indonesia; PT. Findeco Jaya, Pulogadung, Jakarta). Poster session included 17 presentations about some aspects of plant biotechnology and renewable resources. Particularly there was the poster of Russian scientistsŚ ―Mechanism of stable radical generation in lignin under the action of nitrogen dioxide‖ (E. Ya. Davydov, I. S. Gaponova, S. M. Lomakin, G. B. Pariiskii, T. V. Pokholok, Gennadi E. Zaikov, N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia). The problems which were discussed during this conference are very important for pure as well as for practical application. The Editors would like to thank Ms. Jana Wittwer for her help during the preparation of this manuscript. Gennady Zaikov Frank Pudel Grzegorz Spychalski

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 1

SORGHUM AS BIOGAS CROP Anja Hartmann, Karen Zeise and Maendy Fritz Technology and Support Centre in the Centre of Excellence for Renewable Resources (TFZ) Schulgasse 18, 94315 Straubing, Germany

ABSTRACT Within the last years sorghum has been discussed as a prospective energy crop with a high yield potential. By means of an extensive screening of up to 305 sorghum cultivars we identified the most promising and climatically well adapted cultivars. Results show that forage type sorghum of S. bicolor is superior in terms of dry matter production. Thus highest methane yields per hectare can be expected from these cultivars. On poor soils and under drought stress conditions, some S. bicolor cultivars produced dry matter yields even comparable to maize. Regarding dry matter content S. bicolor x S. sudanense as well as grain types of S. bicolor showed best performance.

Keywords: Biogas crop, Sorghum, dry matter yield, dry matter content, nutrient composition, methane yield

1. INTRODUCTION Increasing problems due to CO2 emissions arising along with the energetic use of fossil fuels and the need to meet requirements of climate protection as well as the awareness of the finiteness of this energy source have lead to a growing importance of plant biomass as energy and fuel source. Besides the growing of oilseeds (e.g. rape) the cultivation of energy crops for biogas production has become more and more important. From 2007 to 2010, the area used for biogas crops rose by 60 % from 400 to approximately 650 thousand hectares [6]. Maize is still the major biogas substrate with highest biomass yields. But in order to create alternatives to extremely maize-based energy crop rotations and to avoid problems arising from

2

Anja Hartmann, Karen Zeise and Maendy Fritz

monoculture such as decreasing biodiversity and growing pest and disease pressure it is necessary to focus on a wider range of crops. One of the future crops used in biogas crop rotations could be sorghum [1].

2. AIM AND BACKGROUND Sorghum is an interesting biogas crop for several reasons. It has a high yield potential and so far it is no host for the Western Corn Rootworm [2] and thus can be an alternative to maize in areas with high Rootworm infestation where crop rotation is a legal obligation. Besides, its fine root branching system advantages this plant in using water and nutrients from the soil that are less available for other crops [7]. Due to its ability to cope with many types of stresses, including heat and drought, sorghum can be an especially valuable crop in areas where water deficiency in summer is a problem. In view of the predicted global warming and the generally increasing risk of droughts [5][9] sorghum could gain importance in the future even in agricultural areas where till now adequate climatic conditions predominate. Field trials conducted in Saxony with sorghum showed that on light soil and under water deficiency, yields of sorghum can exceed yields of energy maize. Some sorghum cultivars produced 30 to 40 % more biomass than the tested maize cultivars [10]. In order to identify promising and climatically well adapted cultivars the Technology and Support Centre in the Centre of Excellence for Renewable Resources (TFZ), Straubing, accomplished a vast screening of sorghum cultivars. The cultivars were tested regarding their yield potential, dry matter content, which is critical for ensiling, their nutrient composition and the resulting theoretical methane production. The most promising sorghum cultivars and maize as a reference were grown at different experimental sites with different soil properties in order to compare the growth performance under varying water and nutrient conditions.

3. MATERIAL AND METHODS 3.1. Study Site The screening of sorghum cultivars was conducted over four years (2006 - 2009) in Straubing, Germany (longitude 48° 54´ N, latitude 12° 32´ O) at an altitude of 350 m above sea level. The average annual temperature is 8,3 °C and the average annual precipitation 783 mm. The soil (loess sediments) consists of a fertile silt loam with good water holding capacity. To compare growth performance under different nutrient and water conditions, in 2009 four sorghum cultivars were grown at an additional site (Aholfing) under similar climatic conditions but on a poorer and fast draining soil (loamy sand). Due to its low water holding capacity plants regularly suffer from water deficit during summer.

Sorghum as Biogas Crop

3

3.2. Experimental Design The screening comprised 305 sorghum cultivars/strains of the species Sorghum bicolor (L.) Moench ssp. bicolor and ssp. drummondii (sudanense) or a cross-bread of both as shown in Table 1. In the following these three subspecies are referred to as S. bicolor , S. bicolor x S. sudanense and S. sudanense. The cultivars or strains of S. bicolor were further divided into four types referring to their main intended use:    

forage (whole plant is used for grazing, ensiling as forage or as raw material in biogas plants) sweet (habitus resembles forage type but with a higher sugar content which is extracted from the stems and is used for producing syrup or ethanol) dual (used either as whole plant for biogas/feeding or for grain production) grain (starch is used for ethanol production or the grains as aliment).

The experiments were conducted in a randomized block design with 3 (Straubing) and 4 (Aholfing) replicates, respectively. Row spacing in Straubing was 37,5 cm with a seed density of 25 seeds/m². In Aholfing row spacing was 75 cm with a seed density of 20 seeds/m². The sample area used to calculate biomass yield per hectare was 10,8 m² surrounded by two rows of the same cultivar. Sorghum and maize were sown between end of May and beginning of June. Harvest was carried out from beginning to middle of October, the latest possible date to maximize biomass yield and dry matter content, but before first frost to avoid yield loss due to lodging. Table 1. Number of cultivars/strains included in the specific testing group Subspecies

Type

Number

S. bicolor

forage

117

sweet

14

dual

18

grain

79

S. bicolor x S. sudanense

66

S. sudanense

11

3.3. Crop Measurements During the growing season growth stage, plant height and the occurrence of diseases and pests were monitored continuously. At harvest samples of 1 kg were taken to obtain dry matter content (desiccation at 105 °C) and for the analysis of nutrients (desiccation at 60 °C).

4

Anja Hartmann, Karen Zeise and Maendy Fritz

Nutrient analyses were carried out according to standard protocols [14]. Based on the nutrient contents and their digestibility, the theoretical methane yield was calculated according to the formula published by Schattauer and Weiland [12], using digestibility coefficients from feed nutrient value tables for ruminants [4].

4. RESULTS AND DISCUSSION 4.1. Yield Performance and Dry Matter Content of Different Sorghum Types

35

30

t/ha

%

25

20

20 15

15

10

10

a

a

b

b

a

5 Dry matter yield

b

Dry matter content

S. x

co S.

bi

co

l.

bi S.

su da S. n. su da ne ns e

ai n)

lo r(

r( lo co bi

gr

du al

t) ee sw S.

S.

bi

co lo

r(

fo or ( bi co l

)

0

ra ge )

0

S.

5

Dry matter content

Dry matter yield

Dry matter yield differed significantly between the subspecies and among the S. bicolor types. Highest yield was achieved by the cultivars of S. bicolor classified as forage type. In average forage sorghum had a biomass production of 18 t of dry matter (DM) per hectare. The large standard deviation shows its high potential (see Figure 1). A maximum yield was produced in 2006 by cultivar ―Goliath‖ (26 t/ha). Dry matter yields of sweet types and of S. bicolor x S. sudanense cultivars were comparable (nearly 18 t/ha). Dual (15 t/ha) and grain type cultivars (14 t/ha) as well as S. sudanense (15 t/ha) cultivars showed significantly lower yields. Regarding the yield performance it becomes evident that S. bicolor cultivars (forage and sweet type) and S. bicolor x S. sudanense are most suitable as raw material for biogas plants since they had highest biomass production. Yield performance observed in the present trials are similar to results found in other trials conducted in Southern Germany where highest dry matter production was 23 t/ha (forage type) [3]. Under warmer climatic conditions and sufficient water supply dry matter yields of even more than 32 t/ha are possible [8].

Figure 1. Dry matter yield of different sorghum types grown between 2006 and 2009 in Straubing. Bars and dots represent the mean values ± SD per group. Different letters indicate significantly different mean values P < 0.05.

Sorghum as Biogas Crop

5

However, besides a good yield performance the dry matter content at harvest has to be taken into account when choosing appropriate cultivars. For loss-free ensiling the dry matter content should exceed 27 %. Lower dry matter contents can result in excessive effluent, a lower silage quality, and in a greater dry matter loss in the silo. Dry matter content was mostly critical for all cultivars tested in our trials. Grain and dual type of S. bicolor as well as S. sudanense showed highest dry matter contents (see Figure 1). Lowest dry matter contents were found in sweet and forage type cultivars. A study of Röhricht et al. [11], testing sorghum cultivation at several experimental sites within the scope of a German joint research project, confirms this conclusion. The best cultivars out of the 305 which were tested between 2006 and 2009 are shown in Figure 2. ―Goliath‖ and ―Herkules‖ are promising cultivars with outstanding yields and just acceptable dry matter contents. However, lodging was a severe problem since these high-yielding plants can grow up to more than 4 m. The early maturing ―Lussi‖ showed best results among S. bicolor x S. sudanense hybrids with a dry matter content which was comparable to maize.

24 Goliath

Dry matter yield

t/ha

Herkules

20 Green Grazer

18

Grazer N

Latte

Mithril

Zerberus Wotan Grazex II

Bovital

Lussi

Maja Inka

16 S. bicolor (forage)

S. bicolor x S. sudanense

0 0 22

24

26 28 30 32 Dry matter content

%

36

Figure 2. Dry matter yield and content of the best cultivars tested in Straubing between 2006 and 2009.

4.2. Yield Performance of Sorghum and Maize under Different Growing Conditions In the scope of our cultivar screening sorghum has shown its yield potential at our experimental site in Straubing, on fertile soil with sufficient water supply. Nevertheless, it is mostly discussed as an alternative to maize for regions less suitable for maize production due to heat or summer droughts. Thus, we compared the results obtained from our trials conducted in Straubing with data obtained from a second experimental site on poor sandy, fast draining soil (Aholfing). From Figure 3 it becomes evident that both crops, sorghum and maize, suffer clearly from inferior soil fertility and reduced water supply. Maize produced about 26 % less biomass in Aholfing than in Straubing. Yield loss of sorghum cultivars varied

6

Anja Hartmann, Karen Zeise and Maendy Fritz

between 24 % and 12 %. Yield differences between maize and sorghum were more distinctive in Straubing than in Aholfing, where even the S. biocolor x S. sudanense cultivars produced biomass quantities nearly comparable to maize. Other studies show that in warm and dry conditions sorghum can produce even 30 to 40 % more biomass than maize [10] though in that case climatic conditions were even less suitable for maize production than in Aholfing, where the crops suffered only from a temporary dry period in August. Another point to be taken into consideration is the sowing date. Maize and sorghum were sown at the same time (end of May) which enables us to compare their yield potential. Though in practice, chilling tolerance of maize allows the farmer to sow maize already at the end of April which results in superior yields. 30

30 Maize Reference S. bicolor (forage) S. bic x S. sud

20 15 10 100

80

99

84

Maize Reference S. bicolor (forage) S. bic x S. sud

t/ha

75

5

Dry matter yield

Dry matter yield

t/ha

20 15 10 100

98

102

101

93

5

Straubing

0

M KS aiz H e 83 0 W 1 ot an M aj a In ka

M KS aiz H e 83 0 W 1 ot an M aj a In ka

Aholfing

0

Figure 3. Dry matter yields of sorghum cultivars and a maize reference grown on two experimental sites (Straubing and Aholfing) with different soil properties and water supply. Bars represent the mean values ± SD per group. Values on the bars indicate the relative biomass yield given in percent in relation to the maize reference, which was set 100 %.

4.3. Nutrient Composition and Theoretical Methane Production and Yield Nutrient composition differed among the sorghum types. Crude protein and starch tended to be highest in grain type of S. bicolor whereas sugar content was lowest (see Table 2). Sweet type of sorghum which is preferably used for syrup production had highest sugar accumulation. Crude fiber content did not vary remarkably among all types though sweet, grain and dual types contained slightly less fiber than forage type, cross-breads or S. sudanense. The reduced fiber content of sweet sorghum can probably be explained by its retarded state of maturity at harvest. Such cultivars are bred to mature late in order to avoid that the panicle becomes a sink for nutrients, especially sugars, which would be transferred from the stem into the developing grain. Crude fat content is generally low in this crop and does not contribute to a considerable extent to its biogas potential. Despite the differences in nutrient composition the calculated theoretical methane production per kilogram of dry matter was nearly equal among all sorghum types (see Figure

Sorghum as Biogas Crop

7

4). Results varied between 280 and 284 Nl methane/kg oDM. The calculated methane production exceeds slightly the results shown by Röhricht et al. [11] and Schittenhelm [13] though it has to be taken into account that their studies only included a reduced number of cultivars whereas our data is based on an extensive screening. Compared to the methane potential of maize silage, which is estimated between 236 and 367 Nl/kg oDM [12], sorghum can achieve nearly equivalent amounts. Table 2. Nutrient composition [% DM] of sorghum types. Results represent mean values ± SD per group Subspecies/Type

Crude protein

Crude fat

Crude fibre

Starch

Sugar

S. bicolor (forage)

7,9 ± 1,2

1,2 ± 0,2

28,6 ± 2,8

0,2 ± 1,0

12,0 ± 2,8

S. bicolor (sweet)

7,5 ± 0,7

1,1 ± 0,1

25,6 ± 1,6

0,1 ± 0,3

18,6 ± 1,3

S. bicolor (dual)

9,1 ± 2,2

1,5 ± 0,2

26,6 ± 2,4

2,0 ± 3,0

11,4 ± 1,3

S. bicolor (grain)

10,1 ± 0,8

1,6 ± 0,4

24,8 ± 3,0

8,0 ± 10,5

5,4 ± 3,6

S. bicolor x S. sudanense

8,3 ± 0,8

1,4 ± 0,3

29,0 ± 2,6

2,8 ± 4,0

11,4 ± 4,2

S. sudanense

9,2 ± 0,6

1,5 ± 0,2

30,2 ± 2,9

3,0 ± 2,6

12,8 ± 5,3 350

8000 Methane yield

Methane production

Nl/kg oDM

6000

250

5000

200

4000 150

3000 2000

ac

a

bc

b

ac

ac

1000

ne ns e

su da

su d. x

. ic S. b

S.

S.

gr

ai

n)

) r(

co bi S.

S.

bi

co

lo

lo

(s or ol ic

S. b

r( du al

t) ee w

ra (fo or ol ic S. b

50 0

ge )

0

100

Methane production

Methane yield

m³/ha

Figure 4. Calculated methane production and yield of sorghum types (results from 2006 to 2009). Bars and dots represent the mean values ± SD per group.

8

Anja Hartmann, Karen Zeise and Maendy Fritz

In contrast to the lack of difference in methane production, the sorghum types showed significant differences in methane yield per hectare, which is the most important criteria evaluating a biogas crop (see Figure 4). Results were between 3730 and 5077 Nm³/ha. This range differs from results shown by Jäger [7] who estimated 3750 to 4100 Nm³/ha for forage sorghum. This disparity can probably be ascribed to lower dry matter yields underlying his data due to different growing conditions. Lowest methane yields were observed in dual and grain type of S. bicolor . This is attributed to the lower dry matter yield of these cultivars since harvested biomass has a greater influence on the methane produced per hectare than the nutrient composition.

CONCLUSION Results of a four year screening including altogether 305 sorghum cultivars or strains proof the high yield potential of this crop under the climatic conditions of Southern Germany. S. bicolor and the cross-bread of S. bicolor x S. sudanense comprise high-yielding cultivars with at least acceptable dry matter contents though this seems generally to be a critical point. Grain/dual type of S. bicolor and S. sudanense cultivars had higher dry matter contents but lower biomass yields and therefore seemed less suitable for biogas production. Outstanding S. bicolor cultivars like ―Goliath‖ or ―Herkules‖ can produce more than 20 t/ha DM. However, lodging was a problem with these tall plants. Consequently, besides increased yields a lower susceptibility towards lodging should be a focus of future sorghum breeding. Under optimal climatic condition maize is still superior in terms of dry matter yield and dry matter content. But under conditions less suitable for maize cultivation, with dry and hot periods, sorghum can have comparable or even superior biomass yields being a valuable alternative. In addition, problems arising from maize monoculture will favor an increasing cultivation of sorghum. Nutrient composition differed among the sorghum types but without significant impact on the calculated theoretical methane production per kg dry matter. Results varied between 280 and 284 Nl/kg oDM. Thus methane yield per hectare is mostly influenced by the harvested dry matter yield. Our results showed a possible methane yield range from 3703 to 5077 Nm³/ha. Cultivars for cultivation should consequently be chosen regarding their yield potential, their susceptibility towards lodging and their dry matter content at harvest to ensure loss-free ensiling. In conclusion we evaluate sorghum as a promising energy crop. Nevertheless, breeding efforts should focus on early maturing to achieve sufficient dry matter contents at harvest and on chilling tolerance to enable earlier sowing. An extended growing period would possibly result in higher biomass yields and in an advanced maturity stages at the end of the growing season.

ACKNOWLEDGMENTS The financial support by the Federal Ministry of Education and Research (BMBF) and the Bavarian State Ministry of Food, Agriculture and Forestry is greatly acknowledged. The

Sorghum as Biogas Crop

9

authors gratefully thank the technicians Alois Aigner, Josef Sennebogen, Benno Sötz and Michael Kandler for their excellent work and high motivation.

REFERENCES [1]

[2] [3] [4] [5]

[6] [7] [8]

[9]

[10]

[11]

[12]

[13]

[14]

Adam, L. (2008): Geeignete Rohstoffpflanzen zur Biogaserzeugung – Neue Fruchtarten: Sudangras und Zuckerhirse. Bauernblatt Schleswig-Holstein, vol. 34, iss. 62, p. 28-31. Berenji, J.; Dahlberg, J. (2004): Perspectives of Sorghum in Europe. J. Agronomy and Crop Science, vol. 190, p. 332-338. Böhmel, C. Jäger, F. (2007): Sorghum eine Ergänzung zu Mais?. Mais, vol. 34, iss. 4, p. 138-142. DLG (1997): Futterwerttabellen Wiederkäuer. 7. Auflage. DLG-Verlag, Frankfurt a. M., Germany. Ejeta, G.; Knoll, J.E. (2007): Marker-assisted selection in sorghum. In: Varshney, R.K. and R. Tuberosa (eds.), Genomic-assisted Crop Improvement Genomics Applications in Crops, vol. 2, p. 187–205. FNR (2010): Anbau nachwachsender Rohstoffe in Deutschland. Gülzow: FNR. URL : http://www.fnr.de/ (13.09.2010). Jäger, F. (2009): Vielfalt im Fermenter – Sorghum als sinnvolle Ergänzung im Energiepflanzenanbau? Neue Landwirtschaft. vol. 3, p. 98-101. Mastrorilli, M.; Katerji, N.; Rana, G. (1999): Productivity and water use efficiency of sweet sorghum as affected by soil water deficit occuring at different vegetative growth stages. European Journal of Agronomy, vol. 111, p. 207-215. Mehl, G.; Stocker, T.F. (2007): Global Climate Projections. In: Solomon, S.; Qin, D.; Manning, M.; Chen, Z.; Marquis, M.; Averyt, K.B.; Tignor, M.; Miller, H.L. (Hrsg.): Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA. Röhricht. Ch.; Zander, D. (2008): Anbau und Nutzung von Energiehirse. Schriftenreihe der Sächsischen Landesanstalt für Landwirtschaft. Dresden: Sächsische Landesanstalt für Landwirtschaft, vol. 2, 144 pages. Röhricht, C.; Zander, D.; Barthelmes, G.; Martin, M.; Knoblauch, S. ;Wagner, M.; Fritz, M.; Hartmann, A. (2010): Sorghumhirsen. Fortschritt. Joule - Agrarenergie, Technik, Politik, Wirtschaft, vol. 3, iss. 2, p. 80-83. Schattauer, A.; Weiland, P. (2009): Grundlagen der anaeroben Fermentation. In: Handreichung Biogasgewinnung und –nutzung. Fachagentur Nachwachsende Rohstoffe e.V. (Hrsg.), Gülzow, Germany; p. 30. Schittenhelm, S. (2010): Effect of Drought Stress in Yield and Quality of Maize/Sunflower and Maize/Sorghum Intercrops for Biogas Production. J. of Agronomy and Crop Science, vol. 196, iss. 4, p. 253-261. VDLUFA (1976): Die chemische Untersuchung von Futtermitteln. Methodenhandbuch. Band III, 4. Ergänzung 1997. VDLUFA-Verlag, Darmstadt, Germany.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 2

ENZYME MODIFIED GRAIN BY-PRODUCT REINFORCED POLYPROPYLENE COMPOSITES Abdullah Al Mamun, Andrzej K. Bledzki and Jürgen Volk1 Institut für Werkstofftechnik, Universität Kassel,Mönchebergstrasse- 3, 34125 Kassel, Germany 1 IGV GmbH, Arthur-Scheunert-Allee 40/41, 14558 Bergholz- Rehbrücke, Germany

ABSTRACT Rice husk reinforced polypropylene composites were fabricated using a high speed mixer followed by injection moulding with 40% of fibre load. Prior to composite production, the fibres were modified by protease, lipase and laccase enzymes. The modification process has been optimized regard as dosing rate and temperature. To observe the changes of the fibres surface functionality and morphology, the fibres were characterised by FT-IR. The effect of modification of the fibre was assessed on the basis of moisture resistance, mechanical, thermal properties of the resulting composites. It was found that the moisture absorption of the composites was reduced 35% due to modification. Tensile and flexural strength of composites were found to be better due to modification. The viscoelastic properties and melt flow rate have also been measured.

Keywords: Grain by-product, enzyme, moisture absorption, mechanical properties, rheology

INTRODUCTION In recent years, a special concern has been manifested towards ―green composites‖. Some of the effort has been introduced based on the use of new waste sources, with the aim to obtain biologically active compounds which can be applied in different fields and applications. These natural lignocellulosic cereal residues (by-products) are compatible with

12

Abdullah Al Mamun, Andrzej K. Bledzki and Jürgen Volk

the environment and could provide the sources for specialty chemicals [1, 2]. Grain byproducts are an annually renewable fibre and are available in abundant volume through out the world. According to April 2009 the world production of rice husk is 89.6 million metric tons [3]. The use of the cereal residues or by-products as a filler or reinforcement in the production of plastic composites alleviate the shortage of wood resources and can have the potential to start a natural fibre industry in countries where there are little wood resources left. The composite industries are looking into alternative low cost lignocellulosic sources, which can decrease overall manufacturing costs and increase properties of the materials [4, 5]. Wood fibre is the most widely used lignocellulosic natural fibre for reinforcing plastics. The demand of wood plastic composites (WPC) is increasing steadily with new application window in North America as well as Europe. Considering economic and ecology, wood fibre plastic established itself as standard material. Therefore, scientist from all over the world are searching new source which could be the proper alternative of wood fibre. According to source and availability, grain by-products are getting interest in the region of Asia, Europe and North America. The abundance of grain by-product is eco-friendly, available, cheap and which is complicated in term of cell geometry, morphology and chemical composition. It also has created an environmental issue such as fouling and attraction of pests [6]. Rice (Oryza sativa) is the seed of the monocot plant of the poaceae grass family. As a cereal grain, it is the most important staple food for a large part of the world's human population. It is the grain with the second highest worldwide production. Rice is probably the most important grain with regards to human nutrition and caloric intake, providing more than one fifth of the calories consumed worldwide. Rice husk is lignocellulosic agro waste which is 20 % of rice [7, 8]. The chemical composition of grain by-product fibre includes cellulose, hemicellulose, lignin, pectin, protein, fat, waxes and water soluble substances [9]. The greater proportion of cellulose lies within the elementary fibre in the form of crystalline and amorphous cellulose. Therefore, it can be expected that the fibre surface will be enhanced in lignin and hemicellulose material, which helps bind the fibres together. Grain by-products content relatively higher amount of fat and protein on the surface. The portion of wax and protein bonded together by covalent bond and which acts as a natural barrier to the environment. The plant cell barrier restricts intensively wettability and adhesion in composites and responsibles for odour and emission. The final properties of composites material depend on fibre properties (morphology, surface chemistry, chemical composition and crystalline contents) as well as matrix properties (nature and functionality). The adhesion between the reinforcing fibre and the matrix in composite materials plays an important role. The wetting of the fibre is an integrated step in the adhesion process. The interface depends on complex thermodynamics of fibre and matrix. Fibre properties i.e. composition, surface roughness and surface polarity have important contribution to fibre wettability. The use enzyme technology could increase substantially in the processing of natural fibre and the use of enzyme in the field of textile and natural fibre modification is also rapidly increasing. A major reason for embracing this technology is the fact that application of enzyme is regard as environmental friendly and the reactions catalysed are very specific with a focussed performance as a consequence. Other potential benefits of enzyme technology

Enzyme Modified Grain by-Product Reinforced Polypropylene Composites

13

include cost reduction, energy and water saving, improved product quality and potential process integration [10]. The application of enzymes to modify the surface of natural fibre, such as helm, flax, wool, cotton has been widely researched by industry. Most of the industrial applications are aimed to improve surface properties by removing adsorbed components, such as lignin, fats, vaxes, proteins and non crystalline parts. In the textiles processing areas, such as deseizing, scouring and bleaching of cellulose and woollen fabrics are some examples of successful biotreatments of textiles [11]. Not only unwanted adsorbed material may be removed but also modification of the fiber surface may also be accomplished by enzymes [12]. Besides defurring and antifelting treatments of textile, the so called biopolishing of cotton fabrics and garments is a good example. Cellulases are commonly used industrial enzymes to finish cotton. There are others industrial enzymes hemicellulases and pectinases are active on native cellulosic fibres (cotton, flax, hemp, jute, etc.) for bio scouring which is able to remove unwanted elements from fibre surface and simultaneously enhances wettabity and machinability. Some others industrial enzymes for instance protease, lipase and laccase which are active on the removing unwanted protein lipid, fat and wax form the fibre surface. Additionally the enzyme laccase could able to break down the aliphatic and aromatic ring molecules [10]. Structural materials (cellulose and starch) contents of rice husk are about 40% to 50 % and rice husk contents 3% fat, 4% protein and 11% lignin. According the chemical compositions of grain by-product enzymes protease, lipase and laccase enzymes are used for this research work. The main aim of this research was to study the potential of enzyme modification of rice husk on its properties and as reinforcements for thermoplastics.

EXPERIMENTS Materials Polymeric Matrix Polypropylene (Sabic PP 575P) was provided as granules by Sabic Deutschland GmbH and Co. KG, Duesseldorf, Germany. Its melting temperature was 173°C and melting index 3 was 10.5 g/10 min at 230°C. Its density at room temperature was 0.905 g/cm . Rice Husk Rice was grown up in Italy, 2006. Rice husk were collected via IGV institute, Potsdam, 3 Germany. The average particle size is 180- 350 µ and bulk density is 0.825 gm/cm .

Fibre Treatment The fibres were placed in an autoclave with demineralised water (1:6). The autoclave temperature was optimized and pH 7.5 was also adjusted using phosphate buffer and sodium

14

Abdullah Al Mamun, Andrzej K. Bledzki and Jürgen Volk

hydroxide. Enzymes protease (Liqanase), lipase (lipex) and laccase (lecitase) were collected from Novozymes A/S, Denmark. The experiment was carried out with different dosing rate and different duration. At the end of fibre treatment the enzyme were deactivate. The fibres were washed with water and dried in oven for 24 hours at 80°C.

Fibre Characterization Fourier-Transform Infrared Spectroscopy (FT-IR) A Nicolet 6000 FT-IR, Thermo Scientific, UK was used to obtain spectra for the rice husk. KBr disk sample preparation method was followed in taking infrared spectra. Fibres were ground and mixed with KBr at the ratio 1:99 then the mixer was pressed under vacuum -1 to form pellets. FT-IR spectra were recorded in a range of 4000 - 400 cm at a resolution of 4 -1 cm with 256 scans. Composites Preparation Rice husk with polypropylene were mixed by high speed cascade mixer (Henschel heatcooling mixer system, type HM40-KM120). Fibres were dried at 80°C in an air circulating oven for 24 hours (moisture content < 0.5%) before mixing. The fibre at 40% proportion and polypropylene was placed into hot mixer and heated up till to the melting temperature of polypropylene and then hot agglomerate granules were transferred to the cool mixer where hot agglomerate granules were cooled down to room temperature by the cold water. Then cold agglomerate granules were dried again (80°C, 24 hours) before the sample preparation by injection moulding process. Test samples were prepared from dried agglomerate by injection moulding process. Composites Characterization Tensile and flexural tests were performed at a test speed of 2 mm/min according to EN ISO 527 and EN ISO 178 using a Zwick UPM 1446 machine. All tests were performed at room temperature (23°C) and at a relative humidity of 50%. Charpy impact test was carried out using 10 notched samples according to EN ISO 179 using Zwick Charpy impact machine. In each case a standard deviation < 5% (drop weight) was used to calculate the Charpy impact strength. The melt flow rate of rice husk fibre composites were investigated using a Meltmixer 2000, Thermo Haake, Germany at 2.16 kg load and 230°C. Three specimens have been considered for each type of composites. The Vicat softening temperature of wheat husk, rye husk and soft wood fibre composites were investigated using a Heraeus, W.C. Heraeus GmbH, Germany at 50N load and heating rate 50°C per hour. The test was performed according to EN ISO 306. Three specimens have been considered for each type of composites.

Enzyme Modified Grain by-Product Reinforced Polypropylene Composites

15

RESULTS AND DISCUSSION To get the optimum enzyme activity on modification of the rice husk, the process temperature, dosing rate and duration have been optimised.

Temperature A certain percent of enzyme and two different treatment durations were considered for temperature optimisation. Three different temperatures (40°C-60°C) were taken with respect to weight loss of fibre which is shown in figure 1. It was observed that for the both treatment durations the weight loss is higher at 50°C. It may be concluded that 50°C is the optimum temperature for this system.

Figure 1. Enzyme activity on temperature.

Duration and Dosing Rate Figure 2 showed the enzyme activity on treatment duration and on enzyme content. It can be seen that the activity approximately linearly increased with treatment duration at the lower content of enzyme (till 1wt %). After then the enzyme activity increased a little bit with respect to treatment duration. On the other hand the enzyme activity increased slightly after treatment duration four hours. So it may be summarised that the optimum enzyme dosing rate is 1 wt% and the optimum treatment duration is four hours.

Figure 2. Enzyme activity on treatment duration and on enzyme content.

16

Abdullah Al Mamun, Andrzej K. Bledzki and Jürgen Volk

Fibre Characterisation -1

A shoulder peak at 1723 cm in the non treated rice husk spectrum is assigned to the C=O stretching of the acetyl and uronic ester groups of hemicellulose or to the ester linkage of carboxylic group of the ferulic and p-coumaric acids of lignin. On the other hand for the treated rice husk the shoulder nearly absent which indicates the reduction of hemicellulose or -1 lignin. The sharp peaks at 1643 cm for non treated rice husk were reflected for amide I. The amide I band represents 80% of the C=O stretching of the amide group, coupled to the inplane N-H bending and C-N stretching modes. The exact frequency of this vibration depends on the nature of hydrogen bonding involving the C=O and N-H groups and the secondary -1 structure of protein. The broadening of peaks at 1643 cm for enzyme treated rice husk were indicated reduction of protein content. -1 -1 -1 Sharp peaks at 1170 cm , 1047 cm and 730 cm for non treated rice husk were reflected CO-O-C asymmetric stretching, C-O-P stretching and CH2 rocking respectively which indicates the fat and lipids contented. On the other hand for enzyme treated fibre those peaks were absented. So due to modification the protein, fat, lipid and lignin were removed from the fibre surface.

Figure 3. FT-IR spectrum of treated and non treated rice husk.

Composites Properties Physical Properties Water absorption of the composites is examined by placing in conditioning cabinet at 50 o ± 3 C and 95% RH for the periods of 50 days. Samples are periodically removed from cabinet and were measured the weight gained in a balance. It was observed (figure 4) that the moisture absorption was reduced 30% to 35% due to modification and the composites containing fibre treated with laccase showed superior moisture absorption properties than other modified rice husk composites.

Enzyme Modified Grain by-Product Reinforced Polypropylene Composites

17

Figure 4. Moisture absorption of treated and non treated rice husk composites.

Mechanical Properties Flexural strength (Figure 5) is the ability of the material to withstand bending forces applied perpendicular to its longitudinal axis. The stresses induced due to the flexural load are combination of compressive and tensile stresses. The flexural strength of modified rice husk reinforced PP composites found to be 30 % to 40% better than rice husk composites. The tensile strength (Figure 5) of rice husk composites found to be 25% to 35% improved due to modification. The flexural and tensile strength properties of soft wood fibre composites are 39 MPa and 23 MPa respectively which comparable with rice husk composites. There may be the reason of removal of unwanted and amorphous materials [13]. The FTIR result proved that due to both modifications certain percent of protein, fat and lipids were removed from fibre surface. It was also scrutinized that a little small portion of lignin was removed from rice husk by the laccase treatment

Figure 5. Strength of treated and non treated rice husk composites.

The impact strength of a composite is influenced by many factors, including the toughness properties of the reinforcement, the nature of interfacial region and frictional work involved in pulling out the fibre from the matrix. The Charpy impact test is a standardized

18

Abdullah Al Mamun, Andrzej K. Bledzki and Jürgen Volk

high strain-rate test which determines the amount of energy absorbed by a material during fracture. This absorbed energy is a measure of a given material's toughness and acts as a tool to study brittle-ductile transition. Figure 6 shows the Charpy impact strength of rice husk composites. The Charpy impact strength of treated rice husk composites found to be a bit improvement but considering standard deviation there is insufficient changes. This could be explained by brittleness and local internal deformation exhibit relatively more for non treated rice husk composites [14]. On the other hand the Charpy impact strength of wood fibre composites is 3.1 mJ/mm2, which is lower than the rice husk composites.

Figure 6. Charpy strength of treated and non treated rice husk composites.

Rheology From the figure 7, it can be seen that the vicat softening temperature of rice husk composites increased 33°C with compare to control polypropylene. On the other hand due to modification the vicat softening temperature was further improved about 10°C.This is because of strong interfacial interaction.

Figure 7. Softening temperature of treated and non treated rice husk composites.

Enzyme Modified Grain by-Product Reinforced Polypropylene Composites

19

The melt flow rate of treated and non treated rice husk composites is shown in figure 8. It is seen that the melt flow rate reduced for all cases of fibre composites. This is because of the fibre content retards the molecular mobility of polymer. On the other hand slightly reduction of melt flow rate of treated rice husk composites was observed. It may be the further retard of molecular mobility due to strong interfacial bond.

Figure 8. Melt flow rate of treated and non treated rice husk composites.

CONCLUSIONS This study inspected the effect of enzyme modification of rice husk on fibre properties and its reinforced polypropylene composites properties. The following conclusions could be drawn;  

The following fibre modification parameters were observed; optimum temperature (50°C), enzyme dosing rate(1 wt%) and treatment duration ( 4hours) Due to modification, o Moisture absorption resistance increased 30% to 35%. o Strength properties increased 25% to 40%. o Vicat softening temperature increased about 10°C

REFERENCES [1]

[2]

Suddel B. C., Evans W. J.Ś Chapter 7 in ―Natural Fibers, biopolymers and biocomposites‖ (Eds. Mohanty A. K., Misra M., Drzal L. T.), Taylor and Francis, USA 2005, p. 231—259. Mohanty A. K., Misra M., and Drazel L.T.: Sustainable bio-composites from renewable resources: Opportunity and challenges in the green materials world. J. Polym. Evirn., 10, 19-26, (2002).

20 [3] [4]

[5]

[6] [7] [8] [9] [10] [11]

[12]

[13]

[14]

Abdullah Al Mamun, Andrzej K. Bledzki and Jürgen Volk World production data sheet, Foreign Agricultural Service, United States department of Agriculture, USA, www.fas.usda.gov. Panthapulakkal, S., Sain, M., Agro-residue reinforced high-density polyethylene composites: Fiber characterization and analysis of composite properties, Composites: Part A, 2007; 38, p.1445–1454. Bledzki A. K., Faruk O., Mamun A. A., Abaca fibre reinforced PP composites: Influence of fibre length and compounding processes on the mechanical properties, Journal of Polimery, 2008, 53, 2. Puglia D., Biagiotti J., Kenny L. M.: Journal of Natural Fibers 2004, 1, No. 3, 23. International Rice Research Institute The Rice Plant and How it Grows Retrieved, January 29, 2008. Duke, J.A. Handbook of Edible Weeds, CRC Press, Boca Raton, FL, 1992. Xue Li, Lope G., Tabil S. P., Chemical treatments of natural fiber for use in natural fiber-reinforced composites: A review, J. Polym. Environ, 15, 25–33, (2007) Aehle, W., Enzyme in industryś production and application, ―industrial enzymes‖, Wiley-VCH verlag, Germany, 2004. Fischer, H., Mussig, J., Bluhm,C., Marek, J., Autonov, V., Enzymatic modification of hemp fibres for sustainable production of high quality materials, 11th international conference on STRUTEX, Liberec, December, 2004. Saleem, Z., Rennebaum, H., Pudel, F., Grimm, E., Treating bast fibres with pectinase improves mechanical properties of reinforced thermoplasric composites, Composites science and technology, 68, 471-476, 2008. Bledzki, A. K., Mamun, A. A., Lucka, M., Gutowski, V. S., "The effects of acetylation on properties of flax fibre and its polypropylene composites‖, eXPRESS Polymer Letters Vol.2, No.6, 413–422, 2008. Bledzki, A. K., Mamun, A. A., Lucka, M., and Michalski, J., "Biological and electrical resistance of acetylated flax fibre reinforced polypropylene composites," BioRes. 4(1), 111-125, 2009.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 3

BIOMASS AS COMBUSTION FUEL – EXPERIENCES AND FINDINGS Grete Bach1*, Stefanie Bachmann, Daniel Kolb, Peter Kosel2† and Wendy Franke3‡ 1

Institut für Neuwertwirtschaft FTZ GmbH, Elsteraue OT Tröglitz 2 Kosel GmbHDorfstrasse 3, D-02991 Lauta 3 BEM Umweltservice GmbH Ludwigsburg

1. INTRODUCTION Biomaterials, especially wood, have become interesting as combustion fuel sources in the last years to save environment and to reduce greenhouse gas emissions and fossil fuel consumption. Special furnaces of small and large capacities have been constructed and wood is a common combustion fuel now [1]. But there are a lot of other renewable biomaterials that could be used as raw material for combustion, for instance agricultural and wild plants such as straw, miscanthus, plant mixtures, rapeseed and linseed straw and others. Agricultural crops are in concurrence with the food industry and therefore their use for combustion is under discussion in the last time, so that only residues of such crops, as e.g. straw of cereal crops and corn, linseed and rapeseed, husk of rice and sunflower but also corn and wheat residues from mills and oil mills are accepted as sustainable fuel for combustion. For wild plants these discussions doesn‘t play any role - so miscanthus, vetiveria-grass or jatropha should be an interesting alternative to the above mentioned raw materials here, especially in tropical and subtropical countries [2]. In addition also bio residues from production processes of the agriculture and food industries and bio waste are possible sources of combustion raw materials, among them sieve-

*

Institut für Neuwertwirtschaft FTZ GmbH, Dr.-Bergius-Strasse 19, D-06729 Elsteraue OT Tröglitz, Phone: ++49(0)3441- 53 88 45, e-mail: [email protected]. † Kosel GmbHDorfstrasse 3, D-02991 Lauta, Phone: ++49-(0)35722 – 3 69 10, e-mail: [email protected]. ‡ BEM Umweltservice GmbH Martin-Luther-Strasse 26, D-71636 Ludwigsburg. Phone: ++49-(0)7141-702 98-0, email: [email protected].

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Grete Bach, Stefanie Bachmann, Daniel Kolb et al.

remnants from composting are resources, which are under research and development now with the aim to widen combustion raw material resources. Up to now the German ecological legacy doesn‘t allow using such materials as fuel for little furnaces up to 50 kW except wood and straw, but an amendment for widening the accepted biomass-fuels is in preparation. For larger furnaces biomass residues can be used as th th substitute fuel without limitations if the emissions are according to 4 or 17 BImSchG, depending on the capacity of the furnace. So biomass has great chances to get a desired fuel in the future (Table 1). Table 1. Growth of biomass potentials in the European Community 2010-2030 /3/

Mio t

Use of biomass 2003

Potential 2010

Potential 2020

Potential 2030

Wood

59

43

39 - 45

39 - 72

Bio residues

3 biogas / 5 com.waste

100

100

102

Energy crops

2

43 - 46

76 - 94

102 - 142

Summary

69

186 - 189

215 - 239

243 - 316

There have been developed a great variety of furnaces for different kind of biomass fuels. Table 2. Dosation- and Combustion Systems for Biomass-Fuels Kind of fuel

max. diameter of particles

suitable dosation systems

suitable combustion systems

bulk material

< 5 mm

pneumatic dosage

Dust combustion, circul. Fluid bed

bulk material

< 50 mm

worm conveyer

dosage from below grate combustion, stat. cycl. Fb

bulk material

< 100 mm

dosage in pieces

grate combustion, BFB

bulk material

< 500 mm

dosage in pieces. Stoker dosage

grate combustion, BFB

bales

< 50 mm

pneumatic dosage worm conveyer

Dust combustion stat. and circul. Fluid bed

bales

whole

crane, Stoker dosage

grate combustion, Cigar bumer

Pellets

< 30 mm

worm conveyer

dosage from below grate firing, stat. and circ. Fluid bed

Pellets bed

< 120 mm

dosage in pieces Stoker dosage

grate combustion, station. Fluid

Biomass as Combustion Fuel – Experiences and Findings

23

To collect data and experience about the combustion behaviour of the above mentioned potential biomass-fuels we carried out research work in this field, concerning the analytical composition of different biomaterials with regard to their combustion properties, experimental lab scale studies of the combustion behaviour, analysing emissions and slag formation in connection with the respective environmental legality and also burning runs in 35 kWh- and 150 kWh- furnaces.

2. ANALYTICAL PROPERTIES OF DIFFERENT BIOMATERIALS AND BIO RESIDUES Biomaterials have specific properties different from those of common fuels: they have middle heat values, a high nitrogen content, leading to high NOx-emissions, high concentrations of K- and Na-chlorides, bearing the danger of corrosion, high amounts of minerals leading to high dust emissions and to slag formation, both highly undesired processes in combustion. We have characterized selected kinds of biomass analytically and got the below shown results (Tab. 3, 4) Table 3. Characterization of different kinds of biomass

Value

Wood

Compost remnants

Wooden dust

Miscanthus

Bio mix

Rapeseed Sunflower expeller husk

Heat value MJ/kg

18,46

18,32

19,16

17,62

17,52

17,03

17,37

Ash content % OS

0,45

2,87

0,52

2,87

4,06

6,95

1,50

Si % OS

5,52

6,70

3,81

20,75

4,0

22,3

2,07

K, Na % OS

4,67

7,61

3,78

17,75

14,7

13,19

21,05

Ca % OS

18,33

18,30

18,61

4,78

18,6

8,31

16,49

Table 3 shows, that the heat value of all kind of biomass is between 17,5…18,5 MJ/kg, i.e. somewhat lower than for fossil fuels with heat values clearly above 20 MJ/kg, but high enough to be used as combustion fuel, with the lowest permitted level of 11 MJ/kg. It is well known, that the overall content of minerals in energy crops is much higher than in fossil fuels, so a higher output of ash and a diminished melting temperature of ashes with the danger of slagging is to be expected when using them for combustion. Conspicuous is the extremely high content of silica in energy crops, especially in miscanthus and in rapeseed residues from the oil mills, which are much higher than those for wood and very much higher than for fossil fuels. So high dust formation during burning must be expected, which have to be diminished to the accepted limits, when using those kinds of biomass as combustion fuel.

24

Grete Bach, Stefanie Bachmann, Daniel Kolb et al.

A similar situation occurs with the sodium and potassium content, which is in wooden biomass 2 times, in energy crops even 10 times higher than in coal, causing a high risk of corrosion, having in mind, that both anions are chlorides, as can be considered from the relatively high content of chlorine in energy crops (Table 4). Table 4. Characterization of different kinds of biomass Value

Wood

Compost remnants

Wooden dust

Miscanthus

Biomix

Rapeseed expeller

Sunflower husk

Cl % OS

< 0,02

< 0,02

0,04

0,24

0,16

0,53

0,12

S % OS

0,05

0,05

0,09

< 0,05

0,16

0,12

< 0,05

The analytical studies confirmed the results of other authors, that energy crops and bio residues are quite good combustion fuels if their specific properties are taken into account. To realize this the combustion has to be led so that the emissions agree with the possible limits. The allowed emissions for combustion units of different capacity are given by the 1st, 4th and 17th BimSchV (Table 5) Table 5. Allowed emission limits for biomass fuels in Germany Capacity of combustion unit

Legacy

Rel. to O2

Emission limits

Vol.-%

CO g/Nm³

Summ…-C g/Nm³

NOx g/Nm³

Dust g/Nm³

Combustion of natural wood 50 - 50%) 3. Determining optimum harvest date for Silesia cultivar. Samples were taken on three dates: 1st harvest date – beginning of inflorescence formation (>50%), 2nd harvest date – full bloom of inflorescence (>50%), 3rd harvest date – full seed maturity (>50%). 4. Determining optimum sowing density of hemp cultivar Silesia. The following densities were tested [kg ha-1] 5, 10, 15, 20, 30, 40, 50 and 60. Samples were taken at full bloom stage. 5. The effect of time of day when inflorescence is harvested on the content of essential oils in Białobrzeskie cultivar. Samples were taken according to the following schedule: a)before sunrise b)between 800 - 900 a.m. c)early afternoon, between 1300 - 1400 d)right before sunset. 6. The effect of nitrogen fertilization level on essential oil content: nitro-chalk (NH4NO3 + CaCO3) applied once, before sowing. Samples of Biało-brzeskie cultivar were taken at full bloom of inflorescence. The following fertilization levels were applied: a)no fertilizer b)70 kg·ha-1 c)140 kg·ha-1 d)210 kg·ha-1.

Efficiency of Hemp Essentials Oil Depending on Sowing Density and Time…

33

Efficiency of essential oil (L·ha-1) was determined based on the content of essential oil in inflorescence and yields of panicles harvested at three harvest dates from experiments conducted in the Research Station in Pętkowo by Burczyk et al. [5]. Similarly, the efficiency of essential oil upon the sowing density was determined based on the content of essential oil in inflorescence and the yields of panicles harvested from the very same experiments [5]. The experiments mentioned above were carried out on the brown soil formed on clay with winter cereals being grown in previous season. The content of nutrients was as follows (calculated in mg/100g of soil): P2O5 = 10.0 - 25.5; K2O = 10.5 - 24.5; MgO = 5.0 - 6.2. Soil reaction varied from pH 5.50 to 6.50. Tillage and post tillage treatments were conducted according to the good agricultural practice, beginning from winter plough; in spring only necessary post tillage soil treatments were applied to reduce soil drying. Mineral fertilizers were applied right before sowing in the -1 -1 -1 following amounts: N- 90 kg·ha , P2O5- 80 kg·ha and K2O- 120 kg·ha . Seeds were planted in the third decade of April. Applied row spacing was 15 cm. No plant protection chemicals were applied. Harvest and sampling of inflorescence was conducted according to the experimental schemes described earlier. Samples were supplied to the chemical laboratory where dry weight of samples was determined and samples were prepared for essential oil content analysis. Evaluation of obtained results was carried out using variance analysis for split-plot scheme.

RESULTS The effect of genetic and agronomic factors on the content and efficiency of essential oils in hemp was evaluated using statistical analyses. The results of chemical analyses show the tendencies rather then a clear effect of particular elements compared in the field experiments [5]. The results presented in Table 1 and 2 confirm an important role of plant genotype in the content of essential oil in selected cultivars of hemp and prospective new lines. It was interesting to observe that a tendency for increasing content of hemp essential oil in Bialobrzeskie was stronger than in remaining cultivars and lines (Table 1). Among tested prospective lines only two of them (IWN- 304 and 608) were characterized by higher content of essential oils (Table 2). A significant effect on the essential oil content was determined by the harvest date. The results presented in Table 3 show that the content was the highest when inflorescences were harvested at full bloom stage. Worse results were obtained when the harvest was carried out at the beginning of inflorescence formation. Harvesting panicles at full seed maturity stage was connected with the lowest content of essential oils (Table 3). Sowing density of hemp showed no significant effect on the content of essential oils in inflorescences. However, the results presented in Table 4 show, that optimum sowing density -1 is 10-30 kg ha of seeds. The content of hemp essential oil was also determined as effect of time of day in which the panicles were harvested. The results of chemical analyses (Table 5) do not confirm this dependency.

34

H. Burczyk, R. Kaniewski, W. Konczewicz et al. Table 1. Essential oils content in panicle depending on harvest dates and cultivar of hemp [ %] Harvest time

Cultivar

2004

2005

2006

2007

2008

Average

Date 1

Białobrzeskie Beniko Silesia – Tygra

0,87 0,71 0,44

0,37 0,35 0,33

0,30 0,23 0,27

0,41 0,39 0,38

0,46 0,32 0,39*

0,48 0,40 0,36

0,41

Date 2

Białobrzeskie Beniko Silesia – Tygra

0,44 0,33 0,58

0,29 0,31 0,21

0,34 0,31 0,28

0,42 0,43 0,37

0,40 0,51 0,44*

0,37 0,38 0,38

0,38

0,40

0,42

Average

0,56

0,31

0,29

Average for harvest dates

LSD (0,01) for time of harvest, cultivar and years – irrelevant

* Tygra – only in 2008 year.

Table 2. Essential oils content in panicle of the new hemp lines [%] New hemp line

2004

2005

2006

2007

Average

IWN-104

0,45

0,32

0,46

0,44

0,42

IWN-204

0,58

0,21

0,59

0,53

0,48

IWN-304

0,50

0,38

0,61

0,51

0,50

IWN-507

0,70

0,37

0,29

0,39

0,44

IWN-608

0,75

0,44

0,50

0,51

0,55

Average

0,60

0,34

0,49

0,48

LSD (0,01) for years 0,04 LSD (0,01) for new line 0,01 LSD (0,01) for interaction 0,01

Table 3. Essential oils content in panicle depending on harvest dates of hemp [%] Years

I date

II date

III date

Average

2004

0,36

0,45

0,39

0,40

2005

0,31

0,34

0,23

0,29

2006

0,33

0,39

0,25

0,32

Average

0,33

0,39

0,29

LSD (0,01) for years 0,02 LSD (0,01) for harvest dates 0,01 LSD (0,01) for interaction 0,02

Efficiency of Hemp Essentials Oil Depending on Sowing Density and Time…

35

Table 4. Essential oils content in panicle depending on sowing rate of hemp [ %] kg∙ha-1 Years

5

10

15

20

30

40

50

60

Average

2004

0,36

0,35

0,44

0,43

0,46

0,39

0,37

0,44

0,40

2005

0,23

0,26

0,30

0,34

0,33

0,33

0,31

0,27

0,30

2006

0,29

0,42

0,27

0,28

0,37

0,30

0,39

0,28

0,32

Average

0,29

0,34

0,34

0,35

0,39

0,34

0,36

0,33

LSD (0,01) for years 0,06 LSD (0,01) for sowing rate 0,01 LSD (0,01) for interaction 0,01

Table 5. Essential oils content in panicle depending on the time of day of hemp harvest [%)] The time of day of panicle harvest

2004

2005

2006

2007

Average

a) Before sunrise

0,60

0,24

0,17

0,26

0,32

b) In the morning (700 - 800)

0,59

0,26

0,22

0,30

0,34

c) At noon (1200 – 1300)

0,56

0,30

0,20

0,29

0,34

d) Before sunset

0,53

0,25

0,29

0,28

0,34

Average

0,57

0,26

0,22

0,28

LSD (0,01) for years 0,01 LSD (0,01) for harvest time of day 0,01 LSD (0,01) for interaction 0,01

A positive effect of nitrogen application was found only when 70 kg·ha-1 dose was applied (Table 6). Content of essential oil in inflorescences was clearly different in different year of experiment (2004-2006), regardless to the compared factors. The highest content of essential oil was found in 2004 and the lowest in 2005 and 2006. Observations show, that this can be caused by the weather pattern during vegetation. In 2004 much less rainfall was recorded (238 mm) than in 2005 (270 mm) and 2006 (275 mm). Obviously, learning the weather pattern effect (rainfalls, temperature and air humidity) on the content of essential oils requires further and in-depth research. Despite the fact that inflorescence harvest dates and sowing densities have little effect on the content of essential oils in panicles, they do however, have a significant effect on panicles yields [5]. Due to the fact that efficiency of essential oils per ha depends on essential oils

36

H. Burczyk, R. Kaniewski, W. Konczewicz et al.

content in inflorescence and on the yield of panicles, the optimum harvest date and sowing density of hemp have a significant importance for the ultimate results. Table 6. Essential oils content in panicle depending on the nitrogen level of fertilization [%] The nitrogen level of fertilization in kg∙ha-1

2004

2005

2006

2007

Average

a) Without nitrogen

0,52

0,32

0,29

0,21

0,33

b) 70

0,57

0,34

0,37

0,23

0,38

c) 140

0,57

0,34

0,22

0,25

0,34

d) 210

0,49

0,31

0,23

0,20

0,31

Average

0,54

0,33

0,28

0,22

LSD (0,01) for years 0,02 LSD (0,01) for the nitrogen level of fertilization 0,01 LSD (0,01) for interaction 0,02

The highest efficiency of essential oils was obtained when panicles were harvested at full bloom stage as in this harvest date both the content of essential oil and the panicle yields are the highest. Delaying the harvest of inflorescences up to full maturity of seeds reduces the efficiency of essential oils by 18%. On the other hand, earlier harvest, at the stage of forming inflorescences reduces this efficiency by 26% (Figure 1). Efficiency of essential oil proved to be also significantly dependent on sowing density of hemp. The highest efficiency of essential -1 oils was obtained at sowing density 10-30 kg·ha . This range of sowing density secured good content of essential oils and the highest efficiency reaching 10 L per hectare (Figure 2).

Figure 1. Essential oil yield depending on the harvest time of panicle in years 2004-2006 [ l·ha-1].

Efficiency of Hemp Essentials Oil Depending on Sowing Density and Time…

37

Figure 2. Essential oil yield depending on sowing rate of hemp in years 2004-2006 [l·ha-1].

DISCUSSION Several years of experiments confirmed the hypothesis of slight effect of selected agronomic and genetic factors on of hemp cultivars on the content of essential oil in hemp inflorescences. Similar research was conducted by Ch. Meier and V. Mediavilla [8]. They were looking for the connection between hemp cultivars, sowing density and essential oil content. Authors found that no such correlation exists. They found, however, that the optimum date of inflorescence harvest is full bloom stage at which the content of essential oil reaches its maximum. Therefore, it can be assumed that the content of essential oil per area unit depends, most of all, on the yield of hemp panicles which is significantly influenced by both date of harvest and sowing density, and to the lower extent, by a cultivar. These findings have strong practical importance when selecting the direction of hemp application. When growing hemp for fibre and setting optimum date of harvest it is possible to obtain significant amounts of essential oils. Since 2007 obtaining essential oils has been implemented in Research Station in Pętkowo near roda Wielkopolska on the industrial scale. Implementation covered construction of distillery which allows to obtain the essential oils by water steam distillation. The method covers loading fresh inflorescences into distillation apparatus through the hatch located in the upper cover. After hatch is being closed, the distillation process begins. A distillate vapours go to a condenser and are directed from there to a receiving tank where essential oils are separated from the water. Obtained raw essential oil requires further purification and standardization in laboratory (Photo 1).

38

H. Burczyk, R. Kaniewski, W. Konczewicz et al.

Photo 1. Distiller for steam distillation of hemp essential oil in Experimental Farm Pętkowo N / roda Wlkp.

The efficiency of essential oil obtained by this method is by about 30% lower than figures obtained in laboratory from material obtained from experimental fields. Both, the results of research on what is the effect of selected factors on essential oil content and the efficiencies obtained from a two-year implementation project (2008-2008) in commercial scale (2-3 ha) show that these results can be implemented in the following two variants. First – used in hemp grown mainly for inflorescence harvesting and essential oils. The yields of essential oil can reach 6-8 litres per ha. The straw in this case will be an additional product and can be processed for fibre. Second – when hemp is grown for fibre with panicles usually being useless by-product, allows to obtain 3-5 litres of essential oils per ha. Both variants allow to improve, depending on what is the main goal of hemp cultivation, economical results of hemp cultivation by inclusion of additional direction of fibre or essential oil usage.

CONCLUSIONS 1. Based on the content of essential oil in inflorescences and essential oil efficiency per area unit, the results of many year research confirmed the use of hemp as a source of essential oil. 2. Research showed that there is no strong dependency between essential oil content and compared cultivars, new prospective lines, sowing densities, harvest in different time of day and nitrogen doses. However, some tendencies were observed when higher essential oil content (by about 20%) appeared in two new lines (IWN-304 and 608) and in inflorescences harvested at full bloom stage.

Efficiency of Hemp Essentials Oil Depending on Sowing Density and Time…

39

3. The efficiency of essential oil from area unit is more dependent on yields of panicles than on their content in inflorescences. Hence the highest efficiency of essential oil (about 11 L ha-1) was obtained when harvesting inflorescences at full bloom stage at sowing density 10-30 kg∙ha-1. 4. When hemp is grown for essential oil as the preliminary harvested commodity, it is strongly recommended to apply suggested sowing densities and harvest dates of panicles. On the other hand when growing hemp mainly for fibre, it is possible to harvest inflorescences separately and process them additionally into 3-5 L ha-1 of essential oil which allows for improving the economical results of hemp cultivation.

REFERENCES [1]

Burczyk H., 2003, Production of Hemp Sowing Seed in Poland. Journal of Industrial Hemp. Vol. 8 (1), 81-88. [2] Burczyk H., Kowalski M., Pławuszewski M., 2005, The trends and methods of hemp breeding in Poland. Journal of Natural Fibres, Nr. 2/ 2005. [3] Burczyk H., Grabowska L., Kowalski M., 2006, Industrial hemp as an alternative to wood pulp. Centre of Exellence in Plant Agrobiology and Molecular Genetics. PAGEN, PAN, Poznań, Vol.5, 159-168. [4] Burczyk H., Grabowska L., Kołodziej J., Strybe M., 2008, The industrial hemp as a raw material in the energy production. Journal of Industrial Hemp. Vol. 13 (1), 37-48. [5] Burczyk H., Grabowska L., Strybe M., Konczewicz W., 2008. Wpływ gęsto ci siewu i terminu zbioru konopi włóknistych na wydajno ć biomasy oraz elementów składowych plonu. Pamiętnik Puławski (w druku). [6] Łubkowski Z., 1968, Metodyka do wiadczalnictwa rolniczego. PNR i L, Warszawa. [7] Kołdowski M., Wysocka - Rumińska A., Tałałaj S., Wiszniewski J., 1955, Ro liny Olejkowe i Olejki Naturalne. PWR i L. Warszawa. [8] Maier Ch. and Mediavilla V., 1998, Factors influencing the field and the quality of hemp (Cannabis sativa L.) essential oil. Journal of the International Hemp Association, Vol. 5 (1), 16-20. [9] Malingre‘ th H., Hendriks S., Baterman R., Bos J., Visser J., 1975, The essential oil of Cannabis sativa L., Planta Medica , Nr. 28, 56-61. [10] Me Partland J. M., 1997. Cannabis as repellent and pesticide. Journal of the International Hemp Association.Vol.4(2), 89-94. [11] Mediavilla V. and Steinemann I., 1997, Essential oil of Cannabis sativa L. strains., Journal of the International Hemp Association.Vol. 4 (2), 82-84.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 5

IWN-11 THE NEW BIOSTYMULATOR FOR INCREASING CROPS RESISTANCE TO DROUGHT STRESS Krzysztof Heller1*, Małgorzata Byczyńska2 and Zenon Woźnica2†

1

Institute of Natural Fibers and Medicinal Plants, Poznań, Poland 2 Agricultural University of Poznań, Poznań

ABSTRACT IWN-11 is a new bio stimulator for plant protection (enhancing the resistance of crops to drought stress) formulated in Institute of Natural Fibres and Medical Plants Poznań, in cooperation with Poznań University of Life Sciences. IWN-11 active ingredient is acetylsalicylic acid (20 %) + adjuvants mixture (surfactants + emulgators) (80%). IWN-11 effects on water plants industry at the cellular level. It is not toxic, not absorbed and is biodegradable. Application of IWN-11 in conditions of controlled drought stress had a beneficial effect on growth, development and yields of fibre flax, corns, sugar and beet. The best results in winter triticale and spring wheat experiments were obtained when IWN-11 was applied at 1,0 l/ha and at BBCH 37 growth stage. In winter barley experiment better results gave IWN-11 applied at the same rate but at the earlier growth stage (BBCH 32). The corns grain yield increase resulted from positive influence of IWN-11 on number of ears/m2. IWN-11 modified fibre efficiency of flax in relation to straw yield and improved fibre quality.

Keywords: biostimulator, IWN-11, ASA, fibra flax, corns, drought stress, plant protection

*

Institute of Natural Fibers and Medicinal Plants,ul. Wojska Polskiego 71 B, 60-630 Poznań, Poland , [email protected]. † Agricultural University of Poznań, ul. Mazowiecka 45/46, 60-623 Poznań, [email protected].

Krzysztof Heller, Małgorzata Byczyńska and Zenon Woźnica

42

INTRODUCTION Water, often called the „the life solvent‖ determines the growth and development of all living organisms. Most physiological and biochemical reactions undergoes in water environment. Water plays the essential role in plant life as the substrate for biochemical reactions (photosynthesis), their product (breathing), takes part in catalysis of chemical reactions (e.g. of hydrolase). Water molecules stabilize the structure of proteins, nucleic acid, saccharides, determine cell and whole plant turgidity, control the moves of stomata, development of cells and tissues, proper temperature of organs, transport of metabolites, products of photosynthesis and other metabolites. It is very difficult to overestimate the importance of water (5, 7). In numerous regions of the globe water deficit in the habitat is the main factor limiting yielding of crops (2, 8). Water shortage, as a result of global climatic changes (4, 6), affects also Poland. The deficit of water in the habitat is the reason of inhibiting most processes that determine the growth and development of plants. Gradually as the shortage increases the following processes become hindered: cell growth, protein synthesis, activity of nitrate reductase, increase of ABA level (abscisic acid), decrease of cytokine level, closing of stomata, lowering photosynthesis rate, breathing disorders, proline and sugar accumulation, wilting, protoplasmic circulation inhibition. The most profound plant response to moisture deficit is the inhibition of cell growth, what directly affects the quantity and quality of yielding (2, 5). At the Institute of Natural Fibres and Medicinal Plants since 2002 the studies on increasing crop plants resistance to drought stress have been carried out (1,3); the studies include: Basic research:   

Drought effect on morphogenesis and ontogenesis of flax plants Determination of critical water periods in plant development Evaluation of drought tolerance of flax genotypes from INFandMP collection

Applicative research: 

Biological assessment of the effect of biostimulators, that increase plant tolerance to drought

As a result of the applicative studies on compounds that improve flax resistance to drought, a new formula for a biostimulator named IWN-11 was developed. The preparation IWN-11 (active substance – acetylsalicylic acid) resulted from mutual cooperation between INFandMP and Poznan University of Life Sciences in Poznan. The paper presented physical and chemical properties of IWN-11 and preliminary results of usefulness of the tested compound for crops.

IWN-11 the New Biostymulator for Increasing Crops Resistance to Drought Stress

43

MATERIALS AND METHODS Objectives The study was aimed at developing the formula and biological assessment of the biostimulator IWN-11 used for increasing plant resistance to water shortage in the habitat and determination of its effect on growth, development and yielding of plants.

Scope of Research In the laboratory conditions the formula of IWN-11 containing acetylsalicylic acid (ASA) was developed. In pot and field experiments the biological assessment of its usefulness for crops was tested. The pot experiments were made in randomized block method, in four replications at the Experimental Farm of INFandMP in Petkowo (Wielkopolska). In the experiment spring wheat, barley and rape and flax were cultivated. The biological assessment of usefulness in crop cultivation of the following biostimulators was done: ASA -1 -1 -1 -1 (in dose 0,4 kg ha ), Asahi SL (0,6 l ha ), IWN-11 (1,0-2,0 l ha ), IWN-21 (1,0-2,0 l ha ). For each crop two levels of Filed Water Capacity in the soil (FWC) were tested according to the method developed by Wanschaty. 45 % FWC was marked as optimal while 25 % FWC of soil indicated drought. The tested compounds were applied to the plants cultivated under water deficit conditions (FWC 25%) in the following periods of plant development:    

Spring wheat: BBCH 29 (end of flowering), BBCH 37 (visible flag leaf) Spring barley: BBCH 29 (end of flowering), BBCH 37 (visible flag leaf), Spring rape: BBCH 30 (before rape started shooting), BBCH 50 (flower buds closed inside leaves), Fibre flax: BBCH 32 (beginning of fast growth phase – plant height 20 cm).

Field experiments were done in Experimental Farm in Pętkowo and Experimental Farm in Stary Sielec (Wielkopolska). The usefulness of IWN -11 for the following crops was assessed:

    

Winter wheat – application time BBCH 29 (end of branching), BBCH 37 (visible flag leaf) Winter triticale - BBCH 29 (end of branching), BBCH 37 (visible flag leaf) Winter barley – application time: BBCH 29 (end of branching), BBCH 37 (visible flag leaf) Winter rape – application time: BBCH 30 (before rape started shooting), BBCH 50 (flower buds closed inside leaves), Sugar beet: application time: BBCH 14 (4 proper leaves), BBCH 18 (8 proper leaves).

The following observations and measurements were conducted during the experiment:

Krzysztof Heller, Małgorzata Byczyńska and Zenon Woźnica

44    

general data – soil, weather observations – temperature and moisture measurements, growth and morphological development of plants (ontogenesis and morphogenesis), phyto-toxic effect of the studied compounds on the plants, yields and their quality (seeds, corns, sugar beet roots and leaves) yields of fibre flax (straw, quality of straw, fibre yield and its quality).

RESULTS Laboratory Experiments The laboratory tests resulted in preparing the formula for IWN-11 (active substance ASA), characterized with good water solubility, high durability and homogeneity and low surface tension of the spraying liquid (Figure 1).

Figure 1. Surface tension (ml/m) of spray liquid (200 l/ha).

Chemical-physical characteristics of IWN-11    

Composition of IWN-11 – active substance ASA (acetylsalicylic acid) - 20% + 80% mixture of supplementary substances (emulsifier + surfactant), pH – 5,75, density of the solution – 1,142 [g/ml] surface tension of spray liquid with using the dose of IWN-11 – at 1,0 l/ha in 200 l/ha of water (30,67 ml/m). (for ASA solution – surface tension was twice as high (61,68 ml/m) (Figure 1).

Pot Experiments (EF Pętkowo) Spring Wheat Better results at increasing wheat tolerance to drought were achieved with using biostimulators in the later stage i.e. BBCH 37 (visible flag leaf) – growth in grain yield was

IWN-11 the New Biostymulator for Increasing Crops Resistance to Drought Stress

45

by 32,8 % - 74,1 %. Among the compounds used in that period, the most effective proved to -1 -1 be ASA (0,4 kg ha ) (increase in grain yield by 74,1 %) and IWN-21 (1 l ha ) (increase in grain yield by 57,1 %). Using the compounds at the earlier stage (BBCH 29 – end of -1 branching) resulted in higher grain yield by 8,1 % - 26,0 % (IWN -11 in dose 1 l ha ). Introducing drought stress (FWC of the soil at 25 % – compared to optimal FWC of 45%) caused decrease of grain yield by 53,2 %. Spring Barley During the pot experiment conducted in 2008 in Petkowo, spring barley turned out to be more resistant to drought stress than spring wheat. The drought stress at 25% FWC of the soil resulted in yield drop by only 2,8 %. Under those conditions the best yields of grain were -1 achieved after application of IWN -21 (2,0 l ha – in the phase BBCH 37). Spring Rape The tested biostimulators IWN-11 and IWN-21 were more efficient in improving plant tolerance to drought when used in the earlier time i.e. at the phase BBCH 30 (beginning of elongation of the shoot) as compared with the later time i.e. BBCH 50 (flower buds closed inside the leaves). Among the compounds used in that time applying IWN-21 in dose of 2,0 l -1 ha turned our to be the most successful (grain yield increase by 29,3 %). Fibre Flax -1 IWN-11 in dose of 1l ha applied at BBCH 32 (beginning of fast growth phase) was the most efficient in protecting fibre flax against the effect of drought.

Preliminary Field Trials Winter Triticale Better results were obtained when the biostimulator IWN-11 was used at the later time i.e. at BBCH 37 (visible flag leaf) – then the growth of grain yield reached 20,2 % - 23,6 %. The same compound applied earlier, at BBCH 29 (end of branching), resulted in the increase by 10,8% – 13,3 %. Yield generating effect of IWN-11 involved its beneficial influence on 2 average number of straws bearing ears (item/m ) and also mass of thousand seeds in triticale yield (g). Winter Wheat In the experiment at EF Pętkowo, the biostimulators tested were more efficient, when they were used in the earlier stage of wheat development - BBCH 29 (end of branching). The highest increase of grain yield (by 15,0 %) was obtained with the use of IWN-11 in dose 1,0 l -1 ha for wheat at BBCH 29. During the experiment conducted at EF Stary Sielec the biostimulators did not show yield generating effect on winter wheat.

Krzysztof Heller, Małgorzata Byczyńska and Zenon Woźnica

46

Winter Barley Better results were achieved with IWN-11 applied in the earlier stage of growth i.e. at BBCH 29 – end of branching. The yield growth varied between 31,0 % (IWN-11 in dose 2,0 l/ha) and 46,8 % (IWN-11 – 1,0 l/ha). Yield increasing effect of the compound is linked to its 2 beneficial influence on the number of ear bearing straws (items/m ) in winter barley. Winter Rape In both experiments at EF in Pętkowo and Stary Sielec no beneficial effect of the tested biostimulators on growth, development and yielding of winter rape was observed. Sugar Beet -1 -1 -1 Biostimulators: ASA (0,4 kg ha ), Asahi SL (0,6 l ha ), IWN-11 (1,0-2,0 l ha ) were applied in two different stages: BBCH 14 (4-leaf phase) and BBCH 18 (8-leaf phase). The latter time proved to be more promising i.e. BBCH 18. At EF Pętkowo the highest increase of -1 root yield (+ 21,1 %) was obtained when IWN-11 (2,0 l ha ) was applied at BBCH 18 (Tab. 1).

Table 1. The effect of biostimulators on sugar beet field (2008, EF Pętkowo) Treatment

Dose (kg, l/ha)

Untreated ASA * Asahi SL * IWN-11 * IWN-11 * ASA ** Asahi SL ** IWN-11 ** IWN-11 ** LSD

0,4 0,6 1,0 2,0 0,4 0,6 1,0 2,0

Yield (dt/ha) Total 1219,0 1090,0 1269,0 1160,0 1146,0 1289,0 1397,0 1320,0 1412,0 277,2

Roots 852,4 734,5 856,2 814,9 808,7 873,8 1028,1 929,0 1032,5 209,7

Leaves 366,6 355,8 412,7 345,2 337,6 415,1 369,2 389,1 379,0 96,6

* growth stage of sugar beet (BBCH 14). ** growth stage of sugar beet (BBCH 18).

Table 2. The effect of biostimulators on sugar beet field (2008, EF Stary Sielec) Treatment

Dose (kg, l/ha)

Untreated ASA * Asahi SL * IWN-11 * IWN-11 * ASA ** Asahi SL ** IWN-11 ** IWN-11 ** LSD

0,4 0,6 1,0 2,0 0,4 0,6 1,0 2,0

* growth stage of sugar beet (BBCH 14). ** growth stage of sugar beet (BBCH 18).

Yield (dt/ha) Total 1050,0 1050,0 1140,0 1097,5 1275,0 1297,5 1090,0 1432,5 1365,0 64,06

Roots 815,0 675,0 845,0 800,0 1000,0 870,0 800,0 1045,0 1030,0 62,99

Leaves 235,0 285,0 295,0 297,5 275,0 427,5 290,0 387,5 335,0 31,44

IWN-11 the New Biostymulator for Increasing Crops Resistance to Drought Stress

47

In the field experiment at EF Stary Sielec the best results were achieved for IWN-11 in dose 1,0 l/ha used at BBCH 18 (increase by 28,2 %) (Tab. 2)

CONCLUSIONS 1. The compound IWN-11 (active substance - ASA) is a compound environmentally safe, characterized with good water solubility, high durability and homogeneity and low surface tension of spraying liquid. 2. Applying IWN-11 resulted in improvement in growth, development and yielding of corns, sugar beet, spring rape and fibre flax. 3. Yield generating effect of IWN-11 in corn cultivation was linked to its beneficial influence on the number of ear bearing straws.

REFERENCES [1]

[2] [3]

[4]

[5] [6] [7] [8]

Byczyńska M. and Heller K. 2004: The influence of drought stress on morphogenesis of fibre flax cultivars. The Proc. of the 3th Global Wokshop „Bast Fibrous Plants for Healthy Life‖. Banja Luka, Bosnia and Hercegovina, Republik of Srpska, October 2428. CD Gupta U.S.2007: Physiology of Stressed Crops. Vol. V, Membrane System. Ed.Science Publishers, Georgia, USA: 403 Heller K., Rólski St. Byczyńska M. 2006: The application of ASA (acetic salicic acid) for increasing fibre flax plants resistance to stress of drought. Vol. 7. Ed. Górecki H., Dobrzański Z., Kafarski P. Ed. Czech-Pol. Trade. Czech Republic: 293-302 Houghton J. T., Ding Y., Griggs D.J., Noguer M., van der Linden P.J., Maskell K., Johnson C.A. 2001: Climate Change: The scientific Basic. Cambridge University Press: Cambridge. Kozłowska M. 2007: Fizjologia ro lin. Wyd. PWRiL. Poznań: ss.544 Lipa J.J. 1997: Zmiany klimatu ziemi – konsekwencje dla rolnictwa i ochrony rolin. Progress in Plant Protection/ Postępy w Ochronie Roślin. Vol. 37, No 1: 27-35. Singh S.K. 2005. Plant Physiology. Ed. Campus Books Int. New Delhi, pp. 342 Widtsoe J. A. 2007. Dry Farming for Sustainable Agriculture. Ed. Agrobios, India:361

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 6

YELLOW NATURAL DYESTUFFS AND THEIR LIGHT FASTNESS PROPERTIES AND UV PROTECTION ON NATURAL FABRICS Katarzyna Schmidt-Przewoźna and Jakub Kowalinski Institute of Natural Fibres and Medicinal Plants, Poznan, Poland Laboratory of Natural Dyeing

The Institute of Natural Fibers and Medicinal Plants has been carrying out complex research connected with application of natural dyes on fabrics. Colors of nature, obtained from various plants, have contributed to creating a collection of clothes produced from linen and silk fabrics. The aim of the studies is evaluation and comparison of possibility and properties of natural yellow colours obtained from natural plants. In our study we use: Weld Reseda luteola L., Dyer‘s chamomile Anthemis tinctoria L., Dyers corepsis Coreopsis tinctoria L., Coreopsis Coreopsis grandiflora L., French marigold Tagets species L., Dyer's greenweed Genista tinctoria L, Safflower Carthamus tinctorius L. Tumeric Curcuma longa L., Cuth Accacia catechu, Henna Lawsonia inermis L., Kamala Mallotus philippinensis Muell. Common belief that these dyes have poorer color fastness is not justified. The results of tests have shown that naturally dyed linen and hemp fabrics are characterized with good resistance to sweat, washing and light. Common belief that these dyes have poorer color fastness is not justified. The results of tests have shown that many naturally dyed fabrics are characterized with good resistance to sweat, washing and light.

EXPERIMENTAL Materials Historical collection of dyeing plants was established in Institute experimental plantation

50

Katarzyna Schmidt-Przewoźna and Jakub Kowalinski

Petkowo in 2003. The garden has been used as a place for trainings and experiments directed for agriculture, environmental protection students, artwork conservators and artists. Natural dyes are the sources of a huge palette of colors with few limitations of fastness and brilliancy of shade. All but one plants cultivated in our experimental station are mordant dyes.

1. The Sources of Natural Dyestuffs Cultivated in Experimental Farm INF Yellow Colors Weld Reseda luteola L., Dyer‘s camomile Anthemis tinctoria L., Dyers corepsis Coreopsis tinctoria L., Coreopsis Coreopsis grandiflora L., French marigold Tagets species L., Dyer's greenweed Genista tinctoria L, Safflower Carthamus tinctorius L.

2. Natural Sources of Color Used in Our Study of Colors are Weld Reseda luteola L., Tumeric Curcuma longa L., Dyer‘s camomile Anthemis tinctoria L., Dyers corepsis Coreopsis tinctoria L., Coreopsis Coreopsis grandiflora L., French marigold Tagets species L.,Common knotweed Polygonium aviculare L., Dyers Corepsis Corepsis tinctoria L., Annatto Bixa orellana L., Cuth Accacia catechu, Henna Lawsonia inermis L., Sappanwood (red wood) Calsapinia sappan,

Experimental farm, Petkowo. Figure 1. Dyer‘coreopsis Coreopsis tinctoria L.

Experimental farm, Petkowo. Figure 2. Safflower Carthamus tintorious L.

Yellow Natural Dyestuffs and their Light Fastness Properties and UV Protection…

51

3. Method of Dyeing 3.1. Extraction of Dye The plants are crushed to small pieces and soaked in hot water overnight, boiled one hour and filtrated. 3.2. Mordants In our methods we used: Oak galls, Sodium carbonate anhydrous, Copper sulphate, Citric acid ,iron - ferrous sulphate and Alum - potassium aluminium sulphate. 3.3. Development of Color on Linen, Hemp and Silk Fabrics 3.4. Equipment Laboratory dyeing machine: EASYKROME UGOLINI

MEASUREMENT OF COLOR FASTNESS 1. Light Fastness Color Fastness to Sunlight To observe the effect of sunlight on the color fastness linen and silk samples were tested on Laboratory Machine Xenotest 150. The test was carried out according to the standard PNISO 105-B02:1997. 30 naturally dyed samples were exposed to sunlight for 200 hours. After time of light exposure, the samples were graded for color fastness.

2. Measurement of Color Fastness to Wash The changes of color linen and silk samples were assessed in the Grey scale [1-5]. Testing Washing Fastness with the Laboratory Dyer Ugolini

       

according to the standard PN-ISO 105-C06:1996 Preparation of washing bath: 4g of washing agent per 1 l of water Preparation of the samples of naturally dyed and reference fabrics. For Tests A and B Linen: reference fabrics- linen and wool Silk: reference fabrics - silk and cotton Test conditions A1M: temp 40ºC, time: 45 minutes, For natural silk crepe and silk shantung temperature of 30ºC and duration of 45 minutes have been applied.

Katarzyna Schmidt-Przewoźna and Jakub Kowalinski

52

Table 1. Light and washing fastness naturally dyed linen and silk samples No

Fabric

Natural dyestuff

mordant

color

1

3

Linen

Green yellow Green yeallow yeallo

4

Knitted silk Knitted silk linen

French marigold Tagets species (kwiaty bordo) French marigold Tagets species French marigold Tagets species (yellow flowers) Kamala Mallotus philippinensis Weld Reseda luteolaL.

alum

2

Silk Shantung Linen

Cuth Accacia catechu

Copper sulphate Alum = copper sulphate alum

5 6 7

Silk Shantung

Dyer‘s chamomile Anthemis tinctoria L

8

Linen

9

Silk

10

Silk

11

Silk

12

Linen

13

Silk

14

Knitted silk Linen

French marigold Tagets species (yellow flowers) French marigold Tagets species (yellow flowers) Safflower Cartamus tintorius L Dyer‘s chamomile Anthemis tinctoria L French marigold Tagets species (kwiaty bordo) French marigold Tagets species (kwiaty bordo) Annato Bixa orelana

15

Annato Bixa orelana

Wash fastness 5

Light fastness 4-5

3 change of shade 4

5

Gold yellow Light khaki brown

5

3

4

5

3

5

Green yellow

3

7

yellow

3

4

alum

yellow

3

3

Citric acid Citric acid ferrous suplhate Ferrous suplhate Washing soda Washing soda

Peru brown beige

5

3

3 change of shade 4 chamge of shade 4

4

4

2-3

4

5

alum alum alum alum

gray beige Dark orange orange

5-6

2 5

3. Results and Discussion Color fastness to sunlight on average was fair to good. Light fastness of dyed samples were found to be good [5] for 6 samples, very good [6] for 1 sample. The washing fastness tests according to the standard used for man-made dyestuffs have shown that this standard is not suitable, especially for silk fabrics. The natural silk fabrics are not washed in automatic washing machines but they are only hand-washed. Natural silk is sensitive to temperature higher than 30°C, differences in temperature during rinsing and to friction. Improper washing of fabrics and knitwear made of silk can totally destroy it e.g. cause complete discoloration. Silk must be laundered very delicately (without pre-soaking) in water at 25-30°C, with the use of mild liquid detergents or soap flakes. The fabrics dyed with natural dyestuffs are hand-washed in lower temperatures with mild washing agents otherwise the change of environment can change the hue of the fabric. The study on washing fastness of

Yellow Natural Dyestuffs and their Light Fastness Properties and UV Protection…

53

silk was done in temperature of 30ºC and a mild detergent. In top class washing machines there are special programs for silk (man-made or viscose) for 1kg of dry clothes, temperature of 30ºC and time of washing at 35 minutes. The results indicate that silk and linen samples from our study naturally dyed posses good to very good wash fastness evaluated as 4 and sometimes 5 on the grey scale in temperature of dyeing 30ºC for silk and 40ºC for linen.

UV PROTECTION FACTOR OF NATURALLY DYED LINEN, HEMP AND SILK The study comprised also determining Ultraviolet Protection Factor (UPF) of linen, hemp and silk fabrics dyed by natural dyestuffs. It also described the influence of fabrics structure, color, method of dyeing on level UV protection. Fabrics: Fabric A - 43002 thin linen Silk A – silk knitwear 100% Fabric B - 30187 thick linen Silk B –silk shantung 100%

UPF PROTECTION ON LINEN AND SILK The Laboratory of Physiological Influence of Textiles on Human Body has done research to compare the result of UPF protection on linen and silk samples dyed with natural dyes. There are many ways increasing the UPF barrier effect. The transmission, absorption, and reflection of UV radiation are in turn dependent on the fibre, fabric construction (thickness and porosity) and finishing. Many dyes used in finishing process, absorb UVR. Darker colors of the same fabric type (black, navy, dark red) will usually absorb UVR more than light pastel shades and consequently will have a higher UPF rating. The finishing of textiles plays a very important role in eco-production. Natural fibres show good sun protection thanks to contents of natural pigments like lignin, waxes and pectins that act as UFR absorbents. UPF barrier effect can be obtained also with the use of special UV blockers, which are generally used in medicinal products and cosmetics. There is a very wide list of UV blockers presented in ―Chemical@Engineering News (April, 2005). [6] In this study numerous colours applied on silk and linen were analyzed. Determination of the UVR transmission of a dry textile was done in accordance to Australian/New Zealand Standard and British Standard for sun protection clothing with the use of Cary 50 Solascreen apparatus. Table 2. UPF classification system (according to the Australian Standard)[1] UPF RANGE 15-24 25-39 40-50, 50+

UVR protection category Good but insufficient protection Very good protection Excellent protection

UPF Ratings 15,20 25,30,35 40,45,50,50+

Katarzyna Schmidt-Przewoźna and Jakub Kowalinski

54

Table 3. The results of UPF on linen and silk samples dyed by natural red dyestuffs. [4,5] No

Samples

1 2 3

Linen hemp linen hemp linen

4

Natural Dyes

Mordant

Color

UVA

UVB

UPF

Tumeric Tumeric Tumeric

No mordant No mordant Pre-mordant Pre-mordant No mordant

10,069 11,696 4,456 3,713 3,543

5,455 8,4 3,036 3,567 3,305

10 5 25 20 20

hemp

Tumeric

No mordant

6,592

5,470

15

5

linen

Tumeric

2,907

2,011

30

6

hemp

Tumeric

3,758

3,614

25

7

linen

Tumeric

Pre-mordant+copper sulphate Pre-mordant+copper sulphate Pre-mordant+soda

3,302

2,53

30

8 9

hemp linen

Tumeric Tumeric

2,789 4,022

2,477 3,364

25 30

10

hemp

Tumeric

5,33

5,08

15

11

linen

Tumeric

White white yellow yello Light yellow Sun yellow Olive yellow Dark yellow Sahara yeallow beige Sun yellow Bright yellow olive

4,235

2,614

30

12

hemp

Tumeric

2,772

2,532

30

13

linen

1,646

1,979

30

14

Linen

Soda

Old gold

1,854

2,137

40

15

linen

Citric acid

gold

2,237

2,939

35

16

linen

Copper sulphate

Old gold

0,967

1,255

50

17

linen

Ferrous sulphate

1,117

50

Linen

No mordant

Dark brown brown

0,881

18

1,816

2,178

35

19

linen

Dyers Coreopsis Dyers Coreopsis Dyers Coreopsis Dyers Coreopsis Dyers Coreopsis Common knotweed Henna

Olive brown Old gold

No mordant

rust

1,739

2,438

35

Pre-mordant+soda Pre-mordant+citric acid Pre-mordant+citric acid Pre-mordant+ferrous suplhate Pre-mordant+ferrous sulphate No mordant

The study was concluded according to: PN– EN ISO 13758-1:2002

COMMENTS 1) The value of UPF linen and silk fabrics depends on: product structure, density of thread, thickness, kind of used dyestuffs, color and kind of fabrics 2) The result of the comparison of UPF on linen and silk fabrics:

Yellow Natural Dyestuffs and their Light Fastness Properties and UV Protection…

55

Excellent UVR Protection We Obtained on Samples 1. 2. 3. 4.

Dyers Coreopsis (linen ) – soda 50+ Dyers Coreopsis (linen ) - copper sulphate 50+ Dyers Coreopsis (linen ) - ferrous sulphate 50+ Dyers Coreopsis (linen ) – Soda 40

Very Good Protection We Obtained on Samples    

Henna (linen) - no mordant Common knotweed (linen) -no mordant Dyers Coreopsis (linen) – citric acid Dyers Coreopsis (linen) - no mordant:

35 35 35 30

CONCLUSIONS A vision of the world of natural dyes renewed both by the awareness of threads on natural environments and recent interdisciplinary research gives a new opportunity for designing unique collection of clothes. Recently, considerable attention has been paid to the barrier properties of textiles designed for clothing as a protection against UV radiation, while also taking into account the trends of current fashion. The findings reported in the literature concerning the barrier properties of fabrics in relation to UV radiation show that attention has been focused on the physical aspect of barrier properties of fabrics or yarns used for fabric production. Linen and silk fabrics with good UV properties are healthy eco-product.     





Fabrics used in the summertime apparels often provide poor protection against UV because they are usually made from light–to-medium weight fabrics. Dyeing and finishing process improve sun protection properties, irrespective of chemical nature of the fibres. The study of UPF on linen and hemp samples dyed by natural dyestuffs showed that many samples have very good and excellent sun protection. The main focus in ―eco product‖ production is placed on finishing methods. However, such processes may not be considered without relation to the materials, yarn manufacturing, knitting and weaving. In our research we are interested eco technology in natural dyeing and creation of modern, ecological textile products. The aim of the Institute‘s research is to create ecological clothing with the best parameters and high comfort which also protects from harmful UV radiation. Apart from its traditional functions (protection against changeable weather conditions and mechanical damage of skin, fashion and creating self-image) clothing must now perform other functions – i.e. must act as a barrier against harmful UV radiation. Results of our work were patented and we got trade mark ―Color of nature‖.

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Katarzyna Schmidt-Przewoźna and Jakub Kowalinski

Figure 4. ―Color of nature‖ – patent submission. no. Z-3363353 for naturally dyed fabrics and collection.

Figure 5. Linen and silk fabrics naturally dyed.

Figure 6. Linen and silk fabrics naturally dyed.

Yellow Natural Dyestuffs and their Light Fastness Properties and UV Protection…

57

Linen fabrics are cool, show anti-bacterial activity, do not collect electrostatic charges, do not cause skin irritations or allergic reactions – they create the microclimate best for human skin. In summertime linen clothing feels the best. Colours of naturally dyed fabrics add to the esthetics of linen and, as our studies have shown, a large group of such fabrics protects very well against UV radiation which is their added value.

REFERENCES [1] [2] [3] [4]

[5]

[6]

Sun protective clothing – Evaluation and classification – Australian / New Zealand Standard. rd M.T. Pailthorpe: ―Sun Protection and Apparel Textiles‖ – The 3 Asian Textile Conference, (1995). J. Rupp., A Bohringer., A Yonenaga., J. Hilden: „Textiles for protection against harmful ultraviolet radiation― – International Textile Bulletin no 6, November (2001). nd M. ZimniewskaŚ ―Linen and Hemp Fabrics as a Natural Way of Sun Protection‖ 2 Global Workshop of the FAO European Cooperative Research Network on Flax and Other Bast Plants, ―Bast Plants in the New Millennium‖, 3-6.06. Borovets, Bułgaria. (2001). K. Schmidt-Przewoźna, M. Zimniewska: The Effect of natural Dyes Used for linen Fabric on UV-Blocking In: Renewable Resources and Plant Biotechnology (NOVA Science) 110-117. USA, New York (2006). M.S. Reisch: ―New –wave sunscreens‖, ―Chemical@Enginiering News (April, 2).

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 7

MULTIPURPOSE WHITE MULBERRY (MORUS ALBA L.) Malgorzata Lochynska 1*and Grzegorz Oleszak2† 1

Department of Silkworms Breeding and Mulberry Cultivation Institute of Natural Fibres and Medicinal Plants, Poznan, Poland 2 Experimental Farm in Petkowo, Institute of Natural Fibres and Medicinal Plants, Petkowo, roda Wielkopolska, Poland

ABSTRACT In 2006 Institute of Natural Fibres and Medicinal Plants began research on an old Polish cultivar of white mulberry – ―Zolwinska‖. The initial studies have shown great potential of this plant in energy, food and pharmaceutical industries. The leaves and fruits contain very valuable active substances, which may be used in health care. What is more, very fast growth of mulberry causes that biomass of the plant may be used as biofuel. The aim of the presentation is promoting the white mulberry and presenting its possible various uses, which may be very useful for several sectors of the European economy.

Keywords: mulberry, cultivar, energy industry, food industry, active compounds

INTRODUCTION Morus alba Linnaeus, 1753 is one of the numerous species in the family Moraceae, which comprises trees, bushes and herbs. Most of the species are native to Asia with warm climate. These plants are characterized with milky sap in shoots. They are both monoecious and *

Department of Silkworms Breeding and Mulberry Cultivation, Institute of Natural Fibres and Medicinal Plants, Wojska Polskiego 71b, 60-630 Poznan, Poland. e-mail: [email protected]. † Experimental Farm in Petkowo, Institute of Natural Fibres and Medicinal Plants, Petkowo, 63-000 roda Wielkopolska, Poland. e-mail: [email protected].

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Malgorzata Lochynska and Grzegorz Oleszak

dioecious, their flowers are inconspicuous and odorless and the small, sweet fruits are multiple: drupelets or nuts. The leaves are alternately arranged, simple, often lobed and serrated on the margin (Litwinczuk 1993, Butt et al. 2008). The taxonomy of Morus genus is very complex and complicated because of widespread hybridization, wherein the hybrids are fertile. So far, over 150 species of mulberry have been described, however only 10–16 species are widely recognized by botanists (Datwyler et al. 2004). All mulberries grow very fast for 40-50 years reaching 60-80 cm in diameter at breast height, then their growth rate falls. Mulberry tree lives for 200-300 years.

MATERIAL AND METHODS Present work is based on old, Polish cultivar of white mulberry ―Zolwinska‖, collected from a plantation in Experimental Farm in Petkowo INFandMP. This cultivar was bred in 1950‘s in Milanowek, near Warsaw, Poland. It characterizes with huge leaves and rapid growth, because ―Zolwinska‖ cultivar was designed for silkworm breeding. What is more, black-coloured fruits of Polish mulberry were used in producing jam and wine. Decision of the Director of Central Research Center for Cultivar (COBORU) of 26 April 2010 Institute of Natural Fibres and Medicinal Plants has received the exclusive right to the variety ―Zolwinska‖. The variety was registered in the Census Reported Variety of granting an exclusive right under the number is 1792. The energy value of white mulberry ―Zolwinska‖ was examined at the Department of Environment Protection INFandMP. The dried annual shoots collected in July 2009 were used in the examination. The research was carried out in the oxygen bomb calorimeter type KL-5.

THE POTENTIAL OF WHITE MULBERRY There are numerous uses of white mulberry known for centuries. First of all, both white and black fruits were very valuable and sought-after because of their taste and possible use in the kitchen. In the literature there are many of recipes for juice, jam, wine, cakes etc. (Stasinski 1957). The seeds contain 25-35% of a yellow oil, so mulberry was used as an oilbearing plant (Sharma et al. 1994). What is more, infusion and tea of leaves are healthy because of high content of valuable active compounds. A decoction of leaves is still considered as diaphoretic and emalliorent and applied for gargling in inflammations of throat. The fruits give cooling effect and are used as a laxative, the roots possess anthelmintic activity and astringent properties and the bark is used as a purgative and vermifuge (Sharma et al. 1994). However, white mulberry was mainly known for its excellent antidiabetic action. The flavonoids contained in the leaves and bark of mulberry, especially quercetin and 1deoxynojirimycin (DNJ) lower significantly blood glucose levels by inhibiting enzyme activity, such as: -glucosidases, sucrase and maltase (Oku et al. 2006). Literature data reports that alcoholic extract from the bark , administered to diabetic rats for 10 days, decreased glucose levels by 59%, thereby raising insulin levels by 44% compared with the control group (Singab et al. 2005). Moreover, protein Moran 20K, derived from the extract of

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the bark of mulberry roots, has a huge potential for lowering blood sugar levels in streptozotocin-induced mice model with hyperglycemia (Kim et al. 1999, Andallu et al. 2003). Concluding, the leaves and bark of mulberry and their components have unique properties useful in the fight against type II diabetes. It has been used in medicine for centuries, and repeatedly new food supplements are created including obtained from the white mulberry, are very popular on the market these days. However, white mulberry has also other equally important for human health properties. First of all, the leaves and fruits contain 15-31% high quality protein, crude fiber, 200-300 mg/100g of ascorbic acid, of which over 90% is present in the reduced form, vitamin B, folic acid, folinic acid and vitamin D, -carotenes and trace minerals (iron, zinc, calcium, potassium, phosphorous, magnesium) (Sharma et al. 1994, Srivastava et al. 2006, Ercisli et al. 2007). Moreover, there is a lot of flavonoids (quercetin, rutin, isoquercitrin, astragalin, kuwanon G and C, catechin, mulberrofuran G, albanol B, morusin, sanggenon B and D), which exhibit strong antioxidant activity (Kofujita et al. 2004), antifungal and antibacterial potential against harmful strains of bacteria Bacillus, Staphylococcus, Escherichia and Streptococcus (Shirata et al. 1982, Park et al. 2003) and against viruses Herpes simplex type 1 (HSV-1) (Kimura et al. 2007, Butt et al. 2008). The flavonoids, such as rutin, isoquercitrin, astragalin and quercetin 3-(6-malonylglukoside) and anthocyanins are the most important antioxidants obtained from mulberries (Kim et al. 1999, Doi et al. 2001). What is more, flavones isolated from mulberry leaves show cytotoxic activity against liver cancer cells in rats, human leukemia and melanoma cells in mice (Nam et al. 2002, Kofujita et al. 2004). The polysaccharides from the bark of mulberry roots stimulate lymphocyte proliferation and reduce the production of antibodies (Kim et al. 2000). Both these properties play a significant role in effective treatment against allergies and are currently used in allergic diseases (Butt et al. 2008). In addition, the cyanidin extracted from mulberry fruit protects the brain against endothelial dysfunction and reduces the likelihood of Alzheimer's disease (Serraino et al. 2003). The literature data also reported considerable capacity of mulberry for local whitening of skin, so called depigmentation. The extract from the leaves and bark of mulberry roots causes strong inhibition of DOPA oxidase and exhibits activity against tyrosynase. This in turn inhibits the overproduction of melanin and causes its degradation in local hyperpigmentations (eg. melanoma, ephelide, lentigo) (Everett et al. 1993, Fang et al. 2005). Undoubtedly, one of the most interesting properties of mulberry is the prevention and inhibition of atherosclerosis. The flavonoids (anthocyanins, quercetin) obtained from the white mulberry leaves extract, have strong inhibitory effects on LDL oxidation and increase resistance to blood cholesterol deposits (Chen et al. 2007, Butt et al. 2008). Thus these substances prevent two major causes of atherosclerosis: accumulation of LDL deposits in the vessels and its oxidation. Moreover, the extract from the mulberry bark achieves good results in relieving the state of atherosclerosis, oxidation, aggregation and retention of LDL (Katsube et al. 2006). The mulberry shows also great potential in fuel and energy production. In India all annual shoots, available after silkworm breeding season, are cut, dried and used as the main renewable source of fuel (Chinnaswamy et al. 1995). There is no doubt, that fast-growing mulberry, rich in cellulose (57,4%), hemicellulose (16,3%) and lignin (24,6%) (Sharma et al. 1994) possess huge energy value – 17,9 MJ/kg (Lochynska, unpublished). In addition, the mulberry shows a considerable resistance to disease and pests and has relatively low soil

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Malgorzata Lochynska and Grzegorz Oleszak

requirements. Mulberry plantations provide a low utilization of productive or degraded agricultural land. Therefore, it may be used to produce heat, electricity and in fuel production: both liquid and gas. These energy crops can be burnt in bulk, or in the form of pellets and briquettes. Moreover, it is possible to obtain 14-17 tones/ha of biomass (Sharma et al. 1994, Lochynska, unpublished). All these properties of white mulberry show that it may be much better than energy willow (Salix viminalis), rape straw, mallow (Sida hermaphrodita) and kenaf (Hibiscus cannabinus) (Stolarski et al. 2002, Niedziolka et al. 2006, Kolodziej et al. 2007, Burczyk et al. 2009). The comparison of energy value of selected materials is given in the figure 1. The comparison of annual biomass yield is given in the figure 2.

(after Stolarski et al. 2002, Niedziolka et al. 2006, Kolodziej et al. 2007, Burczyk et al. 2009 and own studies). Figure 1. The energy value of selected, dried plant materials in MJ/kg.

There are other ways of using this multipurpose tree. certainly, it may be cultivated as fodder for the mulberry silkworm, which eat only mulberry leaves. It is obvious that high quality of fresh leaves is related with high quality of cocoons. Moreover, mulberry may be used as a natural dyeing plant, because orange-yellow or golden brown wood contains 32% of tannins (Sharma et al. 1994). The mulberry wood is much valued by the sport equipment industry because of its elasticity and flexibility when steamed; it is easy to burnish and varnish. Hockey sticks, tennis and badminton rackets are mainly made form mulberry wood, mulberry planks may be used in the furniture industry – manufacturing furniture, beautiful veneers and wood accessories. The stem bark of white mulberry is very fibrous, so it is used for paper making in China and Europe. It is interesting that first banknotes in ancient China were made from mulberry bark and phloem.

Multipurpose White Mulberry (Morus Alba L.)

63

(after Stolarski et al. 2002, Burczyk et al. 2009 and own studies). Figure 2. The annual biomass yield of selected plant materials in tones/ha.

The branches and twigs are amazingly flexible. Therefore they are used as binding and cooperage materials, for making baskets and wheels for wood wagons. Furthermore, they may be used as a good substrate for oyster mushrooms (Pleurotus sp.) (Madan et al. 1992). These fibrous materials, fruits and leaves may be applied as a source of humus, organic fertilizer and soil conditioner or slurry in anaerobic digesters. Mulberry leaves (not only fruits) are eaten by vegetarians, both leaves and fruits after drying are applied as fodder for birds, reptiles and rodents. In addition, mulberry may be also used in gardening. Young plants formed into dense hedges protect excellently gardens against wind, noise, fumes and rodents. They are also resistant to air pollution. Trimmed mulberry gives no fruits, so may be planted along the sidewalks. What is interesting, sweet fruits of mulberry attract starlings and other birds. It is advisable to plant mulberry near the cherry trees, because some birds choose sweet mulberry fruit instead of cherries. It may be also planted at the field margins – birds, attracted by sweet mulberry fruits, eat pests in adjacent fields.

SUMMARY White mulberry as a multipurpose plant may be used:    

in pharmaceutical industry and medicine, in food industry, in energy and fuel industry, in sport equipment industry,

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Malgorzata Lochynska and Grzegorz Oleszak           

in furniture industry, as slurry in anaerobic digesters, as a tanning and dyeing plant, as an oil-bearing plant, in sericology, in paper industry, in production of oyster mushrooms, as organic fertilizer and humus in gardening, as binding and cooperage materials, for making baskets and wooden wheels, as fodder for birds, reptiles and rodents, as tree-protector against pollution, wind, noise, pests, and birds.

REFERENCES Andallu B., Varadacjaryulu N., 2003, Antioxidant role of mulberry (Morus indica L. cv. Anantha) leaves in streptozoticin-diabetic rats. Clinica Chimica Acta , 338: 3-10. Burczyk H., Kolodziej J., 2009, Porownanie plonow i warto ci energetycznych konopi wloknistych, kukurydzy i sorga z ro linami egzotycznymi. Len i Konopie. Biuletyn Informacyjny Polskiej Izby Lnu i Konopi, 12: 22-36 (in Polish). Butt M.S., Nazir A., Sultan M.T., Schoёn K., 2008, Morus alba L. nature‘s functional tonic. Trends in Food Science and Technology, 19: 505-512. Chen P.N., Chu S.C., Chiou H.L., Kuo W.H., Chiang C.L., Hsieh Y.S., 2006, Mulberry anthocyanins cyanidin 3-rutinoside and cyaniding 3-glucoside exhibited an inhibitory effect on the migration and invasion of a human lung cancer cell line. Cancer Letter , 235: 248-259. Chinnaswamy K.P., Hariprasad K.B., 1995, Fuel Energy Potentiality of Mulberry. Indian Silk 34 (4): 15-18. Datwyler S.L., Weiblen G.D., 2004, On the origin of the fig: phylogenetic relationships of Moraceae from Ndhf sequences. American Journal of Botany 91(5): 767–777. Doi K., Kojami T., Makino M., Kiura Y., Fujimoto Y., 2001, Studies on the constituens of the leaves of Morus alba L. Chemical and Pharmacology Bulletin, 49: 151-153. Hansawasdi C., Kawabata J., 2006, Alpha-glucosidase inhibitory effect of mulberry (Morus alba ) leaves on Caco-2. Fitoterapia , 77: 568-573. Iozumi K., Hoganson G.E., Pennella R., Everett M.A., Fuller B.B., 1993, Role of tyrosinase as the determinant of pigmentation In cultures human melanocytes. Journal of Investigative Dermatology, 100: 806-811. Katsube T., Imawaka N., Kawano Y., Yamazaki Y., Siwaku K., Yamane Y., 2006, Antioxidant flavanol glycosides in mulberry (Morus alba L.) leaves isolated based on LDL antioxidant activity. Food Chemistry, 97: 25-31. Kim S.Y., Gao J.J., Lee W.C., Ryu K.S., Lee R.R., Kim Y.C., 1999, Antioxidative flavonoids from the leaves of Morus alba . Archiv der Pharmazie, 22: 81-85. Kimura T., Nakagawa K., Kubota H., Kojami Y., Goto Y., Yamagishi K., et al., 2007, Foodgrade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of

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postprandial blood glucose in humans. Journal of Agricultural and Food Chemistry, 55: 5869-5874. Kofujita H., Yaguchi M., Doi N., Suzuki K., 2004, A novel cytotoxic prenylated flavonoid from the root of Morus alba . Journal of Insect Biotechnology and Sericology, 73: 113116. Kolodziej J., Mankowski J., Kubacki A., 2007, Wla ciwo ci energetyczne odpadow z przerobu lnu i konopi w porownaniu z innymi surowcami ro linnymi. Len i Konopie. Biuletyn Informacyjny Polskiej Izby Lnu i Konopi, 8: 35-43 (in Polish). Litwinczuk W., 1993, Charakterystyka, rozmnazanie i zastosowanie morwy bialej (Morus alba L.). Biuletyn Ogrodow Botanicznych, 2: 27-35 (in Polish). Nam S.Y., Yi H.K., Lee J.C., Song C.H., Park J.W., et al., 2002, Cortex mori extract induces cancer cell apoptosis through inhibition of microtubule assembly. Archives of Pharmacological Research 25: 191-196. Niedziolka I., Zuchniarz A., 2006, Analiza energetyczna wybranych rodzajow biomasy pochodzenia ro linnego. Motrol., 8a: 232–237 (in Polish). Madan M., Sharma S., Vasudevan P., 1992, Mineral content of Pleurotus sajor-caju and organic substrates used. Microbios., 69 (279): 113-118. Oku T., Hamada M., Nakamura M., Sadamori N., Nakamura S., 2006, Inhibitory effects of extractives from leaves of Morus alba on human and rat small intestinal disaccharidase activity. British Journal of Nutrition, 95: 933-938. Park K.M., You J.S., Lee H.Y., Baek N.I., Hwang J.K., 2003, Kuwanon G: an antibacterial agent from the root bark of Morus alba against oral pathogens. Journal of Ethnopharmacology, 84: 181-185. Serraino I., Dugo L., Dugo P., Mondello L., Mazzon E., Dugo G., et al., 2003, Protective effects of cyanidin-3-Oglucoside from blackberry extract against peroxynitrite-induced endothelial dysfunction and vascular failure. Life Science, 73: 1097-1114. Singab A.N., El-Beshbishy H.A. Yonekawa M., Nomura T., Fukai T., 2005, Hypoglycemic effect of Egyptian Morus alba root bark extract: effect on diabetes and lipid peroxidation of streptozotocin-induced diabetic rats. Journal of Ethnopharmacology, 100: 333-338. Srivastava S., Kapoor R., Thathola A., Srivastava R.P, 2006, Nutritional quality of leaves of some genotypem of mulberry (Morus alba ). International Journal of Food Science and Nutrition, 57: 305-313. Stasinski K., 1957, Zbior i przechowywanie nasion morwy. Wydawnictwa Instytutu Jedwabiu Naturalnego, Biblioteczka dla praktykow 3, pp. 8 (in Polish). Stolarski M., Szczukowski S., Tworkowski J., 2002, Produktywno ć klonow wierzb krzewiastych uprawianych na gruntach ornych w zalezno ci od czestotliwo ci zbioru i gesto ci sadzenia. Fragmenta Agronomia , 2: 41-48 (in Polish). Yen G.C., Wu S., Duh P.D., 1996, Extraction and identification of antioxidant components from the leaves of mulberry (Morus alba ). Journal of Agricultural and Food Chemistry, 44: 1687-1690.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 8

AGRICULTURAL RESIDUES AS A RENEWABLE SOURCE OF BIO-ENERGY WITH SPECIAL FOCUS ON CEREAL STRAWS S. Narra1*, C. Glaser2†, H. J. Gusovius3‡, C. Stollberg # and P. Ay2^ 1

Brandenburg University of Technology Cottbus, Siemens-Halske-Ring 8, 03046, Cottbus, Germany 2 Processing of Biogenous Resources, Brandenburg University of Technology, Cottbus, Germany, 3 Department of Post Harvest Technology, Leibniz Institute for Agricultural Engineering Potsdam-Bornim, 4 Process Technology of Biogenous Resources, Hochschule Wismar – University of Applied Sciences, Technology, Business and Design; Faculty of Engineering, Poel, Germany,

ABSTRACT Utilization of the agricultural residues (cereal straws) is often difficult due to their uneven and troublesome characteristics and also due to their low density ranging from 40 -3 -3 kg m to 120 kg m including the baled straw. This drawback can be overcome by means of densification, i.e. compaction of the residues into products of high density and regular shape. The higher bulk density and higher energy density results in lower transportation costs and higher energy efficiency. The particle size characteristics indicate an ideal distribution of the particles for the combustion in furnaces. The durability and the *

Correspondence Author: Satyanarayana Narra. Position title: Scientific Co-worker, Chair of Mineral Processing Organisation name: Brandenburg University of Technology Cottbus. Adress: Siemens-Halske-Ring 8, 03046, Cottbus, Germany. Phone / Email: +49 (0) 355 - 693635 / [email protected]. † E-mail: [email protected]. ‡ E-mail: [email protected]. # E-mail: [email protected]. ^ E-mail: [email protected].

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S. Narra, C. Glaser, H. J. Gusovius et al. mechanical stability of the pellets communited with the twin-screw extruder were comparatively better then the pellets prepared after communition with impact mill. Particle size characteristics, abrasion values and the strength characteristics showed a good co-relation. The pellets also displayed optimal combustion characteristics.

Keywords: Agricultural residues, rye straw, wheat straw, straw pellets, particle size distribution

INTRODUCTION Reduction and off-setting of the anthropogenic emissions of CO2 and other greenhouse gases (GHGs) are important strategies of mitigating the risk of global warming. Thus, the need for developing CO2 neutral and renewable sources of energy is more than ever before. Use of crop residue as a possible source of feedstock for bioenergy production must be critically and objectively assessed because of its positive impact on soil C sequestration, soil quality maintenance and ecosystem functions. The advantages of agricultural and forest plants production can be increased by complete sustainable utilisation of the raw materials and residues. The available agricultural and forest residues are husks, straws, saw dust, etc. The annual availability of these residues is comparatively high, which has been ignored for decades and only in the recent past have gained attention due to sky rocketing of the fossil fuel prices. Agricultural Residues are used for many purposes and such uses often are site specific. Besides being used as fuel, residues are also used as Fodder, Fertiliser, Fibre, Feedstock, soil conditioner, etc. Though thousands of tonnes of agricultural residues are turned out annual y, none of it has been harnessed with significant importance. The residues are utilized by some industries based on their characteristics of holding high moisture contents and not as a source of energy. All the agricultural residues are bulky and in addition have high moisture contents which make them unattractive as a fuel. Currently there is a tremendous interest in using biomass as an energy source through out the world and agricultural resources are playing an important role. Biomass as an energy source would replace the fossil fuels and also reduce the greenhouse gas emissions. Agricultural residues are becoming an increasingly important energy source for the future due to their yearly production. Woody biomass is still considered as a main source of bio-energy. Agricultural residues are considered as a substitute for woody biomass due to the difficulties arising with wood in the balance ratio of the growth to burning rates and the availability of resources for the future. The utilization of agri-cultural residues as a substitute for woody biomass shows a great potential due to its surplus yearly production. Biomass is an important fuel for heating and power generation because it is a readily available renewable energy source that reduces carbon dioxide emissions (Petrou and Pappis, 2009). Agricultural residues are very difficult to handle due to their irregular shape and size, high moisture content, and low bulk density. These problems can be overcome by densification of biomass into regular size and shape (briquettes and pellets). Pelletisation is employed in many industries to form a more durable substance and to enhance the material handling characteristics (Finney et al., 2009). The primary reason for pelletisation is to

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69

increase the bulk and energy densities of the material. Pellets of various agricultural residues can be used for energy production in a broad range from private house hold appliances to full scale power plants (Obernberger and Thek, 2009). There are several advantages of densified fuel pellets compared to direct incineration of raw materials. The higher bulk density and higher energy density results in lower transportation costs and higher energy efficiency (Holm et al., 2006). Further the reduced moisture content increases the energy efficiency and long term storage capability (Kaliyan and Morey, 2009). The physical and chemical quality of the fuel pellets are evaluated based on the characteristics of the raw materials. The quality of the fuel-pellets are standardized in DIN 51 731, DINplus and ÖNORM M 7135. The standard values of the quality parameters are given in table 1. The standards are specified especially for wood pellets. There are no specific standards for agricultural residue pellets. Further the results of the fuel-pellets from cereal straws were compared with agricultural residue standards Agro and Agro+ (table 1). The physical characteristics include particle size distribution after communition processes, bulk density and durability of the pellets, etc. The chemical characteristics include ash content, composition of chemical elements (C, H, N, S, Cl, K), the heavy metals concentrations (Cd, Pb, Zn, Cr, Cu, As, Hg, Sb, Ti), water content, lignin content etc. These properties influence the suitability of the raw material as a fuel. Chemical properties mainly influence the burning and heating suitability and the heating value. Physical properties are of highest importance for the binding mechanisms which occur during the biomass densification (Hartmann, 2007). Table 1. Standard quality values of the wood pellets as specified in DIN 51 731, ÖNORM M 7135 and DINplus Norms for Pellets

DIN 51 731

ÖNORM M 7135

DINplus

* Agro

* Agro+

Diameter

4 - 10 mm

4 - 10 mm

--

6 - 16 mm

6 - 8 mm

Length

≤ 50 mm

≤ 5*d

< 5*d

10 - 30 mm

10 - 30 mm

Abrasion

--

≤ 2.3 %

≤ 2.3 %

≤8%

≤5%

Heating value

17.5 - 19.5 MJ/kg

> 18 MJ/kg

> 18 MJ/kg

≥ 14.7 MJ/kg

≥ 15.5 MJ/kg

Water content

≤ 12 %

≤ 10 %

≤ 10 %

≤ 15 %

≤ 11 %

Ash content

< 1.5 %

< 0.5 %

< 0.5 %

≤7%

≤5%

Density

> 540 kg/m³

> 540 kg/m³

> 540 kg/m³

≥ 650 kg/m³

≥ 650 kg/m³

Binders / Additives

Not allowed

≤2%

≤2%

No limit

No limit

S

< 0.08 %

< 0.04 %

< 0.04 %

≤ 0.2 %

≤ 0.2 %

N

< 0.3 %

< 0.3 %

< 0.3 %

≤ 1.5 %

≤ 1.5 %

Cl

< 0.03 %

< 0.02 %

< 0.02 %

≤ 0.3 %

≤ 0.2 %

* Agricultural residue pellet standards, France.

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Agricultural residues require a product specific size reduction and liberation before pelletisation. An investigation on Rye and Wheat straw as a raw material for pelletisation has been carried out. Due to low bulk density of straw, straw has to be ground and compacted into dense and durable pellets in order to facilitate handling, storage and transportation (Adapa et al., 2007; Mani et al., 2003). In addition, because of uniform shape and size, densified products can be easily adopted in direct-combustion or co-firing with coal, gasification, pyrolysis, and in other biomass-based conversions (Kaliyan and Morey, 2006a). The objective of this work is to produce 8 mm pellets which fulfil the quality standards as described in the norms. The straw was ground to particles having a size less than or equal to 6 mm, such that the particles do not block the pellet mould having a diameter of 8 mm (Obernberger and Thek, 2009). The size reduction of the Rye and Wheat straw was accomplished with the help of an impact mil / Hammer mill (HM) as well as a twin-screw extruder (EX). The aim of this work is to investigate 1) particle size distribution of the Rye and Wheat straws after different communition processes, 2) optimisation of water content as a binding material in pellet production, 3) quality of pellets with the parameters abrasion and strength.

MATERIALS AND METHODS The raw materials used in this study are straw of winter varieties of Rye straw (Secale cereale) and Wheat straw (Triticum vulgaris). The Rye and Wheat was grown in year 2005 and was harvested in the year 2006 in Goßmar, Brandenburg, Germany. The impact mil / hammer mill (HM) employs a high speed rotating disc to which the hammer bars are fixed (figure 1a). The hammer bars are swung outwards by centrifugal force. The material is fed into the mill through a feeder. The material is downsized by being beaten by the hammer bars in order to reduce the particle size and fall through the sieve having an aperture size of 6mm. The straw was communited in the industrial impact mill by the company Futtermittel und Dienstleistungs GmbH, Sonnewalde, Brandenburg.

a) Figure 1. a) Impact mil / hammer mil (HM). b) Twin-screw extruder (EX).

b)

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The twin screw extruder (EX) works with the principle of defibration (figure 1b). The straw is fed into the twin screw extruder, where it is taken by the rotating screws. The material is brought with the rotating screws through the barrel and compacted against a die. This helps with building up of a pressure gradient along the screws. The material gets ground in close contact between the barrel walls and the rotating screws which causes frictional effects and leads to shearing forces. A destruction of the material‘s cell occurred through the processing of moisture enriched material at high temperatures ranging in between 80°C and 130°C under pressure. Through high mechanical energy and high shear forces, the materials physical size (particle size) and chemical properties were changed. Through the extrusion process the straw surface gets partial y destroyed which has influences on the contained lignin content and on the wax surface. Both Rye and Wheat straw are processed in the industrial twin-screw extruder by the company Lehmann Maschinenbau GmbH, Pöhl, Saxony. The determination of particle size distribution can be carried out using different techniques (sieving, image analysis and laser diffraction). For quality control not only the particle size is of importance, but also the particle shape is an important characteristic. With the help of image analysis, the complete dimensions of the single particles can be analyzed. Image analysis was carried out with the help of Fibreshape from Innovative Sintering Technologies at a resolution of 2400 dpi (figure 2). These settings enable to detect the particles having a size equal to or higher then 10 µm. The particle size distribution was carried out with three representative samples for straw after two different communition processes, each sample on average was having a minimum of 16,000 particles in each variant. Within image analysis different size characteristics (e.g. particle length, particle width) for each particle are measured.

a)

b)

Figure 2. Images of the Particles after different communition processes a). Hammer mill / b. Twinscrew extruder) were scanned with a flat-bed scanner and analysed with Fibreshape from Innovative Sintering Technologies.

The moisture content was determined using ASAE Standard S358.2 (ASAE, 2006a), where oven drying of the samples was carried out at 103°C for 24 hours

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In order to achieve the optimized pellets, the raw materials Rye straw and Wheat straw were prepared with increasing water contents starting from 16 volume %. This was carried out by first measuring the water content of the raw materials and the additional water was supplied in the mixing process. The water was supplied as a binding agent in the pelletisation of the raw materials. The moisture level greater then 14 volume% was used based upon the literature review as this moisture level would produce high density and quality pellets from various straws (Kaliyan and Morey, 2006b; Mani et al., 2006a; Obernberger and Thek, 2004; Shaw and Tabil, 2007). The water content was varied in percentage to develop the optimal mixture conditions for pelletisation and through which achieve lower abrasion and higher strength. The water content of the straw measured was in between 6.5 and 8.7 volume% after the respective communition processes. The additional water ranging from 7.3 to 9.5 volume% has been supplied in to the mixing apparatus for obtaining a total water content of 15 volume%, and respectively repeated the procedure for the remaining water contents investigated. Pelletisation was carried out with the help of a laboratory compactor (Hosokowa Bepex, Type L200/50G+K). The working principle of the compactor is similar to that of a hollow roller press. The material is auger fed towards the working area of the roller moulds, where it is pressed and the materials passes through the mould openings (figure 3). Densification of the raw materials takes place in the moulds. The mould openings are 25 mm long and have a diameter of 8 mm. The temperature measured during the pelletisation process was in between 80°C to 100°C. The pellet size and the range of variation influence the selection of the conveying systems as well as the combustion behaviour of the pellets. Pellets should be homogeneous in size and shape, which is recommended by the small scale pellet furnace manufacturers. The development of automatic biomass heating systems is only possible when the pellets have uniform size and shape (Obernberger and Thek, 2002). The Rye and Wheat straw pellets were stored for 14 days at a temperature of 20°C such that the water content of the pellets is stabilised. Abrasion tests and strength tests were carried out after the stabilisation of the water content in the pellets.

Figure 3. Hosokowa Bepex laboratory compactor (Type L200/50G+K) with the working principle.

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The abrasion of pellets is one of the most important parameters in the pellet production. Abrasion is an essential criterion to be considered by the end user as lower abrasion value produces less particulate emissions, has lower transportation losses and prevents bridging (clusters of irregular size and shape) of particles during storage (Obernberger and Thek, 2002). A high amount of fines in the pellets can cause failures in the feeding system. Abrasion has been measured using a rotating quadratic crate (Pfost pellet tester) as described in the ASABE standard S269.4 (figure 4). The quadratic crate (LxBxD: 30x30x12.5 mm³) rotates at a speed of 50 rotations/min for ten minutes. The sample was then sieved with a sieve having an aperture of 6.3mm (0.8 * pellet diameter) as suggested by Thomas and Van der Poel, (1996). The difference in the weights of the pellets before and after the abrasion test gives the abrasion value.

Figure 4. Quadratic Abrasion testing device (ASABE standard S269.4).

The strength indicates the quality of pellets. The quality of pellets is noticeable by an exceptionally smooth pellet surface without any fissures. Moreover stronger pellets develop less particulate matter during handling. The strength tests (diametrical pressure test) are carried out with the help of a ZWICK-ROELL (type: ZMART.PRO) material testing (tensile and compressive strengths) machine. The pressure tests were selected as such pressures occur during handling, transportation and storage of the pellets.

RESULTS AND DISCUSSIONS The water content of the Rye and Wheat straw were in between 6.5 and 8.7 volume%. The pellets were prepared with increasing water contents starting from 16 volume%. The water content of the straw pellets measured after stabilisation for 14 days at 20°C was in between 8.3 and 10 volume%. The 10% water content improves the durability of the pellets (Kaliyan and Morey, 2009; Nielsen et al., 2010). The obtained water content is below the value mentioned under the standards for wood pellets. The water content has an influence on the net calorific value, combustion efficiency and the temperature of combustion (Obernberger and Thek, 2002; Nussbaumer and Kaltschmitt, 2001). The higher water content reduces the durability and energy efficiency. The optimum water content for pellets should be less then 10 volume % as specified in DIN 51 731, DINplus, ÖNORM M 7135. Kaliyan and

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Morey, (2006b); Mani et al., (2006a); Obernberger and Thek, (2004); Shaw and Tabil, (2007) state that the 10% moisture content of pellets would result in longer storage, high combustion efficiencies and through which high energy efficiency. The important fuel property which changes with the pelletisation is the bulk density of the raw materials (Ryu et al., 2006; Gilbert et al., 2009). The straws generally have very low bulk density values usually ranging in between 40 and 120 kg m-3 including baled density (Kaliyan and Morey, 2009). The measured bulk density of the Rye straw pellets on average was 560 kg m-3. The increase in bulk density significantly reduces the costs for storage, transportation, handling, feeding of the biomass and also increases the energy density. The particle size and shape distribution analyzed after the communition process are depicted in figure 5. The distribution curves start from 10 µm as the resolution of the scanner was set to 2400 dpi. The particles are classified into different particle size classes which are long particles (length: 800 µm and above, width: 80 µm and above), short particles (length: 200-800 µm, width: 25-80 µm), slime stuff (length: upto 200 µm, width: upto 1 µm) and flour (length: 20-200 µm, width: 1-30 µm). Long particles and short particles are fibrous materials, whereas the slime stuff and flour stuff are fines (Pruden, 2005). Similar particle size distribution was observed with impact mill and also with twin-screw extruder for the Rye and Wheat straws. The fines (slime stuff and flour stuff) make up to 40% of the total particles in the length distribution and 20% in the width distribution. Particle size distribution also affects the combustion process. Small particles and fines have higher burning rates and ignition front speeds (Ryu et al., 2006). Larger particles are thermally thick having slow devolatilization rate and more distributed heat transfer to the nearby particles. Ryu et al., (2006) states that with the increase in particle sizes (from 5 mm to 35 mm) there is a decrease in burning rate and also a decrease in heat influx from larger particles to the smaller particles and fines. The results show that 60-80% of the particles can be classified into smaller particles and fines, indicating that the burning rate and the heat influx would be optimal in the combustion process. 20-40% of the particles are classified as fibrous particles, which intertwine with each other during pelletisation and act as an additional binding feature (Gilbert et al., 2009).

Figure 5. Particle size distribution (length and width) of Rye and Wheat straw after two different milling processes. The black dashed bars represent the borders of different particles classes based on length and width.

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The usage of straw in different forms is dependent on its characteristics. The Carbon (C), Hydrogen (H) and Nitrogen (N) values (table 2) in the Rye and Wheat straws comply with the natural ranges of the raw materials (Obernberger and Thek, 2002). The optimal value of N should be less than 0.3% (DIN 51731). The values of N in the Rye straw analysed were above 0.3%. This indicates that the use of straw would result in increased NOx emissions (Schaffenberger and Stastny, 2008). C, H and Oxygen (O) are the main components of the biomass fuels; C and H are oxidized during combustion by exothermic reactions and therefore influence the calorific value of the fuel. The organic O provides a part of the O for the combustion process. Additional O must be supplied by air injection in the furnace (Obernberger and Thek, 2009). As per the standard DIN 51 731, the values of the Chlorine (Cl) and Sulphur (S) should be less than 0.03% and 0.08% respectively. The values of Cl and S obtained in Rye straw were above the values specified in the standard. The concentrations of Cl and S should be limited as they have negative influences on the combustion processes. The amount of S in the Rye and Wheat straw is very close to the standards mentioned. High amount of S can cause problems regarding emissions (SOx). There is no high risk of SOx emissions as the obtained S values in straw are almost equal to the values mentioned in standards. The Cl content in the straw is very high in comparison to the values mentioned in standards, which would cause problems in depositions and corrosion of the furnace (Schaffenberger and Stastny, 2008). Table 2. Chemical properties of Rye and Wheat straw (Hartmann, 2007) Raw material Rye straw Wheat straw

C H O Volume % of dry mass 46.6 6.0 42.1 45.6 5.8 42.4

N

S

Cl

0.55 0.48

0.085 0.082

0.19 0.19

The heating value of the Rye and Wheat straw can be calculated based on the chemical composition of the raw materials using equation (1) (Kaltschmitt et al., 2009). The water free (wf) heating value of 17.4 MJ kg-1 and 17.2 MJ kg-1 has been calculated for the Rye and Wheat straws respectively. Hu (wf) = 34.8 * C + 93.9 * H + 10.5 * S + 6.3 * N – 10.8 * O

(1)

The pellets were prepared with increasing water contents starting from 15 volume%. The pellets were then tested for abrasion and the optimal water content was determined with respect to the minimum abrasion values achieved. The minimum abrasion values of Rye and Wheat straw pellets were obtained at 17 volume% and 18 volume% water content in the mixture (figure 6). Obernberger and Thek (2002) state that there is a direct correlation between the abrasion and the particle size distribution. The particle size distribution after different milling processes show that there are 40% fines present. The abrasion values achieved with impact milled material (4.7% and 3.7%) and with twin-screw extrusion material (2.6% and 3.0%) from Rye and Wheat straws correlate directly to the amount of fines in the raw material after communition processes.

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Figure 6. Abrasion of the Rye straw (RS) and Wheat straw (WS) pellets prepared with increasing water contents after grounding and liberation of the fibres with Hammer mil (HM) and Twin-screw extruder (EX).

The standards ÖNORM M 7135 and DINplus state that the abrasion value of the pellets should not be greater than 2.3%. The abrasion values of the Rye and Wheat straw obtained are greater than the specified abrasion values. The high amount of fines could be the reason for obtaining such high abrasion values. Higher percentage of fines can cause failures in the furnace feeding systems and also causes higher particulate emissions during combustion. Other parameters such as use of binding agents, additives etc also have an influence on the abrasion characteristics.

Figure 7. The strength of the pellets as measured using three different strength testing methods with increasing water contents for Rye and Wheat straw respectively.

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For the evaluation of strength, the maximum pressure force which a pellet can withstand was analysed. Pellets need to withstand different pressure forces as they are confronted with such pressures during handling, storage and transportation. The results show that maximum pressure which a pellet can withstand was obtained with 17 volume % and 18 volume % water content for Rye and Wheat straw pellets (figure 7). These strength results correlate with the abrasion values obtained.

CONCLUSIONS There are some problems in using straw for heating and energy generation (dust, huge amount of ash, high emissions, etc.). The straw incineration results in high NOx and SOx emissions and also causes depositions and corrosion of the furnace due to high presence of Cl. Special furnaces with filters have to be used especially when straw has to be incinerated. The combustion of straw is profitable in full scale power plants. Regarding CO2-emissions the combustion of straw is CO2-neutral, but the calorific value and the bulk density of straw is low compared to that of woody biomass. Thus the high CO2- emissions from the transport of straw should also be considered. The Rye and Wheat straws can be used in the form of pellets, which have high density (540 kg m-3) and a heating value of 17.2 MJ kg-1 and 17.4 MJ kg-1. The moisture content of the pellets after stabilization was below 10 volume % indicating a longer durability of the pellets. Particle size characteristics showed a good correlation to the abrasion values and favourable combustion characteristics. The minimum abrasion values were obtained with 17 volume% water content for Rye straw and at 18 volume% water content for Wheat straw. The pressure tests also showed a good correlation with the abrasion values i.e. the pellets showed higher pressure resistance with lower abrasion values.

ACKNOWLEDGMENTS The authors would like to acknowledge the project partners ―German Biomass Research Centre Leipzig, Germany‖, ‖Lehmann Maschinenbau GmbH, Pöhl, Germany‖ and ―Futtermittel und Dienstleistungs GmbH, Sonnewalde, Germany‖ for their assistance in the project work and also for giving us the permission to submit the paper.

REFERENCES Adapa P K, G J Schoenau, L G Tabil, E A Arinze, A Singh and A K Dalai (2007) Customized and Value-added High Quality Alfalfa Products - A New Concept. Agricultural Engineering International: the CIGR Ejournal. Manuscript FP 07 003. Vol. IX., 1-28. Brinker M M (2009). Investigation of different image analysis systems for the particle size characterization of straw after different milling operations. Master thesis, Chair of Minerals Processing, Brandenburg University of Technology Cottbus.

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Chen Y, Sharma-Shivappa R R, Keshwani D, Chen C (2007). Potential of Agricultural Residues and Hay for Bioethanol Poduction, Applied bio-chemistry and Biotechnology Part A: Enzyme Engineering and Biology, 276-290. Gilbert P, Ryu C, Sharifi V, Swithenbank J (2009). Effect of process parameters on pelletisation of herbaceous crops, Fuel, 88, 1491-1497. Grover P D, Mishra S K (1996). Biomass Briquetting: Technology and Practices, Regional wood energy development program in Asia GCP/RAS/154/NET, Food and Agriculture Organization of the United Nations, Field document No. 46, Bangkok, Thailand. Hartmann H (2007). Leitfaden Bioenergie, Planung, Betrieb und Wirtschaftlich-keit von Bioenergieanlagen, Fachagentur Nachwachsende Rohstoffe e.V., Gülzow, Germany, p86 (in German). Holm J K, Henrikson U B, Hustad J E, Sørenson L H (2006). Toward an understanding of controlling parameters in the softwood and hardwood pellets production. Energy and Fuels, 20, 2686 - 2694. Kaliyan N, R V Morey (2006a). Factors Affecting Strength and Durability of Den-sified Products. ASABE Annual International Meeting, American Society of Agricultural and Biological Engineers, Portland, Oregon July 9-12, Paper Number 066077, 2950 Niles Road, St. Joseph, MI 49085-9659 USA. Kaliyan N, R V Morey (2006b). Densification Characteristics of Corn Stover and Switchgrass. ASABE Annual International Meeting, American Society of Agricultural and Biological Engineers, Portland, Oregon July 9-12, Paper Number 066174, 2950 Niles Road, St. Joseph, MI 49085-9659 USA. Kaliyan N, R V Morey (2009). Factors affecting strength and durability of den-sified biomass products. Biomass and Bioenergy 33, 337 – 359. Kaltschmitt M, Hartmann H, Hofbauer H (2009). Energie aus Biomasse. Grund-lagen, Techniken und Verfahren, 2. Auflage, Pp 351. Lee D, Owens V N, Boe A, Jeranyama P (2007). Composition of Herbaceous Biomass Feedstocks, Sun Grant Initiative, North Central Center, South Dakota State University, Brookings, USA. Mani S, L G Tabil, S Sokhansanj (2006b). Effects of Compressive Force, Particle Size and Moisture Content on Mechanical Properties of Bio-mass Pellets from Grasses. Biomass and Bioenergy, 97, 1420-1426. Mani S, L G Tabil, S Sokhansanj (2003). An Overview of Compaction of Bio-mass Grinds. Powder Handling and Process, 15(3), 160-168. Nussbaumer T, Kaltschmitt M (2001). Grundlagen der Festbrennstoffnutzung – Begriffsdefinitionen. In: Kaltschmitt M, Hartmann H (Hrsg.): Energie aus Biomasse – Grundlagen, Techniken, Verfahren. Springer Verlag, Berlin, 239 – 247. Obernberger I, Thek G (2002). Physical characterisation and hemical compo-sition of densified biomass fuels with regard to the cumbustion be-haviour. In: proceedings of the first world conference on pellets. Stock-holm, Sweden. Swedish Bioenergy Association (ed). Pp 115 – 122. Obernberger I, G Thek (2004). Physical Characterization and Chemical Composition of Densified Biomass Fuels with regard to their Combustion Behavior. Biomass and Bioenergy, 27, 653-669. Obernberger I, Thek G (2009). Herstellung und energetische Nutzung von Pellets. Produktionsprozess, Eigenschaften, Feuerungstechnik, Öko-logie und Wirtschaftlichkeit,

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Schriftenreihe Thermische Biomasse, Institut für Prozesstechnik, Technische Universität Graz, Bd. 5,Graz, Österreich (in German). Oliveira L, Evtuguin D, Cordeiro N, Silvestra A J D (2009) Structural characteri-zation of stalk lignin from banana plant, Industrial crops and products, 29, 86-95. Petrou E C, Pappis C P (2009). Biofuels: A survey on pros and cons. Energy and Fuels, 23, 1055 – 1066. Pruden B (2005). The effect of fines on paper properties. Paper Technology, 46 (4): 19 – 26. Ryu C, Yang Y B, Khor A, Yates N E, Sharifi V N, Swithenbank J (2006). Effect of fuel properties on biomass combustion: Part I. Experiments – fuel type, equivalence ratio and particle size. Schaffenberger M, Stastny P (2008). Overview of biomass technologies and their assessment. Seminar paper. Interdisciplinary bilateral winter and summer school on energy innovations in Austria and Czech Republic. Schwabe J (2009) Entwicklung von Technologien und Rezepturen für die Herstellung von Biomassepellets aus Roggen- und Weizenstroh, Diplomarbeit, BTU Cottbus (in German). Shaw M D, L G Tabil (2007). Compression and Relaxation Characteristics of Selected Biomass Grinds. ASAE Annual International Meeting, Minneapolis, MN, June 17-20 Paper Number 076183, 2950 Niles Road, St. Joseph, MI 49085-9659 USA. Stieß M (2009). Mechanische Verfahrenstechnik – Partikeltechnologie 1, 3. vol st. neu bearb. Auflage, Springer-Verlag Berlin Heidelberg, Germany, 175-178. Thomas M, Van der Poel A F B (1996). Physical quality of pelleted animal feed 1. Criteria for pellet quality. Animal Feed Science Technology, 61, 89-112. Uslu A, Faaij A P C (2008). Pre-treatment technologies, and their effect on international bioenergy supply chain logistics – techno-economic evaluation of torrefication, fast pyrolysis and pelletisation. Energy 33 (8): 1206 – 1223. Varnaité R (2002). Enzymatic lignin degradation by micromycetes in plant remnants, Biologija , 2, 23-25. White R H (1987). Effect of lignin content and extractives on the higher heating value of wood, Wood and Fibre Science, 19 (4), 446-452.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 9

PREVENTION OF DISPOSAL OF GREENHOUSE GAS FROM DIGESTED RESIDUES BY OPTIMAL USE OF THE NITROGEN FERTILIZER POTENTIAL Ute Bauermeister* GNS - Gesellschaft für Nachhaltige Stoffnutzung mbH, Weinbergweg 23, D-06120 Halle/Saale BM - Verfahrenstechnologie, Weinbergweg 23, D-06120 Halle/Saale

ABSTRACT In Germany about 4.500 biogas plants produce biogas from field crops, residues and manure. The application of digested residues as fertilizer should close nutrient loops. But the ammonium-nitrogen is more volatile then in mineral fertilizer and the application does not take place only at the optimal time of plant growing. This leads to intensified emissions and to a loss of nitrogen fertilizer. To evaluate the contribution of the application method and kind of treatment of digested residues in regard to emissions of greenhouse gases, the disposal of ammonia, nitrous oxide and methane were measured in a research project managed by the Martin-Luther-University Halle-Wittenberg. The results show, that the treatment of digested residues by the ANAStrip ®-process System GNS leads to significantly small emissions and small nutrient losses. A concept was developed to close regional nutrient lops by producing a concentrated nitrogen-fertilizer from digested residues, which can be used as a depot fertilizer with very small nutrient losses under prevention of disposal of greenhouse gas.

Keywords: biogas, digested residue, emissions, greenhouse gas, nutrient loop

*

GNS - Gesellschaft für Nachhaltige Stoffnutzung mbH, Weinbergweg 23, D-06120 Halle/Saale. phone: +049 345 5583-754, e-mail: [email protected], www.GNS-Halle.de and BM - Verfahrenstechnologie, Weinbergweg 23, D-06120 Halle/Saale. phone: +049 345 5583-705, e-mail: [email protected].

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1. ADVANTAGES AND DISADVANTAGES BY FIELD APPLICATION F DIGESTED RESIDUES Digested residues are commonly used as field fertilizer. The primary nutrients N, P, K are contained in concentrations of 0.3 weight percent. The nitrogen is better available to plants because of the mineralization from organic to ammonium nitrogen. Secondary nutrients and micronutrients are also contained in low concentrations. The enclosed not digested organic substance contributes mainly to humus material. These are the advantages by field application of digested residues. But on the other hand, there is also a risk potential. The small concentration of nutrients leads to high costs for field application. So the field application of digested residues is oriented more due to the costs of storage and transportation than to the optimal demand of nutrients and time of plant growth. Additionally the ammonium-nitrogen is more volatile than in mineral fertilizer because of the higher pH-value. As a result, the field application leads to growing emissions of ammonia and, by N-transformations of deposited ammonium in the soil, to growing emissions of nitrous oxide (N2O) and also to leaching of nitrate into ground water. A model for N-transformations and N-gas production in soils is shown in figure 1. Nitrous oxide contributes to the greenhouse effect about 298 times more then CO2. For ammonia a factor of 2,98 can be used, because nearly 1 percent of disposed NH3 can react to N2O [2]. Therefore reduction of ammonia emissions and the following N2O emissions are meaningful. Additionally methane from the digested residues is emitted, which contributes to the greenhouse effect about 25 times more then CO2.

Figure 1. Model for N-transformations and N-gas production in soils, by [1].

2. EVALUATION OF GREENHOUSE GAS EMISSIONS FROM DIGESTED SUBSTRATES To evaluate the contribution of the application method and kind of treatment of digested residues to emissions of greenhouse gases, in a research project, managed by the Martin-

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Luther-University Halle-Wittenberg [3], the disposal of ammonia, nitrous oxide and methane were measured in different lab scale tests and field tests. For some special tests, digested residues were produced in lab scale by GNS using maize plants traced with 13C and/or 15N isotopes during plant growth. To investigate the emission from digested residues in laboratory tests, a defined soil with defined nutrients and defined test plant were used. The application of digested residues was oriented to an N fertilizing of 150 kg N/ha. A part of the digested residues was treated with the ANAStrip®-process system GNS, which allows the removal of NH4-N from digested substrate with an efficiency of 70 to 95 % [4]. To an other part of digested residues, PIADIN® as a nitrification inhibitor was added. As different methods in application were expected: A. B. C. D. E.

without application of digested residue spraying untreated digested residues on the soil surface spraying ammonia-stripped digested residues on the soil surface incorporating untreated digested residues in the soil incorporating digested residues with added nitrification inhibitor in the soil

The realization of the tests and analysis of the emissions were performed by the MartinLuther-University Halle-Wittenberg and the UFZ – Centre of Environmental Research HalleLeipzig. The following figures show the results of these investigations.

Figure 2. Trend of ammonia emissions.

Emission of ammonia was measured only until 2 days after application with highest emissions for spraying untreated digested residues on the soil surface (Figure 2). Incorporating or stripping of digested residues reduces the ammonia emissions significantly (case A compared with B or C) while addition of nitrification inhibitor plus incorporating increases the emissions of ammonia (case C compared with D). The nitrous oxide emissions can also be reduced by incorporating or by stripping of digested residues. In the case of incorporation and addition of nitrification inhibitor, the nitrification to N2O could be clearly prevented (Figure 3).

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Figure 3. Emissions of ammonia and nitrous oxide during the experiment.

The emission of NH3 and N2O leads to a loss of nitrogen for fertilizing. By spraying the ammonia-stripped digested residues in place of the untreated, nearly 74 % N-loss can be prevented (case A compared to B). In comparison to the incorporation of digested residues into soil, addition of nitrification inhibitor reduces the N-loss up to about 45 % (case C to D). But the case of application ammonia-stripped digested residues + incorporation into soil was not tested. The results lead to the conclusion that by incorporation of digested residues treated with the ANAStrip®-process, a better prevention of N-loss can be reached. The estimation of the CO2-equivalents from NH3, N2O and CH4 gives the results seen in figure 4. As can be seen, the emissions of methane could be clearly prevented by the application of ammonia-stripped digested residues. In the ANAStrip®-process, the solved methane in the digested residues is removed and returned to the biogas as an additional effect. In sum, the greenhouse gases emitted by untreated digested residues are the highest. By treating digested residues with the ANAStrip®-process, 80 % of greenhouse emissions can be prevented compared to an untreated application. Additional effects should be achieved by incorporation of these ammonia-stripped digested residues in the soil (addition of case B + C).

Figure 4. Estimated CO2-equivalents from NH3, CH4 and N2O during the experiment.

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An interesting detail of the lab tests is the acceleration of the plant growth. Figure 5 shows the yield of dry matter. In the case (A) the high emissions of ammonia obviously could not be transported away fast enough, so that the plants were harmed. The highest dry matter yield of the plant growth was reached by application of ammonia-reduced digested residues without incorporation in the soil (B). This is more than without fertilizing with N from digested residues.

Figure 5. Yield of dry matter during the experiment.

The uptake of nitrogen by the plants could by determined by analyzing the sum of N and the isotope 15N, whereas 15N comes only from the traced digested residue (level of tracing roughly 10 to 30 %). Figure 6 shows that the amount of 15N-uptake for plant growing from digested residue lies in the range of 3 - 5 %. From the sprayed stripped digested residues (B) with low ammonium content, the rate of 15N-uptake is relatively higher than from untreated digested residues with high ammonium content. The addition of the nitrification inhibitor gives no better N-uptake in relation to the ammonium content.

Figure 6. Uptake of nitrogen by the plants (sum of N, 15N from digested residue).

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Ute Bauermeister

In conclusion, the application of digested residues with more mineralization from organic to ammonium nitrogen does not automatically lead to a better N-uptake. For an optimal use of the mineralized N-fertilizer potential of digested residues, the removal of ammonia by the ANAStrip®-process and production of a concentrated mineral N-fertilizer is a possible way. By treating digested residues with the ANAStrip®-process, about 74 % of N-loss into the atmosphere and 80 % of green house emissions compared to spraying application of untreated digested residues can be prevented. With additional incorporation of digested residues into the soil the potential of prevention of emissions and N-loss is higher.

3. SUSTAINABLE REGIONAL NUTRIENT LOOPS WITH BIOGAS PLANTS During the ANAStrip®-process the removed ammonia will be converted into a mineral fertilizer-solution of ammonium sulfate (25 to 40 weight-%) which can be used as a depot fertilizer with very high N-uptake by the plants. Because gypsum from flue gas desulfurization (FGD-gypsum) can be used as absorbing substance, a solid fertilizer of calcium carbonate (70 % dry substance) is produced in addition. The treatment of the digested substrates in the ANAStrip®-process can be an essential technology for close regional nutrient loops and for further treatment steps to produce a recyclable process water, concentrated mineral fertilizer and dry organic solids with a high energetic value. By use of the solids in combustion or gasification the ash is applicable as a source of nutrients. But the solids are also directly usable as humus soil substrate and fertilizer because further nutrients like P, K, Mg are contained. The N-nutrient loop in agriculture by energetic use of manure and renewable resources like maize silage in a biogas plant by treating digested residues can be demonstrated using the example of a biogas plant with 500 kW electricity (figure 7). To realize such regional nutrient loops, an important task is to find the best economical und ecological ways to combine the technologies and logistics of agricultural cultivation with biogas plant technology and production and application of fertilizer. One of those technologies is the CULTAN (Controlled Uptake Long Term Ammonium Nutrition) method. This is a qualified injection method for application of the ammonium sulphate solution produced by the ANAStrip®-process, based on one-time injection of the whole dose of nitrogen (and additional sulphur) required for the vegetation period. The injection spot in the soil is characterised by high ammonium concentration that is toxic for plant roots and soil micro-organisms. Consequently the microbial conversion of ammonium to nitrate by nitrification is inhibited. Crop roots form dense root nets around these ammonium depots and take up ammonium from the diffusion zone. Machines for liquid fertilizer injections are available, and experimental results show positive effects on yields and quality in most cases compared to conventional surface fertilization [5]. Moreover, nutrient losses by ammonia volatilisation to the atmosphere and nitrate leaching losses are reduced. Together with partner companies in consulting, agriculture and fertilizing, a concept was developed to close regional nutrient lops by producing a concentrated nitrogen-fertilizer from digested residues, which can be used as a depot fertilizer with very small nutrient loss under prevention of disposal of greenhouse gas.

Prevention of Disposal of Greenhouse Gas from Digested Residues…

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Figure 7. N-Nutrient loop for a biogas plant with 500 kW electricity.

REFERENCES [1]

[2] [3]

[4]

[5]

Stange, F., Döhling, F.: 15N tracing model SimKIM to analyse the NO and N2O production during autotrophic, heterotrophic nitrification, and denitrification in soils. Isotopes in Environmental and Health Studies, Vol. 41, No. 3, Sept. 2005, 261 – 274. Mosier A. R.: Exchange of gaseous nitrogen compounds between agricultural systems and the atmosphere. Plant Soil 228, 2001, 17-27. Herbst, F., Gans, W., Martin-Luther-Universität Halle-Wittenberg – Naturwissenschaftliche Fakultät III - Institut für Agrar- und Ernährungswissenschaften, Professur für Pflanzenernährung: Minderung der Freisetzung von klimarelevanten Gasen beim Einsatz von Gärrückständen aus nR-Biogasanlagen durch Zusatzstoffe und Applikationsmethoden. research-project, sponsored by the FNR, Fachagentur Nachwachsende Rohstoffe e.V., Gülzow (FKZ: 22025207, 2008 – 2010). Bauermeister, U., Meier, T., Wild, A.: Stickstoffabtrennung mit dem ANAStrip®Verfahren System GNS. Gülzower Fachgespräche Vol. 30: Gärrestaufbereitung für eine pflanzenbauliche Nutzung – Stand und FandE-Bedarf―, FNR, Fachagentur Nachwachsende Rohstoffe e.V., Gülzow, 2009. Kücke, M.: Effects of N amount and timing of N injection fertilisationto cereals compared to broadcast surface application. Proceedings of the International Symposium Fluid Fertilizer Injection; Braunschweig 09./10.02.2010.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 10

VETIVERIA ZIZANIOIDES GRASS: A USEFUL TOOL G. Bach * and R. Hommel IfN Forschungs‐ und Entwicklungszentrum GmbH, Elsteraue OT Tröglitz

ABSTRACT The use of Vetiver grass, Vetiveria zizanioides, reclassified as Chrysopogon zizanioides, in non subtropical regions was studied over three cultivation periods in respect of environmental, engineering, and agricultural purposes, respectively. One special focus was directed to the use of that grass as sustainable source for producing bioenergy as not competing with food and fodder plants. Final conclusions will be presented in respect to cultivation conditions applicable in Saxony-Anhalt. Studies were carried out with plants originated from three clones designated as winter resistant. Different results had been obtained studying the behaviour of Vetiver on soils rich in nutrients and those ones used in reclamation processes. This covers also the main aspect of settling in Northern regions: the resistance to coldness. Plants have been obtained being now in multiplication, that seems to overcome or tolerate such conditions. This will offer new opportunities to crop this plant. Additionally, results will be presented that demonstrate the usability of Vetiver to function as sole source in fermentation processes to produce bio-gas. Bio-gas productivity was high compared to other grasses. Methane contents were around and above 60%. Both aspects demonstrate the energetic potential and will also offer the opportunity to transfer this technology into regions with a multitude of crops and high crop yields.

INTRODUCTION During the last three vegetation periods (2006 to 2009) the project run in Saxony‐Anhalt that focused on initial studies on planting and use of vetiver grass. Background of these studies lay in ongoing alterations in climate that will strongly affect agriculture in dry zones *

IfN Forschungs‐ und Entwicklungszentrum GmbH, Dr.‐Bergius‐Str. 19, D‐06729 Elsteraue OT Tröglitz, ifnzeitz@t‐online.de.

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of Saxony‐Anhalt on the one hand. And to find alternative non food and fodder competing plants for energy production on the basis of renewable sources. Main topics were: 1. Studies of agro‐technical conditions on agricultural used land and areas obtained after open coal mining, from with the latter ones are object of soil and water erosion, respectively. 2. Characterisation of components of root‘s essential oil. 3. Development of methods to maintain a high quality ensiled grass. 4. Initial investigation in respect to use vetiver grass as sole source for biogas production. Vetiver a perennial graminaceous plant native in India has been reclassified as Chrysopogon zizanoides L. Roberty – the common name Vetiveria zizanoides is still in use [1]. Veriver grass is distributed in more than 100 countries (predominantly in tropical and subtropical areas) and is the basis of the so‐called Vetiver‐System developed for soil and water conservation and protection in India in the mid 1980s. This project strongly supported by the World Bank revealed unique physiological properties of this plant enabling it to be used for reduction of desertification, phytoremediation of contaminated land, waste water cleaning, slope stabilization, as well and many other applications [2]. The use of vetiver oil as source for perfumes and the universal application of this plant make it so interesting. Recently, studies in China, the USA, Australia or Spain, e.g., had been focused to adapt the plant to climate conditions being more different. Frost-resistant clones had been developed, that shall carryrespective resistance genes. Vetiver forms large clumps from stout rhizomes, growing widely with leaves up to 3m length. The root system goes straight down to more than 2 m deepness. All these aspects gave vetiver a mixture of economic, ecological and environ-mental importance: essential oil extracted from roots, insect repellents, and barrier against erosion and soil pollution, respectively. Vetiver (C4 plant) can be grown under extreme conditions – pure soils, extreme temperatures, drought, flooding etc. But it needs radiation. Good development was reported at altitudes up to and over 2,000 m. Root production varies between 2 and 4 t/ha and shoot biomass varies between 20 to 400 tons dry matter per hectare and year. These outstanding properties were the initiation to study the cultivation of vetiver under conditions of the altering climate north of the Alps in Saxony-Anhalt aiming on the potential of this plant as renewable energy plant, that will not compete with food and fodder plants.

MATERIAL AND METHODS The main problem to be overcome before plantation is the stability of plants used in respect to coldness, hardness to frost, respectively. Therefore three clones were selected from which such properties had been reported:

Vetiveria Zizanioides Grass

  

91

Clone Texas (Texas University, Houston, USA) (a respective gene for hardness against frost was indicated) Clone Japan Clone Australia

All these winter resistant clones were multiplied in-vitro and treated with mycorrhiza (AMykor GmbH). Four sites were used for plantation: One farm land (alluvial soil) and three recultivation areas from surface mining dumps. Initial plantations were done in 2006, followed by main plantations in 2007 (see Table 1). The sites differed very strongly in respect to soil quality cf. Hommel et al. 2008 [3]. Table 1. Survey on plantation of vetiver plants in 2006 and 2007, respectively Year Month Site

2006 May

Farm land A Recultivation Sites P1 P2 P3 P4 P5

3400

August

2008 April

20000

1040 600 525 537 1002

Oil contents of roots were estimated according to European Pharmacopoeia (1997) [4]. The composition of essential oil was done by gas chromatography-mass spectrometry after steam extraction. By this extraction more than 90% of the oily components were obtained. Biogas formation in batch was studied in a stirred 5-l-fermenter at 36°C. Methane, carbon dioxide were estimated gas chromatographically.

RESULTS Growth of Vetiver Plants Results of growth of vetiver had been reported at the Narossa 2008³ – it became obvious that under high quality soil conditions best results were obtained in respect of biomass nd production. Representative data are given in Table 5 (2 growth period). In recultivation sites plants displayed a strong root formation that enables the plants to nail themselves into the soil and therewith to protect the soil against wind and also water erosion at sites with a slope of 10%.

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The good growth parameters were contrasted by the survival rate after winter time. Best survival was obtained in recultivation area of surface mining with respectable percentages during both cultivation periods. Table 2. Survival rates of vetiver plants planted in 2006

Farm land

Thai Texas Japan Australia

Open mining P1

Japan

05.07.07 9% 56% 13% 25% 3% 78%

Japan 2006 Japan 2007 Australia 2007 Texas 2007

61% 30% 68 52%

Not cropped Not cropped Not cropped Not cropped Cropped, 13%). This is the acid of khusimol, the main component of the essential vetiver oil, zizaanoic acid (syn. khusimonic acid). Also other components of vetiver oil coud be found like valencene, long-chain alkanes and alkanoles. The composition of oils studied differed strongly from that of the known oil. And even the odour was different. Table 4. Oil content of vetiver roots Clone

Plantation/Harvest

Volume [ml]

Content [% in dm]

Australia

2006/2007

0,25

6,3

2007/2007

0,07

2,8

2006/2007

0,02

0,6

2007/2007

0,13

4,8

open mining

2006/2008

0,07

0,9

Texas

2006/2007

0,12

4,8

2007/2007

0,15

5,6

2006/2006

0,03

0,6

Japan

Thai clone

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Figure 1. Chromatogram of a steam extract of vetiver roots (detail). Compounds at 30.806 min and 32.116 min were identified as 9,10-dehydro-Isolongifolen and zizaanoic acid, respectively.

BIOGAS Hommel et al.³ reported the production of hight yields of biogas ranging between 460 and 500 mlN(g o.d.m.)-1, respectively. Methane contents in the biogas were described above 60%. These data are much higher than those reported for other grasses elsewhere. Table 5 bases on initial results of gas formation reported in 2008 and on the conditions of plantation (4 plants / m²; less fertilization in farm land and no care in recultivation areas, only one cutting). Potentials for bio-gas and methane, respectively, were calculated on the basis of data from harvest 2007. These data demonstrate that vetiver grass will not compete with conventional plants and energy plants as source of biogas production under recent conditions chosen. For different grasses and with up to 5 cuttings 120 dt/ha dry mater, with 6 cuttings 160 dt/ha dry matter may be harvested. That corresponds to methane yields up to 5,000 m³/ha. Table 5. Biogas potential of vetiver clones studied in the vegetation period 2007

Fresh matter [dt/ha] Dry matter [dt/ha] Yield biogas [lN/kg odm] Methan content in biogas [%] Biogas yield [m³N/ha a] Methane yield [m³N/ha a]

Clone Australia 2007 farm land 33 9,74 460 60 450 270

Japan 2007 f.l. 22 6,43 390 65 251 163

Texas 2007 f.l. 24 6,75 500 60 338 203

Japan 2006 (straw, open mining) 2,9 1,25 130 58 16 9,5

Results obtained for vetiver base on limited biomass production by shorten vegetation time what allows only one cutting. There was no intensive usage of the test sites.

Vetiveria Zizanioides Grass

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Increases in yields may be achieved increase the plantation density, qualification of plant material, and even further optimizations in fermentation processes. The main potential will, however, be the selection of adapted clones, winter resistant plants.

CONCLUSIONS In consequence, vetiver is no competing plant for biogas production under actual points of view in Germany. The history of Miscanthus demonstrates that things will change with the time and intensive studies. The useful combination of protection against erosion and its partial energetic use may be a challenge for future applications of vetiver even in Germany. Additionally, solving the energetic problem of the developing countries, in which vetiver is growing very well, is one of the most important duties of the next years. The exploitation of renewable, biogas, is a must to keep the carbon dioxide production from fossil fuels limits and to reduce wood destruction.

ACKNOWLEDGMENTS The project was supported by Investitionsbank Sachsen-Anhalt, grant-No. 600329409.

REFERENCES [1]

[2]

[3] [4]

Adams RP, Dafforn MR (1997): DNA fingerprints (RAPDs) of the pantropical grass, Vetiveria zizanioides L, reveal a single clone, ―Sunshine‖, is widely utilised for erosion control. Special Paper, The Vetiver Network, Leesburg Va.; Adams RP, Zhong M, Turuspekov Y, Dafforn MR, Veldkamp JF (1998): DNA fingerprinting reveals clonal nature of Vetiveria zizanioides (L.) Nash, Gramineae and sources of potential new germplasm. Molecular Ecology 7:813‐818; Greenfield JC (1989). Vetiver Grass: The ideal plant for vegetative soil and moisture conservation. ASTAG The World Bank, Washington DC; National Research Council (1993) Vetiver Grass: A Thin Green Line Against Erosion. Washington, D.C.: National Academy Press. 171 pp; Purseglove JW (1972) Tropical Crops: Monocotyledons 1, New York: John Wiley and Sons; Truong, P.N. (1999). Vetiver Grass Technology for land stabilisation, erosion and sediment control in the Asia Pacific region. Proc. First Asia Pacific Conference on Ground and Water Bioengineering for Erosion Control and Slope Stabilisation. Manila, Philippines. National Research Council (1993) Vetiver Grass: A Thin Green Line Against Erosion. Board on Science and Technology for International Development, National Academy Press, Washington, DC., Truong P (2008) The Vetiver Plant. In Truong P, Tan Van T. Pinners E (eds.), Vetiver System ‐ Applications Technical Reference Manual. The Vetiver Network International. pp.1‐8. Hommel R., Bach G., Mülker Ch. and Schmidt M. 2008, Narossa 2008 Abstracts. European Pharmacopoeia, third ed., 1997 Council of Europe, Strasbourg, 121 pp.

96 [5]

[6]

G. Bach and R. Hommel Adams RP, Habte M, Park S and Dafforn MR (2004) Preliminary comparison of vetiver root essential oils from cleansed (bacteria‐ and fungus‐free) versus non‐cleansed (normal) vetiver plants. Biochem. Syst. Ecol., 32: 1137‐1144. Massardo DR, Senatore F, Alifano P, Del Giudice L and Pontieri P (2006). Vetiver oil production correlates with early root growth. Biochem. Syst. Ecol. 34: 376‐382.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 11

STATE OF THE ART OF THE RENEWABLE RESOURCES IN POLAND R. Kozłowski, K. Seidler-Lozykowska, M. Mackiewicz-Talarczyk*, P. Baraniecki, J. Mankowski, Cz. Ogurkowski and I. Pniewska The Institute of Natural Fibres and Medicinal Plants, Poznan, Poland

ABSTRACT The paper contains the survey of renewable plant resources and information on nonfood and food applications of agricultural crops in Poland. The following areas are covered: oil crops – oilseed rape, linseed; fiber crops – flax and hemp; carbohydrate crops – potatoes, cereals and sugar beet; special crops: medicinal plants, willow, sources of natural dyestuffs and crop protection natural products. The agricultural land area in Poland in 2006 totaled 15,957 thousand ha, while (as June 2007) was 16,177 thousand ha. Arable land in 2006: 12,449 and 11,869 thousand ha in 2007. Agricultural land and forest land designated for non-agricultural purposes: 2007: 1 6,111 ha . Agriculture land area for industrial application: in 2005: 7.9%; 8.2% (2006); 9.6% (2007). According to latest official, CSO – Central Statistical Office of Poland data – in 2007 the cultivation area of major crops in Poland was as follows: cereals: 8, 353 000 ha, potatoes: 570,000 ha, oil crops: 825,000 ha of which 797, 000 ha rape and turnip rape, maize: 629,000 ha*, industrial crops in Poland, besides the oil-bearing sugar beets already mentioned above, are: sugar beets grown on 247,000 ha, and fibrous plants, 2 cultivated on 3,431 ha , while medicinal plants – app. 25,000 ha. The main industrial products obtained from non-food applications of food crops are alcohol and starch. Other crops include fibrous plant: osier, willow and medicinal plants. The paper presents in details the non-food and food products obtained in Poland from the industrial crops considering also identification and application areas with the highest *

The Institute of Natural Fibres and Medicinal Plants, ul. Wojska Polskiego 71b, 60-630 Poznan, Poland, E-mail: [email protected]. 1 Data from Polish Chamber of Flax and Hemp. 2 Central Statistical Office 2008.

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R. Kozłowski, K. Seidler-Lozykowska, M. Mackiewicz-Talarczyk et al. growth potential, including understanding the driving forces behind market fluctuations. The survey is partly an outcome of FP5 IENICA, also FP6 Eurocrop and FP7: 4F CROPS projects.

INTRODUCTION The most important crops in Poland are cereals, particularly wheat and rye, less significant and in order of area are potatoes, fodder crops, sugar beet, oilseeds and pulses. For most crops, production has been lower in recent years than before transition. Self sufficiency in most crops is generally between 90 and 100% except for oilseeds (ranging between 55% and 146%) where the area has fluctuated considerably in recent years and for sugar where there is generally an exportable surplus. Fruit and vegetables account for 3% of the agricultural area and 10.3% of agricultural output. There are strong exports, in particular of fruit and fruit products. (document of the European Commission, available at http://ec.europa.eu/agriculture/publi/peco/poland/summary/sum_en.htm ) The major industrial crops in Poland are sugar beet and oilseed rape. Other crops include fibrous plant: flax and industrial hemp, osier, willow and herbal plants. The paper presents in details the non-food and food products obtained in Poland from the industrial crops.

PERENNIAL GREEN LANDS The area of green lands in Poland is currently 3,271 thous.ha, which is 27.5% of total arable land. In this figure, 2,497 thous.ha (76.3%) is under perennial meadows and 774 thous.ha (23.7%) - pastures (CSO-2008) Meadow plants, like other cultivated plants can also be used as a one of the components in biogas production as it is practiced in other countries in methane fermentation. Grass can produce 0.6 nominal cubic meter (N/m3) biogas, which is as much as from other green farm wastes, potatoes, grain wastes, beet leafs, buttermilk, etc.

OIL CROPS The oilseed rape is the leader on the Polish market of oil crops (after soya it is the second most important oil plant in the world). The other oil crops such as: linseed (oil flax), sunflower, soya, poppy, mustard, have got less significant importance, some of them are just marginal crops in Poland. Germany, France, Great Britain and Poland are the four biggest producers of the raw material for the oil processing industry in Europe. Poland with the medium production (2000-2005) about 1.2 mio tons comprised 8-10% of the rape production in EU-25. (Polish Association of Rape Producers)

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Oilseed Rape (Brassica Napus L.) A major oil crop grown in Poland is oilseed rape (Brassica napus L). Oilseed rape is grown mainly as a winter crop with spring crop being only a marginal case. Only so called "00" cultivars (containing low amounts of eruic acid and glucosinolans) of winter and spring oilseed rape are grown in Poland. Polish requirements for the glucosinolans and eruic acid are far stricter from those in force in the EU. This is a result of the high importance of oilseed cake as a high-protein component of fodder in Poland. Oilseed rape can be harvested by two methods: one- and two-stage. The one-stage harvesting is especially suitable for the industrial crop – for oil production. The two-stage harvesting is used mainly on sowing seed plantations. The average yield of oilseed rape is 2.1 t/ha. Generally the whole crop is used in the food sector, mainly for vegetable oil and margarine. The overproduction of food and increasing prices of mineral fuels has caused growing interest in the production of biofuel from oilseed rape. The potential cultivation area of oilseed rape in Poland is approximately 2 million hectares. In 2001, the oilseed rape in Poland was cultivated on 443.2 thous. ha. In 2006 the acreage of oilseed rape in Poland was only 395.0 thous. ha. In 2007 – 2 130 thous. tons of oilseed rape was harvested from 532 thous. ha (increasing tendency). Table 1. Area of cultivation, yields and harvest of rapeseed and turnip rape Year 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Cultivation area [‗000 ha] 500.4 467.8 417.1 348.5 370.3 606.4 282.6 317.4 466.0 545.3 436.8 443.2 439.0 426.3 538.2 569.0 658.0 825.0

Yield [t/ha] 2.41 2.23 1.82 1.71 2.04 2.27 1.59 1.87 2.36 2.08 2.19 2.40 2.17 1.86 2.77 2.63 2.65 2.67

Production [‗000 t] 1,206.0 1,042.7 758.2 594.4 755.7 1,376.6 449.3 594.9 1,099.1 1,131.9 958.1 1,063.6 952.7 793.0 1,492.6 810.0 962.0 1,350.0

Source: Statistical Yearbook of RP 2008 of CSO.

The average yields of oilseed rapeseed in 2000 were 2.19 t/ha, in 2001 – 2005: 2.46 t/ha, and 2.67 t/ha in 2007. The total harvested amounts of oilseed rapeseed in 2000-2003 were 958.1 thous. t; 1063.6 thous. t; 952.7 thous. t and about 793 thous. t respectively. In 2007 this figure increased to almost 2.13 mio tons. (CSO, Domestic Biofuels Chamber) (Table 1). Thus there are reserves that might be used for cultivation of oilseed rape for fuel. It is predicted 3

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mio tons of rape in 2013, over 3.2 mio tons of seeds annually, esters only 700-750 thous. t. The predicted demand for rape oil to the esters production in 2010: 600 thous. t ( = 1.5 mio tons of seed of rape).(Domestic Biofuels Chamber). The tendencies in rape cultivation in Poland: 

The cultivation of oilseed rape is carried out in more than 58 thousand out of about 2 million farms in Poland. However it is noticed the phenomenon, that in Poland there is less farms of over 300 ha area, which cultivate rape. In 2005, the farms, which cultivated rape on acreage over 300ha consisted only 19% of total amount. The acreage of more that 50% of 2.5 thous. new farms cultivating rape was below 1ha (small farms). Over 50% of the rape cultivating farms had acreage up to 10ha. The medium acreage was 10ha. (Polish Association of Rape Producers)

It is expected that in 2004 about 6 thous. tons of esters were utilized. It is equal to about 6 thous. tons of oilseed rape oil, applied for fuel. This amount was probably produced in the second half-year 2004. This is an increasing tendency in Polish agriculture. Several research centers in Poland, usually with positive results, tested the oilseed rape fuel.

Oil Flax – Linseed (Linum Usitatissimum Var. Oleifera L.) The cultivation area of oil flax (linseed) in 2002 was 622 ha, 740 ha in 2003 (CSO), 600 ha in 2004. In 2005 the linseed cultivated area totaled 840 ha; in 2006: 1,600 ha; in 2007: 1,100 ha, in 2008: 1,300 ha [data of Polish Chamber of Flax and Hemp and Institute of Natural Fibres and Medicinal Plants]. Linseed oil has numerous nutritional applications and can also be used, owing to specific its properties, for the production of paints and varnishes (as linseed oil is a fast-drying oil). The demand for this application is 15,000-18,000 tons of refined linseed oil a year. The research and implementation is conducted on breeding new, highly efficient oil flax varieties (including these with yellow seeds), production of varnishes and oil varnish and new trends in linseed utilization, e.g. the application of linseed in food and pharmacy. The results of this research are implemented in practice: the INF processes ca 600 tones of linseed per year into the following products: 360 tones of cold pressed oil of which 50 tones are applied as pharmaceuticals (treatment of alimentary canal disorders), 36 tones for food applications and the rest as an additive for fodder. Additionally the linseed oil is used in production of paints e.g. for artists and varnishes.

Oil Industry The Polish oilseed rape industry includes three major oil mills: in Kruszwica. Szamotuly and Brzeg. Each mill has the production capacity of ca 300,000 tons of oilseed rape per year. All are privatized mills owned by foreign capital, mainly French and German. There are also some smaller mills, each with a capacity of 30-60 thousand tons of seeds per year. In addition

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there are a number of small-scale production private mills. In total the capacity of the Polish oil industry is about 1 million tons. The production of oil involves two stages: pressing and extraction with naphtha ether. There is a possibility of industrial utilization of oilseed rape straw, which can be utilized as a raw material for production of furfural. Linseed oil is applied for food, pharmacy, an additive for fodder, in the production of varnishes and paints e.g. for artists.

Oil Markets The markets for oilseed rape products are mainly edible vegetable oil and margarine. The by-product obtained in the process of oil pressing is oilseed rape cake used as a high protein component of fodder for livestock. The economical condition of Polish oil industry factories is good although the export of margarine was observed to decline. Some of the rapeseed oil is used in a mixture with linseed oil in production of alkyd resin.

Obstacles in a Non-Food Sector The major obstacles refer to production of liquid biofuel. The development of liquid biofuel production is obscured by economic conditions. The prices of such biofuel are still higher from those produced from petroleum. Additionally, there are two main barriers in the cultivation of oilseed rape: low winter resistance to frosts and shedding of seeds during harvest. Nevertheless, according to the Directive 2003/30/EC and 670/2003 the percentage of esters in diesel fuel in Poland should increase from 2% in 2005 to 5.75% in 2010 (Table 2 and 3.). Table 2. Minimum share of biofuel and other fuels from renewable sources in the total consumption of liquid fuels (as per bioethanol and ethers or esters), which under the provisions of the Directive is supposed to increase steadily in the nearest future Directive provisions – percentage of biocomponents Bioethanol ETBE (as per bioethanol) Ester

2005 2.00%

2006 2.75%

2007 3.50%

2008 4.25%

2009 5.00%

2010 5.75%

By value type: Energy

3.20% 6.82% 2.12%

4.41% 9.37% 2.92%

5.61% 11.93% 3.71%

6.81% 14.49% 4.51%

8.01% 17.04% 5.30%

9.21% 19.60% 6.10%

Volume Volume Volume

ETBE. Ethyl tertiary butyl ether.

With the consumption of diesel fuel at the level of 6 million tonnes per year, this entails a demand for additional 300-900 thousand tonnes of rape. There are few companies in Poland e.g. Wielkopolskie Paliwa Sp. z o.o., involved in processing rapeseed oil for biofuel that look for their chance in the said directive.

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Table 3. Minimum demand for biocomponents in crude oil fuels in 2000, basing on the assumption that in Poland the objectives set forth in the Directive will be realised by means of bioethanol and its derivatives and esters Petrol – consumption in thousand cubic mtres Demand in thousand cubic metres for: Bioethanol Or ETBE Diesel fuel – consumption in thousand cubic metres Demand in thousand cubic metres for: Esters

2005

2006

2007

2008

2009

2010

218 464 2005

300 638 2006

382 812 2007

464 986 2008

545 1160 2009

627 1334 2010

155

213

270

329

386

446

Source: "Agro Serwis" biweekly.

The situation of Polish producers on the bioethanol market; there are no problems with the availability of raw resources or the processing capacity. In 2009 the production of bioethanol in Poland is conducted by 150-200 producers, while still in 2004 – about 300. The production of bioethanol (100% alcohol): in 2004 - 1,900 thous. hl of 100% alcohol; in 2005 – 2,400 thous. hl; in 2006 - 2,700 thous. hl. In the first quarters of 2007 – the production of ethanol totaled 1,200 thous. hl (40% decrease compare to 2006). The decrease of is connected with the increase of the prices of the raw materials. The structure of the raw material for the bioethanol production in 2007: 80% cereals, 13% molasses, 2% potatoes (decreasing tendency), rest other. According to the data of the records of ARR (Agency of Agricultural Markets of Poland) – on 08.02.2008 there were registered 15 producers of bioethanol, who declared the total production capacity 585 mio litters of bioethanol (5,850 thous.hl). Polish government pays attention to the market of biocomponents and liquid biofuels, treated as a significant element of sustainable development , contributing to the improvement of the country energy safety and environmental protection. The government established ―The program of the promotion of biofuels and other renewable fuels in 2008-2014‖ [Ministry of Economy]. Minimum share of biocomponents in crude oil fuels in increased from 0.68% (2007), to 3.38% (first half of 2008) – it is effect of regulation regarding the minimum share of biocomponents. (Ministry of Economy, Department of Energy). On 22.01.2009 – the Polish Ministry of Economy signed the regulation related to the quality requirements of liquid biofuels; regarding fuel in the form of methyl esters, crude oil with 20% of esters as well as mixture of bioethanol with 70% to 85% of bioethanol and engine petrol E85. There is a problem noticed to adjust the engines to the bio fuels. (Ministry of Economy, Department of Energy). Poland‘s largest in 2009 Bioethanol Plant is in Kostrzyn, using Praj Industries Ltd (India) technology; the project envisages use of barley, corn and wheat as feedstock. The project for Green Source, a group Company of Sniace, Spain, will have capacity to produce 740 m3/day ethanol (the brochure of Praj Industries Ltd., IndiaŚ ―Bioethanol projects in Europe‖). The market of methyl esters produced from rape oil in Poland is still in its nascent stage. Under the provisions of the Directive, as early as 2005 Poland will be obliged to add around 150-160 thousand cubic metres to conventional fuels. Around 450-500 thousand tonnes of rapeseed are needed to produce this amount of esters. There will be no problems with increasing the area used for growing rape and the volume of rape grown if the growers receive orders for rapeseed from the buyers.

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FIBROUS CROPS The climatic conditions of Northern Europe allow for growing only two major fiber crops: flax (Linum usitatissimum L.) and hemp (Cannabis sativa L.).

Fiber (Fibrous) Flax (Linum Usitatissimum L.) Flax was a predominant fiber crop grown and utilized industrially in Europe and in Poland until World War II. Flax cultivation and the linen industry lost its importance in most West European countries by the 1970s. Table 4. Fibrous flax cultivated area in the world [ha] Country

2001

2002

2003

2004

2005

2006

1/

6/

5/

6/

1/

1/

AUSTRIA BELARUS BELGIUM BULGARIA CHINA

132 70 000o 16 9903/ 210 100 0006/

171 67 900 15 3155/ 470 80 0006/

142 70 900 19 3065/ 150o 133 0006/

CZECH REPUBLIC DENMARK EGYPT ESTONIA

7 095

5 885

6 003

191/ 7 6499/ 89

01/ 8 9369/ 35

FINLAND FRANCE GERMANY IRELAND ITALY LATVIA LITHUANIA NETHERLANDS POLAND PORTUGAL RUMANIA RUSSIA

3656/ 67 9703/ 2001/ 03/ 13/ o / 9 600 4 4151/ 4 5204/ 03/ 3006/ 127 340 127 3616/ o / 3421/ 323/ 28 280 4 4301/

2025/ 68 4161/ 2006/ o / 05/ o / 9 346 4 0005/ 5 1004/ 01/ 3006/ 110 820 100 0006/ o / 605/ 251/ 28 200 1565/

SLOVAK REP. SPAIN SWEDEN UKRAINE UNITED KINGDOM

2007 10/

2008 est. o / o / 12 030/ o / o /

109 79 000 19 8231/ 70 200 0006/ 130 0007/ 5 500

134 78 500 18 6701/ o / 130 000

129 75 200 16 3541/ o / 118 500

0,02 78 5001/ 14 6301/ o / 110 000

4 3181/

2 73611/

82410/

o

01/ 13 0109/ 17

o

o

o

/ / o /

o

/ 20 0001/ o /

o

975/ 76 4395/x 2246/ o / 205/ o / 9 4441/ 4 6155/ 6 0004/ o / o / 118 060 104 0006/ o / 25/ 01/ 32 4808/ 1755/

675/ 60 0811/ 1806/ o / 80 1 6546/ 5 494 4 5171/ 6 3454/ o / o / 112 300

/ 5 8479/ Fibrous Flax 0, Linseed 91ha 571/ 81 5081/ 381/ o / 181/ 2 0721/ 3 5991/ 4 6911/ 6 8434/ o / o / 95 450

171/ 76 4971/ 30 o / o / 1 420 1 0571/ 4 36611/,1/ 4 22511/ o / o / 86 000

o

/ 75 5231/ 51 o / o / 22010/ 9501/ 3 4581/ 2 05610/ o / 10710/ 75 000

o

o

o

o

o

o

6710/ o / 34 12 0008/ o /

o

o

/ 17 1389/ 0

/ / 301/ 38 2208/ 1 8201/

/ / o / 25 5308/ 1961/

o

/ / o / 16 164 211/

/ / / /

o o

/ 67 0001/ o / o / o / o / o / 2 5001/ 19914/ o / o / 81 000 / / / o / o / o o

SourceŚ Generally, data provided by relevant countries‘ official organizations (see also the country data). Those data are not marked. Another source of information is described below: 1/ A. Daenekindt: Algemeen Belgisch Vlasverbond, Oude Vestingsstraat 15, B-8500 Kortrijk, Belgium, e-mail: [email protected] 2/ FAOSTAT Statistical Database Results 1997 http://apps.fao.org 3/ Mr. Jordi Petchamé Ballabriga, Administrateur, Olives, huile d‘olive et plantes textiles, D.G. VI.C.4 - Loi 130 7/126, European Commission, Rue de la Loi 200, B-1049, Bruxelles, Belgium 4/ Polish Chamber of Flax and Hemp, office at the Institute of Natural Fibres, Poznan, Poland, tel.: +48-61 8 455 851, e-mail: [email protected], data provided by the Ministry of Agriculture and Rural Development.

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54ème Congrès CELC – Berlin, Réunion d'information Générale / Section commune CultureTeillage 6/ CELC/MASTERS OF LINEN, 15, rue du Louvre, 75001 Paris, France, tel.: +33(0)1 42 21 06 83, e-mail: [email protected] 7/ Research Institute of Industrial Crops of Heilong Academy of Agricultural Sciences, Harbin, China, 150086, tel.: (86)0451-55261351, E-mail: [email protected] 8/ Dr Pavel Goloborod'ko, Institute of Bast Crops, Lenina 45, 245130 Glukhov, Sumy, Ukraine, tel.: 3805444 22643 9/ Prof. Dr. D. M. El-Hariri, The Network Representative in the Near East, NRC, Cairo, Egypt, email: [email protected]; acc. to Agricultural Economics Bulletins of the Central Administration for Agricultural Economics and Statistics of Egypt. 10/ Ministry of Agriculture and Rural Development of Poland (basing on European Commission documents) 11/ Data of European Commission, DG AGRI of May 2008, Doc. No 9875/08 note: in all tables the mark °/ means data not available 5/

Total flax cultivated area in EU countries: 103,867 ha3/ (2000), 94,631 ha3/ (2001), in 2002: 88,885 ha1/, in 2003: 98,965 ha1/, in campaign 2004/2005: 118,251 ha, in campaign 2005/2006: 122,379 ha, 2006: 10/ 105,025 ha; in 2007: 10/ 78,500 ha. In entire Europe total flax cultivated area in 2007: 10/ 95,117 ha. Flax in Poland flax maintained its strong position both in agriculture and industry until the 1980s. In the same period, when flax growing and processing declined in countries such as Sweden, Germany and the UK, in Poland the largest cultivation area of flax (over 100,000 ha) was observed in the beginning of the 1970s. In that time the share of Poland as a producer of linen fabric was 8.3% (120 million m2). Poland had 31% of world export of linen and hemp fabric (ranking second in the world). A decline in this field began in the 1990s with a transformation of the Polish economy resulting in a considerable decrease of the production capacity of factories and raw material availability. The following were the key factors in that situation:    

financial reforms of the state including changes in credit policies for factories changes in industrial activity as a result of ownership restructuring opening of the market to high quality raw material and semi–products (yarns) among others from the other EU countries and China changes in the structure of production and connected increase of demand for high quality fiber

As a result, the cultivation area of flax was considerably reduced from 34.3 thous. ha in 1989 to 2.4 thous. ha in 1998. The total flax cultivation area was 5.2 thous ha in 2001 (CSO) and 5.1 thous ha in 2002 (CSO). In 2004 total cultivation area of flax, according to data of Polish Chamber of Flax and Hemp, was 6,345 ha, where fibrous flax 5,745 ha. (Table 4.). The constant decrease of the flax cultivation area in Poland noticed; in 2008 only 1,991 ha were sown. The leading research center involved in research on flax and hemp is the Institute of Natural fibers in Poznan (INF).

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Table 5. Fibrous flax cultivation area [thous. ha] Year 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 est.

Flax cultivation area 34.3 29.6 9.7 5.5 4.0 8.1 13.8 5.9 3.7 2.4 3.1 4.1 5.2 5.1 3.0 5.7 6.5* 4.2* 2.1* 1.99 0.9**

Source: CSO, Poland, * Ministry of Agriculture and Rural Development, ** data by Polish Chamber of Flax and Hemp.

The research topics conducted at INF cover research and development activities, from molecular biology, biotechnology, breeding and cultivation, through processing till final application opportunities. The following are the main research areas in flax conducted at the INF:           

Creative and maintenance breeding of fibrous flax Besides traditional breeding techniques, biotechnological methods are also used, allowing for considerable shortening of breeding process. Modern cultivation technology for obtaining high yields and quality of flax raw material. Harvesting technologies and flax harvesting machines. Technologies of flax straw retting. Modification of fiber and spinning technologies allowing for spinning Weaving and knitting technologies allowing for increasing the efficiency of woven and knitted fabrics production. New finishing technologies of linen and hemp. Production technologies of linen ready-made products with special properties. Pro-ecological utilization of textile materials and by-products. o biocomposites for automotive, construction, furniture and packaging industry Developing new pharmaceutical and nutrition products

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Phytoremediation of degraded areas by fibrous plants Research on physiological effect of bast fibers on human body and comfort of clothes.

Hemp (Cannabis Sativa L.) Hemp has been grown and utilized in Poland for some 1000 years, i.e. from the very beginning of the Polish State. In 2004 the list of cultivated crops cultivars included three fiber hemp cultivars: Beniko, Bialobrzeskie and Silesia. Polish hemp cultivars grown for fiber fully meet the requirements specified in the article 5a of the Regulation 1251/1999 issued by the EU for hemp grown in Europe. In Polish regulations (The Counter Drug Addiction Act of 24.04.1997) only cultivars containing less than 0.2% delta-9-tetrahydrocannabinol in green dry matter can be grown. The Polish cultivars Beniko, Bialobrzeskie and Silesia are listed in the Annex to the IInd Regulation of the Commission No. 206 of 5th February 2004. The only research and development organization in Poland involved in all aspect of hemp research is the Institute of Natural Fibres and Medicinal Plants in Poznan. In 1995 the research has begun to establish conditions for in vitro tissue culture growing. Polish cultivars of monoaecious hemp are resistant to spring ground frosts, which enables early sowing – by the end of sowing time of spring small grains. One of the most promising potential applications of hemp is pulp and paper production. The annual hemp biomass production is 2.5 times higher than that of pine tree forest (in Central European conditions). Hemp pulp is a specialty, long fiber pulp suitable for the production of banknote, cartographic and photographic paper, cigarette tissue and long life document paper (due to stable whiteness of the hemp paper). Recent INF research has shown that hemp and flax fiber contain natural absorbents, among which a special role is played by lignin able to absorb UV radiation. Therefore, there is a great potential for hemp fiber to be used to protect the human body against UV radiation. Additionally, due to the high content of essential oils, hemp can be grown for the cosmetic industry (essential oils used as additive for production of soap lotions, creams, shampoos, perfumes and in aromatherapy). The two compounds found in hemp essential oils, limonene and α-pinene, show insect repelling properties and potentially can be used for plant protection products manufacture. Hemp essential oil also shows bacteriostatic properties to Gram+ bacteria (Staphylococcus and Streptococcus). This effect is comparable to thyme oil. (1A)

Flax and Hemp Industry Processing of flax straw is being carried out by the following organizations: 1) Experimental Plant LENKON in Steszew being the organisational unit of the INFandMP.

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2) STEICO, Czarnkow- 1,151.51 ha of hemp cultivated in 2008 to be processed to into building insulation 3) PPHU ―CELINEN‖ Sp. z o.o. in Radwanice 4) Madex-Malbork Production of yarn and fabric is conducted in 6 mills (linen companies): 1) Zaklady Lniarskie ―ORZEL‖ S.A. in Myslakowice (the spinning is party limited, mainly weaving and finishing) 2) Zaklady Lniarskie ―MADEX‖ in Malbork, 3) Fabryka Wyrobow Lnianych (Linen Products Factory) ‖Zyrardow‖in Zyrardow, 4) Spinning Mill Safilin-Polska in Milakowo, 5) Spinning Mill ―LAMBRECHT‖Sp. z o.o.in Okonek. Hemp cultivation area in Poland had its peak in 1960s when the area under cultivation reached 30,000 ha. Currently, hemp is rather a marginal crop grown on a small area although a considerable increase has been observed: 326 ha in 2003; 909,63 ha in 2004; 129 ha in 2005; 1,007 ha in 2006; 1,376 ha in 2007 (Ministry of Agriculture and Rural Development) and 1,427 ha in 2008 (Polish Chamber of Flax and Hemp, INFandMP). According to the data of the ARMIR (Agency of the Restructuring and Modernization of Agriculture) the hemp cultivation area, registered to obtain the subsidies in 2008 was 1,391.61 ha. The production of hemp yarn and fabrics is not conducted in Poland; only technical yarn in the LENKON – the Experimental Plant located in Steszew (the unit of the Institute of Natural Fibres and Medicinal Plants in Poznan).

Flax and Hemp Markets Markets for flax products cover long and short fiber, so called ―cottonized‖ fiber for blends, pure and blended, dry and wet-spun yarns and woven and knitted fabrics. Table 6. Production and markets of flax products Item 2001 2002 2003 2004* 2008 Dew retting [%] 100 100 100 100 100 Mill consumption of flax [t] 6123 6880 6760 16 000+ Yarn production [t] 5950 6669 7400 7475 Production of linen fabrics 3953 4380 4500 5200 n/a [1000 m] Export of linen textiles 2371 255 3100 4900 (fabrics) [1000 m] Source: Polish Chamber of Flax and Hemp (2004). + Mill consumption includes first time the data from Company Safilin. 75% of flax fibre consumed in Poland in 2004 was imported. n/a not available.

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Table 7. The production of linen yarn and fabrics in Poland in 2008 Linen Company

Linen yarn production [tons]

LAMBRECHT 1,200 SAFILIN 3,000 MADEX 540 ORZEL 160 ZYRARDOW ----------Source: the Linen Companies listed in the table.

Linen fabric production [mio m2]

0.4 1.8 1.5

Table 8. Data on export and import of fibre and linen products in Poland in 2003 Specification Export in thous. USD Scutched flax fibre 2,137 Linen tow and waste products 2,255 Linen yarn total 24,840 Linen fabrics total 21,302 Source: Ministry of Economy, Labour and Social Policy.

Import in thous. USD 10,661 3,789 9,877 31,552

Hemp products sold on domestic markets are: technical yarns (mainly a string), fabrics and non-woven, as well as certified sowing seeds sold to the EU (mainly Germany). In 2004 two factories manufactured hemp string: 1) ―STRADOM‖ Company in Czestochowa. 2) LENKON – the Experimental Plant located in Steszew, in 2009-only LENKON.

Environmental Issues Connected with Flax and Hemp Both flax and hemp are natural, renewable resources and as such do not pose any special burden to the environment unlike in case of materials derived from fossil resources. Some problems occur at the stage of processing:  

problems with sufficient dust removal during flax and hemp processing outdated machinery is the source of noise

Positive issue is that bast fibrous plants extract heavy metals like Cd, Cu, Pb from the soil polluted by industry (related publications listed in reference).

Obstacles in Flax and Hemp Sector   

significant fractionation and dispersion of fiber plant plantations. high price for certified sowing material. outdated machinery in production and processing

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109

lack of funds for modernization competition of cheap chemical fibers average quality of domestic fiber results in low profitability the industry processing natural fibers is highly capital consuming lack of funds for purchase machinery

CARBOHYDRATE CROPS Potatoes In 2002 the potatoes cultivation area in Poland was 1.19 million ha, while in 2003 decreased to 766 thous. ha, which consists almost 55% of EU potatoes cultivation area. In 2004, potatoes area decreased to 713 thousand ha and was of 52,5 thous. ha lower (6,9%) than in 2003 and of 579,1 thous. ha (44,8%) lower compare to mean data in 1996-2000. (CSO). In 2005 cultivation area was 588 thous. Ha, in 2006 totaled 597 ha, and in 2007 decreased to 570 ha (CSO 2008) Table 9. The structure of potatoes production/uses/consumption (Potatoes balance sheet from 2000 to 2007 in thous. t Specification

Amount

Amount

Amount

Amount

2000/01

2004/05

2005/06

2006/07

Resource

24,632

14,153

10,576

9,333

Harvest (production)

24,232

13,999

10,369

8,982

Imports

400

154

207

351

Use

24,632

14,153

10,576

9,333

Exports

367

400

451

393

Domestic uses

24,265

13,753

10,125

8,940

-planting

2,795

1,493

1,480

1,395

-feeding

11,765

4,908

2,013

1,646

-losses

3,390

1,550

1,050

781

-for industry (total for food and nonfood) -of which for starch

1,205

862

762

517

815

760

650

420

-consumption*

5,110

4,940

4,820

4,601

Source: Statistical Yearbook of RP 2008 of CSO; * including potatoes for processing.

The total harvest (production) of potatoes in 2002 amounted to15.5 million tons (down by 20% from the year 2001); while in 2004 was 13.7 million tons. In 2005 production decreased to 10.4 mio tons; in 2006 to 8.98 mio tons; but in 2007 increased to 11.79 mio tons (CSO 2008).

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In Poland the consumption of potatoes is circa 129 kg per person per year, of which 90% would be accounted for ware potatoes. Overall potatoes consumption in Poland in 2005/06 was 4,820 thous. tons, while in 2006/2007 decreased to 4,601 thous. tons (CSO 2008) In 2004 potatoes yield amounted to 193 dt/ha, i.e. 7,8% up from 2003. The yields in the following years totaled as follows: in 2005: 176 dt/ha; in 2006: 150 dt/ha; in 2007 the yields increased to 207 dt/ha. (CSO 2008) The processing of potatoes for starch, dried potatoes and ethanol in 2004/05 season was slightly lower than in previous season: at 0.9 million tons, while the potatoes processing for consumption purposes reaches 600-685 thous. tons. The industrial uses of potatoes in Poland show the decreasing tendency: from 1,205 thous. tons in 2000/01 to 517 thous. t in 2006/07 (table 7). The utilization potatoes for starch decreases as well: from 815 thous. t in 2000/01 to 420 in 2006/07. The structure of potatoes production/uses/consumption in 2000-2007 is shown in Table 9. The potatoes cultivars grown in Poland for starch, included into the Polish National List of COBORU in 2003: – –

Medium early and medium late: Glada, Klepa, Lawina. Late: Bzura, Jasia, Hinga.

Potatoes Industry The industrial processing of potatoes includes two major products: starch and ethyl alcohol. The production of starch and spirit in 2001/02 was 130,000 tons, simultaneously 11,000,000 liters of 100% spirit.

Starch The domestic quota of potatoes starch production is 144,985 tons for the economic year 2004/2005, according to Accession Treaty. In 2003 processing of potatoes for starch was observed to increase up to 950,000 tons. Starch is naturally biodegradable and a renewable biopolymer, present in several plants, but most of all in the potatoes bulb. In the season 2004/2005 the starch production is expected not to be higher than 149,985 thous. tons, in accordance with the contingents, the EU quotas (Accession Treaty of 13.12. 2002). Industrial products made of starch are used in the following industries: paper, Corrugated board production, Textile industry, Foundry, Drilling industry, Glue production. Application of Ethyl Alcohol From Potatoes The addition of ethanol to gasoline started in 1992. The legislation referring to bio-fuels and bio-components is provided in the Regulation of October 2, 2003 (Dz.U. Nr 199, poz. 1934) about biocomponents applied in liquid fuels and about liquid bio-fuels). The processing of potatoes for spirit in 2003 reached 100 thous. tons and the share of potatoes spirit totaled 4% of total spirit (Potatoes Market, November 2003). In 2003/04 - 86 thousand tons of potatoes was processed for spirit, while in 2004/05 the forecast predicts 86 thous. tones.

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Table 10. Industrial processing of potatoes [thousand tons] The product 2000/01 2001/02 2002/03 2003/04 2004/05* For starch 815 690 910 979 725 For dried potatoes 175 85 85 133 130 For spirit 215 100 100 86 86 For other food 450 530 600 543 645 products The total processing 1655 1405 1695 1741 1586 * forecast. Source: CSO-GUS, ―Polziem‖ Sp. Z o.o., Ministry of Finances, information from companies, own elaboration of the Institute of Agriculture and Food Economy, Warsaw.

Table 11. Production of potato spirit Production of spirit [mio liters 100]o In which: potato spirit [mio liters 100o] The share of potato spirit in total spirit production [%] Processing of potato [thous. tons]

1999 167 10,1

2000 173 13,9

2001 181 11,0

2002 210 7,5

2003 220 7,0

2004 240 7,0

6,0

8,0

6,1

3,6

3,2

2,9

113

168

131

87

86

86

Data of the Ministry of Finances, own elaboration of the Institute of Agriculture and Food Economy, Warsaw.

Table 12. Production of chips and “French fries” 2000 28.5 1007.7

2001 36.4 126.4

2002 37.3 151.1

Production of chips Productio n of ―French fries‖ Source: CSO. * the market did not show the decreased demand for ―French fries‖. ** forecast.

2003 40.0 121,0*

2004 45.0 150.0

Markets Potatoes harvest in the year 2004 was 13.7 million tons. Some reduction in potatoes cultivation area occurred as well: in 2004, potatoes area decreased to 713 thousand ha. The processing of potatoes for starch and dry potatoes mass increased in 2001 to over 1.3 million tons, in 2002 up to 1,695 million tons and in 2003 totaled 1,720 million tons. The processing of potatoes for starch, dried potatoes and ethanol in 2004/05 season is expected lower than in previous season: at 0.9 million tons. In the season 2002/03 the potatoes market turnover increased to about 3.7 million tons. The high export of potatoes and their products in 2002/03 reached 820 thous. tons (in equivalent of fresh potatoes), and was of 17% more than in 2001/02. The processing of potatoes for starch in 2001 to 2004 totaled respectively: 690, 910, 979 and 725 thous. tons. Generally, the economical and financial condition of the Polish potatoes processing companies is now better than in the last few years. The decline results

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from production quota for starch, which was lowered to 145 thousand tons after Polish accession to EU on 1 May 2004. The financial standing of potatoes industry has improved, the profitability of production increased, the problems with sales diminished.

Cereals The area of cereals grown in 2001 totaled at 8,882 thous. ha, while in 2002 was 8,295 thousand hectares, in 2003 – 8,163 thousand ha. In 2004 cereals‘ cultivated area was almost 8,400 thousand ha, and increased of 214,0 thous. ha (2,6%) compare to data of 2003. In 2005 the sown area of cereals totaled 8,329 hous.ha, in 2006: 8,381 thous. ha; in 2007: 8,353 thous. ha. The CSO estimation shows, that in 2001-2005 total production of cereals in Poland was 26,758 thous. tons, in 2005: 26,928 thous. tons; in 2006: 21,776 thous. tons; 2007: 27,143 thous.tons. The yields of cereals in Poland in 2001-2005 were 31.9 dt/ha; in 2005: 32.3; in 2006: 26.0 dt/ha; in 2007: 32.5 dt/ha. Table 13. The structure of cereals production / uses / consumption (cereals balance sheet from 2000 to 2007 in thous. t) a) Specification

Amount Amount Amount Amount 2000/01 2004/05 2005/06 2006/07 Resource 25,478 30,668 29,169 18,203 Harvest (production) 22,341 29,635 26,928 21,776 Imports 2,442 1,033 1,020 3,609 Decrease in stocks b) 715 _ 1,221 2,818 Use 25,478 30,668 29,169 28,203 Exports 105 741 1,752 1,330 Domestic uses 25,341 26,122 27,202 27,072 -sowing 1,960 1,831 1,756 1,752 -feeding 15,516 16,043 17,194 17,573 -losses 1,097 1,257 1,223 1,038 -for industry e) 930 1,181 1,255 1,340 -consumption* 5,838 5,810 5,774 5,369 Increase in stocks b) 32 3,805 215 1 Source: Statistical Yearbook of RP 2008 of CSO a) Wheat, rye, barley, oats and cereal mixed, triticale, maize for grain, other cereals (millet, buckwheat). b) Stocks in industrial processing and trade. c) Excluding grain milling.

The industrial application of wheat and rye mainly concerns the production of alcohol, but it is still mainly the alcohol used in the food sector (vodkas). The production of alcohol for non-food purposes is almost entirely based on cereals (96.4%). The industrial consumption (in brewery, starch and alcohol production) for this purpose in season 2002/2003 is estimated at 1,161 tons total for all cereals.

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The example of non-food applications of cereal alcohol is addition to the production of bio-fuels in the form of direct addition to the fuel or as a product used form manufacture ethyl-tert-butiric ester (ETBE). Domestic production of cereals in 2002, including components of fodder, was 27,798 thous. tons. The approximate ratio of grain to straw varies from 1.5-2.0; the annual production of straw in Poland is about 25 million tons (MRRW, 2003). The main use for the straw is animal bedding and feed and fertilizer (95%) (MRRW, 2003). Nevertheless straw can be also a good renewable fuel. The straw harvested from 2 ha (2.5 t/ha) can provide enough energy to heat a 2 house of 70 m

Sugar Beets In 2001 sugar beet crops totaled at 11.36 million tons, harvested from the area of 318,000 hectares. In 2002 the figures were 13.43 million tons and 303,000 ha respectively (CSO). In 2003 – 11.739 million tons, harvested from the area of 286,300 hectares. In 2005 sugar beets were cultivated on 286 thous. ha and the harvest was 11,912 thus.tons. In 2006 sugar beets were cultivated on 262 thous. ha and the harvest was 11,475 thus.tons. In 2007 sugar beets were cultivated on 247 thous. ha and the harvest was12,682 thus.tons. The yield of sugar beet in 2006 was 438 dt/ha as compared to 411 dt/ha average for the period 2001-2005. In 2007 this figure increased to 513 dt/ha. Table 14. The cultivation area, yields and harvest of sugar beet in Poland Specification Cultivation area [thous.ha] Yields from 1 ha [dt] Harvest [thous.t] a

2000 333 411 12,236

2005 286 416 11,912

2006 262 438 11,475

2007 247 513 12,682

Average yearly figures.

The 13.4 million tons of sugar beet was processed in 2002 by 75 mills (one did not operate due to bankruptcy process). 56 sugar mills including 22 grouped in the Domestic Sugar Company LTD and sugar mills owned by foreign capital carried out the sugar beet processing campaign. The non-food applications utilized about 45,000 tons of sugar in 2002, which covered pharmaceutical, chemical industries, cosmetics, fodder as well as unidentified usage e.g. home production of wine, bees feeding etc. In the pharmaceutical industry sugar is used for the production of syrups and for sweetening some preparations. In the chemical industry a saponin made of sugar beet is used for the production of detergents as very good, natural surface active substance; sugar beet molasses (the by-product) is used for the production of potassium and sodium carbonate, alcohol, yeast, citron acid and monosodium glutamate. Dried sugar beet pulp is applied for the production of ruminant fodder.

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SPECIAL CROPS Medicinal Plants This section focuses mainly on medicinal plants, as well as aromatic plants, natural plant protection products and plants used for dyeing. The area of medicinal plants cultivation in Poland is up to 25,000 ha. Table 15. Major medicinal plants cultivated in Poland Species name of medicinal plant Thyme (Thymus vulgaris L.) Valerian (Valeriana officinalis L.) Chamomile (Chamomilla recutita (L.) Rausch.) Peppermint (Mentha piperita) Milk thistle (Sylibum marianum Gaertn.) Caraway (Carum carvi L.) Savory (Safureja hortensis L.) Lemon balm (Melissa officinalis L.) Lovage (Levisticum officinalis Koch.) Sage (Salvia officinalis L.) Evening primrose (Oenothera paradoxa) Purple coneflower (Echinacea purpurea Moench.)

Cultivated area [ha] 3000 2500 2000 2000 1500 1500 400 300 300 300 250 200

Source: Dr. Jerzy Jambor, Phytopharm Kleka S.A.

Herbal medicinal products: the basic herbal pharmaceutical preparations are those applied in preventing diseases and for digestion problems such as Sylimarol and Raphacholin. The leading herbal medicine in geriatric treatment is Geriavit. In pediatrics, the preventive treatment has limited application – mainly immune-stimulating. Aromatherapy: major essential oils applied in Poland are obtained from the following medicinal plants: angelica, thyme, caraway, rosemary, mint, lavender, basil, coriander, fennel, marjoram, chamomile, lemon balm, sage.

Cosmetics Phytocosmetics, containing herbal substances are popular in Poland. The herbs containing flavonoids, polyphenolic acids, anthocyanins, tannins, carotenoids, catechins, saponins, mucilage and polysaccharides are suitable for these products as raw material for phytocosmetics. The most important herbal species for cosmetics are: chamomile (Chamomilla recutita (L.) Rausch.) marigold (Calendula officinalis L.) burdock (Arctium lappa L.) eyebright (Euphrasia officinalis L.) nettle (Urtica dioica L.) purple coneflower (Echinacea purpurea Moench.)

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lemon balm (Melissa officinalis L.) St. Johns Wort (Hypericum perforatum L.) common birch (Betula verrucosa Ehrh.) arnica (Arnika montana L.) oak (Quercus sp.) sweet flag (Acorus calamus L.) ivy (Hedera helix L.).

Aromatics and Condiments (Spices) The main species cultivated for condiments (spices) are: caraway (Carum carvi L.), thyme (Thymus vulgaris L.), marjoram (Origanum majorana L.), savory (Satureja hortensis L.), sweet basil (Ocimum basilicum L.), lovage (Levisticum officinale Koch.), coriander (Coriandrum sativum L.), garlic (Allium sativum L.), angelica (Archangelica officinalis Hoffm.). The commercial cultivation supplies approximately 7,000 tons of dry mass of these herbs per year.

Medicinal Plants Market Data of Herba Polonica (2001, No 2, vol. XLVII) show that every year about 20,000 tons of herbal raw materials are purchased in Poland (about 3,000-5,000 tons collected from the wild, and 15-17,000 tons - from cultivation). It is foreseen that the value of domestic market‘ sales of those raw materials will increase by 10-12 % per year (compared to EU markets where the increase is expected to be 8-10%). The major plant drugs on Polish pharmaceutical market in 2003 (the number of sold packages in million pieces): Raphacholin 5.4, Sylimarol 5.0, Alax 3.9, Plantex 3.7, Venescin 2.5, Calming down pills 2.4, Urosept 1.9, Radirex 1.8, Amol 1.7. The value of the plant drugs sold in 2003 in million PLN (million Euro): Sylimarol 23.7 (5.4), Amol 21.5 (4.9), Raphacholin 20.4 (4.6), Ginkofar 15.2 (3.5), Equisetum 12.5 (2.8), Tanakan 12.0 (2.7), Soyfem 11.0 (2.5), Aescin 10.9 (2.48), Urosept 10.5 (2.4).

Cosmetics There is a growing interest in the Polish society to use more natural and eco- and humanfriendly cosmetics. This means an increased demand for phytocosmetics. Simultaneously, the volume and value of usage of phytocosmetics and natural essential oils increase significantly and it is a stable trend.

Natural Dyestuffs The application of natural dyestuffs in the textile industry could provide opportunities for

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small and Medium Enterprises and creating new jobs. The INF is conducting a grant project involving special workshops in rural areas with high unemployment, aimed at training women in natural art handicraft and natural dyestuff extraction and application. The INF has noticed quite high demand for such production and activities.

Natural Dyestuffs The cultivation of plants for dye extraction could be a brand new and eco-friendly opportunity, because many artificial dyestuffs contain substances, which are aggressive and not human-friendly. Among the research carried out at the Institute of Natural Fibres in Poznan there is a natural dye programme. Research is based on the analysis of the traditional sources, the studying dye manuals and dyeing trials. The main research goal is application of vegetable dyestuffs in natural fabrics and development of a fashion collection dyed with these dyestuffs. The dyestuffs can be found in fruits, shoots rhizomes, roots and bark, trees cores as wall as excrescence and mosses. The INF examined thirty natural dyestuffs to find the most economical and permanent colours. The involved methods use only natural mordants. The sources of tannin were found in tree bark – oak, willow, alder, as well as sumac and rhubarb leaves and oak galls. Tannin used as a premordant helps to improve the absorption of alum and copper. The first industrial trials are performed on the base of the INF experience.

Natural Products for Crop Protection Polish bio-products of production are based on active substances, contained in garlic, rapeseed oil and several other crops (including cereals). Some of products belong to the group of so-called adjuvants, which are most often applied.

Osier (Wicker) and Willow The cultivation area of osier is approximately 2,000 ha. There are special plantations of bush osier, where very good harvesting equipment is used. The species of osier and willow in Poland are: Salix americana, Salix viminalis, Salix puprupea, and Salix amygdalina . Recently the most popular is Salix viminalis. The yield obtained annually can be estimated at 4,000 tons. The yield of bush osier Salix americana is up to 8 tons /ha (average yields vary from 5-8 t/ha in the south-east and between 10-15 t/ha in the western Poland). Osier (wicker) is used mainly for production of different household goods, such as shopping and picnic baskets, traces, furniture (sofas, chairs, armchairs, chest of drawers, garden and furniture sets), lamp shades, table mats, flower stand, sticks, suitcases, trunks, fens etc.

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CROPS AND PRODUCTS FOR RENEWABLE ENERGY Dehydrated Ethanol Since 1993 the ethanol as an additive to fuel has been produced on the industrial scale in Poland. The maximum production for this purpose (110 million liters) was observed in 1997. From that time this production decreased to only 48.3 million liters in 2000. In 2000 the production of dehydrated ethanol was conducted by 13 enterprises. Table 16 shows the utilization of raw materials for ethanol production. Table 16. Utilization of raw materials for ethanol production

Year

1995 1996 1997 1998 1999 2000

Major agricultural raw materials applied for production of ethanol [thous.tons] Raw material Rye Potatoes Molasses [thous.t] Share of total rye [thous.t] Share of total potatoes [thous.t] production [%] production [%] 664.8 10.6 649.0 2.6 0.0 680.0 12.0 640.0 2.4 31.0 630.5 11.9 370.6 1.8 50.0 522.0 9.2 216.0 0.8 76.0 378.0 7.3 112.0 0.6 107.0 176.5 4.4 118.7 0.5 38.8

Source: Ministry of Agriculture and Rural Development, 2004.

Table 17. Production of bioethanol from corn, wheat, potatoes, sugar beet Species

Medium yields in Poland [t/ha]

Yield of ethanol [dm3/t]

Production of ethanol [dm3/t]

Corn Wheat Potatoes Sugar beet

6.1 3.5 19.0 45.0

390 340 140 100

2,379 1,190 2,660 4,500

The consumption of raw material for 100 dm3 ethanol 256 294 714 1,000

Source: Krajowa Rada Gorzelnictwa i Produkcji Biopaliw (Domestic Council of Distillery and Production of Biofuels).

The total production of alcohol from agricultural raw materials is shown in Table 18. Table18. Production of alcohol from agricultural raw materials (1994-2002) [m dm3] Product Raw spirit Ethanoldehydrated spirit

1994 210 27

1995 245 63

1996 278 100.9

1997 240.6 110.6

1998 208 99.8

1999 167.2 88.5

2000 173.3 51.5

2001 181 69.4

2002 210 82.8

Source: Ministry of Agriculture and Rural Development. Department of Land Economy and Rural Infrastructure (MRRW) 2003.

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Solid Biofuel The market of solid biofuel is not regulated and develops spontaneously, being competitive with another fuel markets.   

wood waste products cereal straw bush willow (salix viminalis)

Grasses of Miscanthus genus and Sida hermaphrodita Rusby Species that can be used for production of renewable energy are grasses of large yield potential e.g. species of Miscanthus genus and sida (Sida hermaphrodita Rusby).

SPECIAL PLANTS MARKETS Natural Products for Crop Protection The growing environmental consciousness in Polish society, among farmers and growers, has led to a tendency towards organic production, and increases the demand for natural products e.g. active substances extracted from crops.

Osier (Wicker) The yield obtained annually can be estimated at 4,000 tons. The products obtained from osier (wicker) are exported to West European markets, mainly to Germany, where they are very popular.

Crops for Renewable Energy The data regarding markets for crops for renewable energy in Poland are rather limited yet. Here are some descriptions available to the authors. Dehydrated Ethanol In 2000 the production of dehydrated ethanol for fuel additive aim decreased to 48.3 million liters. (110 million liters in 1997) and was conducted by 13 enterprises. The significant decrease in the production of ethanol for fuel is connected mainly by the limited demand of major fuel producers. Cereal Straw Recently the market of the cereal straw devoted for heating purposes develops dynamically. The production of cereal straw in Poland exceeds 20 million tons per year.

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Legislation All new herbal medicinal products are being tested in National Institute of Public Health and have to be approved by the Office for Registration of Medicinal Products, Medical Devices and Biocides in Warsaw. The cultivation of several special species is regulated or forbidden: Papaver somniferum, Digitalis sp., and Cannabis sativa .

Prioritization The medicinal plants are the most important crops for special uses in Poland. The climate, soil and low levels of pollution in entire country, especially in the east and north–east part of the country are some of the best in Europe for the cultivation of medicinal plants. Also, the high rate of unemployment and overpopulation in rural areas gives great chance for the production of herbal raw material. Additional beneficial factors are:   

tradition of cultivation and usage of herbal plants and herbal medicinal products common acceptance of the society about 100 years of research Table 19. Purchase of herbal raw materials in Poland

Purchasing value Over 1,000 tons 500-1,000 tons 200-500 tons 100-200 tons

50-100 tons 10-50 tons

5-10 tons

Raw Materials Matricariae flos, Menthae piperitae folium, Valerianae radix Carduri mariae fructus Hippocastani semen, Thymi herba, Hypernic herba, Oenotherae semen Tilae flos, Mellisae folium, Boraginis semen Aroniae fructus, Frangulae cortex, Sambuci fructus, Betulae folium, Quercus cortex, Salicis cortex, Hippocastani cortex, Urticae folium, Cynarae herba, Visci albi herba Equiseti herba, Sambuci flos, Crataegi inflorescentia, Taraxaci radix, Salviae folium, Millefolii folium, Farfarae folium, Violae tricoloris herba Agropyri rhizome, Calami rhizome, Levistici radix, Calendulae anthodium, Chelidonii herba, Phaseoli pericarpium, Althaesae radix, Arnicae anthodium, Galegae herba, Origani herba, Angelicae radix Unilae radix, Euphrasiae herba, Cichorii radix, Meliloti herba, Cnici herba, Anthemidis anthodium, Convallariae herba, Malvae arboeae flos

Source: Dr. Jerzy Jambor, Phytopharm Kleka S.A.

The domestic market of herbal medicines with almost 3,000 authorized and implemented products is estimated to be about 200 million USD. Natural products for plant protection, produced in Poland   

Bioczos BR – ProducerŚ PPH ―HIMAL‖ Lodz Biosept 33 SL – Producer: Cintamani Poland – Piaseczno. Polyversum – Producer: Biopartner S. C. Poznan.

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120    

Adpros 850 SL – Producer: Varichem T. Ostrowski- Huta Zabikowo. Olejan 85 EC – ProducerŚ Towarzystwo Chemiczne ― Danmar‖ Lodz. Biochikol 020 PC – Firm Gumitex Poli-Farm sp.z o.o. Lynkowica Grevit 200 SL – Firm Avis Naturall Polska Sp.z o.o.

Table 20. The best sold herbal products in Poland (The annual value of herbal drug sale over 1 mio USD) Name Geriavit Amol

Shape (form) Capsules Liquid

Producer PHARMATION (CH) ROLAND (D)

Sylimarol Raphacholin C Bilobil Wyciag ze Skrzypu Polnego z Witaminami Urosept

Pills Pills Capsules Tablets

HERBAPOL Poznan (PL) HERBAPOL Wroclaw (PL) KRKA (SLO) VITAMEX (S)

Pills

HERBAPOL Poznan (PL)

Oeparol Tablets Uspokajajace

Capsules Tablets

AGROPHARM (PL) LABOFAM (PL)

Poldanen

Tablets coated

Alax

Pills

Sirupus Plantaginis Tadenan 50

Syrup

HERBAPOL Poznnan (PL) HERBAPOL Poznnan (PL) HASCO-LEK (PL)

Cardiol C

Drops

Boldaloin

Tablets

Kalms

Tablets

Esberitox N

Tablets

Echinacea

Tablets

Pyrosal

Syrup

HERBAPOL (PL)

Melisana

Liquid

KLOSTERFRAU (D)

Capsules

DEBAT (Foumier Group) (F) HERBAPOL Wroclaw (PL) HERBAPOL Wroclaw (PL) G.R.LANE HEALTH PRODUCTS (GB) SCHAPER AND BRUMMER (D) RATIOPHARM (D)

Source: Dr. Jerzy Jambor, Phytopharm Kleka S.A.

Wroclaw

The components of plant origin Ginseng extractum sic. Oleum Citronellae, Oleum Myristicae, Oleum Caryophylli, Oleum Cinnamomii, Oleum Citri, Oleum Mentae, Oleum Lavandulae Silybi mariani fructus extractum siccum Raphani radix recens extractum sic., Cynarae herba extractum sic. Ginkgo bilobae extractum siccum Eauiseti extractum sic., Urticae extractum sic.

Extractum sic.: Petroselini radix, Phaseoli pericarpium, Betulae folium, Chamomilae anthodium, Vitis idaeae folium Oenothera paradoxa oleum Melissae folium, Leonuri cardiacae herba, Valerianae radix, Lupuli strobili, Menthae piperitae folium, Lavandulae, flos Pygeu africani cortes extractum sic. Alona, Glycyrrhizae radiz, cortex extr., Atropa radix Plantaginis extractum fluidum

Frangulae

Pygei africani cortex extractum sic. Tinc. Convallariae titr., Tinc Valerianae, Tinc. Crataegi, Colae extractum fluidum Aloe extractum sic., Boldinum Lupuli strob. pulvis, Valerianae extractum sic., Gentianae extractum sic Thujae herba extractum sic., Echinaceae rad. extractum sic., Baptisiae extractum sic. Echimaceae angustifoliae radix extractum sic. Extractum fluidum: Farfarae folium, Sambuci flos, Tiliae inflorescentia, Salicis cortex; Fructus Ribis Concentratum Extractum: Melissae fol., Inulae rad., Angelicae rad., Zingiberis rhiz., Piperis nigri fruct., Gentianae rad., Myristicae sem., Phaseoli pericar., Cinchonae cort., Casiae flos, Cardamomi fruct.

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ACKNOWLEDGMENTS Words of gratitude to all who supported the gaining of the data.

SOURCES OF INFORMATION (REFERENCES IN THE REPORT TEXT) [1A] Publications and materials of the Institute of Natural fibers, Poznan. Among others: EUROFLAX Newsletter (ISSN 1429-8090), Natural fibers- Wlokna Naturalne, Journal of Natural Fibers (edited by INF, printed and distributed by Haworth Press Inc., New York, and Francis and Taylor, Philadelphia, USA) [1] The publications and information of the Institute of Medicinal Plants, Poznan [2] Dr. Jerzy Jambor, PhytoPharm. Kleka S.A., Kleka [3] Dr. Wladyslaw S. Brud, POLLENA-AROMA, Warsaw [4] Wieslaw Dzwonkowski et al. Instytut Ekonomiki Rolnictwa i Gospodarki Zywnosciowej. ―Rynek Ziemniaka‖. "The Potatoes market. Its condition and perspectives. Market reports". [5] publications and the and information of the (CLPZ)–Starch and Potatoes Products Research Laboratory, and Association of Potatoes Starch Producers, Lubon [6] CENTRUM KOSMETOLOGICZNE Sp. z o. o., Warszawa [7] Stefan Szczukowski, Univeristy of Warmia and Mazury, OlsztynŚ ―Evaluation of different plant species for energy production and their cultivation possibility in Warmia and Mazury regions‖, ―Implementation of new technologies for alternative plant production for energy production and herbal preparations‖. Jan Kus ―utilization of renewable energy resources in Poland‖ [8] Prof. Dr. hab. Jan Zastawny, IMUZ Falenty [9] Ministry of Agriculture and Rural Development of Poland

GENERAL SOURCES OF INFORMATION Statistical Yearbook of the Republic of Poland, Warsaw Materials of the Ministry of Agriculture and Rural Development of Poland, 2003 Register of varieties of agricultural plants (Lista odmian roslin rolniczych), COBORU, Slupia Wielka , Poland. ISSN 1231 –8299. 2003. Wieslaw Dzwonkowski et al., Institute of Agriculture and Food Economy.‗The Potatoes market‘ś ‗The Sugar market‘ś ‗The Cereals market‘ś 'The Oilseed Rape market'. Its condition and perspectives. Market reports. ISSN 1231-2762. Warsaw Newsletter of the Polish Chamber of Flax and Hemp ―Flax and Hemp‖(Biuletyn Informacyjny Polskiej Izby Lnu i Konopi ―Len i Konopie‖ ISSN 1731-4828) The publications and information provided by the Institute of Medicinal Plants, Poznan Materials provided by Dr. Jerzy Jambor, PhytoPharm Kleka S.A., Kleka

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Publications and materials of Starch and Potatoes Products Research Laboratory, Lubon (e.g. Marian Maczynski et al. The possibilities of starch utilization and future trends in the potatoes starch production technologies) Materials provided by Dr. Wladyslaw S. Brud, POLLENA-AROMA, Warsaw Information provided by the producers of natural crop protection preparations Information provided by of HERBA STUDIO , Zakrzewo Information provided by of the Drug Institute, Warsaw Information provided by the Polytechnic University of Lodz, Faculty of Food Chemistry and Biotechnology, Institute of Chemical Food Technology, Department of Sugar Industry Chotkowski et al. Plant Breeding and Acclimatization Institute (IHAR), Bonin, Poland: Production of potatoes. Technology–Economy–Marketing. 1997

HEAVY METALS CONNECTED REFERENCES L. Grabowska, P. Baraniecki: Three Year Results on Utilization of Soil Polluted by CopperProducing Industry. Natural Fibres – Wlokna Naturalne, Special Edition, Flax and Other Bast Plants Sympoium, 30.09-01.10.1997 Pozna, Poland. L. Grabowska, P. Baraniecki: Development of Cultivation of Flax, Hemp, Grasses and Rapes on the Areas Polluted with Heavy Metals. INF, Poznan, Poland, 1994. R. Kozlowski, J. Mankowski, L. Grabowska et. al.: Selection of Kinds and Methods of Cultivation in Safety Buffer Zone of Copper Smelter in Glogow. INF, Poznan, Poland, 1993. R. Kozlowski, L. Grabowska, J. Mankowski, J. Mscisz and W. Rynduch: Possibilities of Applying Fibrous Plants Cultivated in Polluted Areas for Pulp and Particleboard Production. Pacific Rim Bio-Based Composites Symposium, 9-13 November 1992, Rotorua, New Zealand.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 12

ENERGY EFFICIENCY OF FOUR CROP SPECIES Jerzy Pudelko *, Jerzy Mankowski and Jacek Kolodziej Institute of Natural Fibres and Medicinal Plants, Poznan, Poland

ABSTRACT The study discussed in the paper aimed at energetic evaluation of the following crops: spring barley, winter wheat, winter rapeseed and sugar beet. The experiments were conducted at Research Station of Agricultural University of Poznan in Dlon. The agronomic treatments in all years of experiment were conducted according to generally used rules of cultivation for given crops. The objective of the study was to determine the net energy efficiency per hectare for the four tested crop species. The net energy was the difference of energetic value of the crop and energy inputs supplied.

Keywords: biomass, net energy, spring barley, winter wheat, winter rapeseed, sugar beet

1. INTRODUCTION The main advantage of biomass as a raw material for energy production, in comparison with fossil fuel is considerable reduction of CO2 emission to the atmosphere. More and more often, it is the biomass produced by agriculture that becomes the source of energy. In 2020, 15% of energy produced in Poland should come from renewable resources and in 2030 – 20%. Selection of crops for energy production must consider the sustainable development approach. The amount of accumulated energy in biomass per area unit varies and depends on the plant species plus natural and agronomic factors [5]. In this study, the energy efficiency of

*

Institute of Natural Fibres and Medicinal Plants, ul. Wojska Polskiego 71B, 60-630 Poznan, Poland.

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the following crops was investigated: spring barley, winter wheat, winter rapeseed and sugar beet. The aim of the study was to determine the net energy efficiency per hectare of the four crops. The net energy was calculated as the difference of energy value of the crop minus energy input for the crop production.

2. MATERIALS AND METHODS The field experiments were conducted at Research Station of the Poznan University of Life Sciences in Dlon. Seeds of spring barley, winter wheat, winter rapeseed and sugar beet were sown in the properly prepared seedbed according to the experimental scheme. The agronomic treatments in all years of the experiment were conducted according to commonly accepted guidelines for particular crop species. The cultivation area of winter wheat in 2006 and 2007 was about 118 ha. In 2008 winter wheat was grown on 110 ha. Cultivation area of spring barley in three consecutive years of the experiment was 108, 115 and 117 ha, respectively. Winter rapeseed in 2006 was cultivated on 97 ha, in 2007 on 125 ha and in 2008 on 117 ha. The lowest cultivation area was covered by sugar beet – 52, 46 and 47 ha, respectively. In 2006 the cultivation area was dominated by winter wheat, in 2007 by winter rapeseed and in 2008 by spring barley and winter rapeseed. Soil tillage covered ploughing, done with a reversible, seven-furrow plough combined with a Campbell roller and Crosskill roller. Using of the drum, which crushed the soil and evened the field surface during the winter plough, no field surface evening was necessary in spring. For pre-sowing cultivation, a compact aggregate K 600 – TS III was used. Using of the aggregate allowed for reduction of the number of runs on the field which had a beneficial effect on the soil by limiting the soil compaction. Reduced number of runs improved also the economics of cultivation by reduction of fuel consumption. The NPK doses varied upon the soil fertility. The study resulted in determination of energetic value of tested plants per hectare and their energy efficiency. Energetic value of crops was determined by calculating the energy content in dry matter of plants. The energy inputs were calculated by the energy consumption coefficients taken from ―Encyklopedia Agrobiznesu‖. The values of the coefficients were as followsŚ work – 40 MJ/wh, tractors and agricultural machinery – 112 MJ/kg, fuel 48 – MJ/kg, sowing material – 7.2 MJ/kg, pest control chemicals – 300 MJ/kg of active ingredient, nitrogen fertilizers – 77 MJ/kg, phosphorus fertilizers – 14 MJ/kg and potassium fertilizers – 10 MJ/kg [6].

3. RESULTS The difference between energy efficiency for rapeseed and winter wheat was lower than the difference in net energy for the two crops. This was the result of relatively high energy wh = working hour

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125

inputs to obtain yields of winter wheat. The highest energy efficiency per cultivated area was obtained for rapeseed – 109.47 GJ/ha. The difference between energy efficiency of sugar beet and winter rapeseed was only 5 GJ/ha. The lowest energy efficiency was observed for cultivation of spring barley – 38.50 GJ/ha. It was lower by 8 GJ/ha than energy efficiency of winter wheat and by as much as about 70 GJ/ha lower than that of winter rapeseed. 120

109,47

103,80

100

[GJ/ha]

80 60

47,37 38,50

40 20 0

spring barley winter wheat

winter rapeseed

sugar beet

Figure 1. Average energetic value of tested crop yields.

100

90,67

90

85,38

80

[GJ/ha]

70 60 50 40 30

26,78

29,69

20 10 0 spring barley winter wheat

winter rapeseed

sugar beet

Figure 2. Average net energy obtained for tested crops.

The highest efficiency of net energy was obtained for cultivation of winter rapeseed. It was over threefold higher than that for spring barley and winter wheat. The net energy from

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cultivation of sugar beet was also high, as it was only by about 5 GJ/ha lower than that of winter rapeseed. Tested cereals produced much lower energetic value then crops mentioned above. Comparable values of net energy were obtained from cultivation of spring barley and winter wheat. However, winter wheat was characterized by higher value of net energy by about 2 GJ/ha. Variability of both energetic value and net energy value in years for particular crops was quite high. These variations resulted from both different energy inputs in particular years and obtained yields. Table 1. Energy inputs, crop energetic value and net energy obtained from tested crops Crop

Years

Spring barley

Winter wheat Winter rapeseed Surgar beet

2006 2007 2008 2006 2007 2008 2006 2007 2008 2006 2007 2008

Energy inputs for yield obtaining [GJ/ha] 13.8 11.17 10.81 17.51 19.19 16.32 21.58 17.24 17.53 26.46 13.28 15.48

Crop energetic value [GJ/ha] 34.20 42.56 38.76 45.60 47.88 48.64 106.80 109.47 112.14 100.44 109.62 101.34

Net energy [GJ/ha] 21.02 31.38 27.94 28.08 28.68 32.31 85.21 92.22 94.60 73.97 96.34 85.85

The highest net energy value from cultivation of spring barley was obtained in 2007. This was due to favorable weather conditions which contributed to relatively high yields. The lowest amount of energy was obtained in 2006 due to high nitrogen inputs and relatively low yields. In case of wheat, the highest net energy value was obtained in 2008 due to high yields and low inputs for production. The highest inputs in cultivation of wheat were connected with nitrogen application. The highest energy inputs for yield were born in 2007. In rapeseed cultivation, the highest net energy value was obtained in 2008. High net energy value in rapeseed cultivation were obtained despite relatively high energy inputs for yield obtaining and resulted from energetic value for this crop. The highest energy inputs were born in sugar beet cultivation. Due to high energetic value of the yield, the obtained net energy was also high. The highest net energy value for sugar beet was obtained in 2007. This was caused by the lowest energy inputs born in that year – twofold lower than in previous year. High energy value was born in nitrogen and potassium fertilizers and in work of agricultural machinery. Table 2. Energy efficiency of tested crops per hectare expressed in tons of coal Raw material Spring barley Winter wheat Winter rapeseed

2006 1,36 1,82 4,27

2007 1,70 1,91 4,37

2008 1,55 1,94 4,48

Mean 1,53 1,89 4,37

Energy Efficiency of Four Crop Species Sugar beet

4,01

4,38

4,05

127 4,14

* according the Mining Institute 1 t of coal = 25 GJ.

In Table 2., the given energetic efficiency was calculated to the coal. According to the Mining Institute 1 t of coal = 25 GJ of energy. The data presented show that barley and winter wheat, due to low energetic value are the crops that rather cannot be used as energy sources. The crops that can be used as energy sources are sugar beet and winter rapeseed. The energetic value of winter rapeseed and sugar beet expressed in tons of coal is over twofold higher as compared to both cereals tested (spring barley and winter wheat).

4. DISCUSSION Successful plant cultivation for energy purposes depends on the inputs born on the production and energy contained in the crop. Studies conducted by the author show that efficiency of net energy obtained for rapeseed and sugar beet is much higher than for wheat and barley. The energy efficiency for winter rapeseed and sugar beet was lower than energy efficiency for hemp, which was investigated in previous studies in which whole hemp plants were grown for energy. The average yield of hemp achieved in the experiment was 13.2 t/ha [3]. 300 249 250

[GJ/ha]

200

150 109

103

winter rapeseed

sugar beet

100

50 0 hemp

Figure 3. Energetic value of two chosen plants compared with hemp.

Hemp is characterized by clearly higher energy efficiency of dry matter as compared to winter rapeseed and sugar beet. The very high energy efficiency is a result of high heat of combustion of hemp which is 18.8 MJ/kg and high yield of dry matter per hectare. Energetic value of rapeseed is also high (17.6 MJ/kg). Thus, the conclusion is that energy efficiency of plants is mainly affected by the level of yield rather than by the heat of combustion. Consequently, the profitability of crops cultivated for biomass depends on the selection of

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proper plant species which should be characterized by high and stable yields of dry matter and relatively easy and low energy consuming agronomic treatments applied. The cultivation costs of crops for energy production purposes cannot be high and the price of raw material very low. If the profitability of crops for biomass production is lower than crops used as fodder or food then the farmers will not decide to grow them [1]. The state policy approaching cultivation of energy crops also becomes important through different types of subsidies. In Poland, the Ministry of Economy is working on a document ―Energy Policy for Poland till 2030‖. The document is to define in details the strategic directions of action in the area of energy policy of the state [4]. It is foreseen that estimated potential of domestic renewable energy resources in agriculture is about 120 PJ in 10 year horizon [2]. According to the strategic provisions related to utilization of renewable energy resources, the main emphasis will be on energy obtained from industrial plants and biogas in farm biogas plants. The share of renewable energy in energy balance of the state is gradually growing: from 1.5% in 2001 up to 7.5% in 2010. It is estimated that the use of renewable energy in 2020 will reach 14%.

CONCLUSIONS 1. Differences in energy inputs between tested crops resulted mainly from the level of inputs connected with application of fertilizers. 2. Higher energy inputs were born for cultivation of winter rapeseed and the highest for cultivation of spring barley. 3. Energy efficiency from cultivation of winter rapeseed and sugar beet was by 60% higher. 4. Higher net energy value was obtained for cultivation of winter rapeseed and sugar beet and lower for winter wheat and spring barely. 5. The net energy from cultivation of winter rapeseed and sugar beet is over threefold higher than that of spring barley and winter wheat. 6. Only winter rapeseed and sugar beet, among tested crops, can be useful for cultivation for energy purposes. The reason for this is high energy efficiency of biomass produced by these crops.

REFERENCES [1]

[2] [3] [4]

Burczyk H., Kolodziej J. (2009): Porownanie plonow i wartosci energetycznych konopi włoknistych, kukurydzy i sorga z roslinami egzotycznymi. Biuletyn Informacyjny Polskiej Izby Lnu i Konopi. Len i Konopie nr 12/2009. Poznan. Grzybek A., Gradziuk P., Kowalczyk K. (2001): Sloma energetyczne paliwo. Akademia Rolnicza w Lublinie. Warszawa. Kolodziej J. (2009): Efektywnosc energetyczna konopi w zaleznosci od czynnikow agrotechnicznych. Praca doktorska. Uniwersytet Przyrodniczy. Poznan. Panczyszyn T. (2008): Polityka energetyczna Polski do 2030 roku. Czysta Energia 9/2008.

Energy Efficiency of Four Crop Species [5]

[6]

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Pudelko J., Skrzypczak W. (2007): Comparision of energy efficiency of some crops in different environmental and agronomic conditions. 13 International Conference for Renewable Recources and Plant Biotechnology. Poznan, Poland. Wos A. (pr. zbiorowa) (1998): Encyklopedia Agrobiznesu. Fundacja Innowacja. Wyzsza Szkola Spoleczno Ekonomiczna . Warszawa.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 13

ENZYMATIC BIOMASS HYDROLYSIS Viktor Antonov1, Josef Marousek², Jan Marek, Stanislav Kuzel² and Tomas Rosenberg³ 1

INOTEX, spol. s r.o. , Dvur Kralove n.L., Czech Republic ²Jihoceska Univerzita, Ceske Budejovice, Czech Republic ³BIOPROFIT, Slapy n.V., Czech Republic

Biomass is a huge renewable source of energy. Such energy is hidden into saccharide chains which are splitable to fermentable monosaccharides. Enzymes with targeted actvities are able to efficiently catalyze this hydrolysis and to speed it up. Enzymes increase the production of fermentable monosaccharides in acid as well as weakly alkaline conditions. Thanks to enzymatic hydrolysis different cellulose plant resources can be utilized better for bioalcohols as well as biogas production. Recent results from labscale testing of specially developed enzymatic products Texazymes from INOTEX product range will be presented. Different types of enzymes as well as an acid, alkaline and high pressure pre‐hydrolysis will be compared. Saccharification of different plant and vegetable sources will be shown and compared.

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1. INTRODUCTION Saccharification of different kinds of biomass sources have been investigated in cooperation of Inotex as an enzyme producer together with one of Czech universities (Jihoceska univerzita) and one of Czech biogass plant engineering companies (Bioprofit). Special attention has been paid to the effect of enzymatic hydrolysis. Effect of different types of enzymes to the saccarification degree of different kinds of biomass has been studied. Enzymatic treatments have been combined and compared with other physical as well as chemical treatments such as thermopressure preparation as such and also in acid and alkaline conditions. The amount of glucose equivalents calculated on dry mass was used as expression of the biomass saccharification degree.

2. MATERIALS Different biomass sources were used such as corn grains, corn silage, beet tubers, potatoes, oat flakes, wheat grains and barleycorns. Different development and market stage enzymes prepared by Inotex were used (see the overview bellow). For comparison several other analytical grade enzymes supplied by Fluka such as alpha‐amylases from Aspergillus oryzae (AO) and Bacillus subtilis (BS) were used. Malt containing diastase enzymes was used too. Table 1. Overview of Inotex enzymes used INOTEX enzymes Texazym APN Inosample CLC Inosample APC Inosample HMP Inosample HMC Inosample ASD Texazym BIO‐TK

Microbial producers Aspergillus oryzae Trichoderma reesei Trichoderma sp. Trichoderma reesei Humicola insolens Bacillus licheniformis Trichoderma sp.

Main activity cellulase cellulase cellulase hemicellulase hemicellulase alpha‐amylase cellulose, hemicellulase

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3. METHODS Saccarification was realised with either enzymatic treatment only or with an physical/chemical pretreatment. As an pretreatment 3 types of thermo-pressure preparation have been tested. Enzymatic hydrolysis: 1) 2) 3) 4)

10% solid fytomass is mixed and the pH is adjusted to the optimal pH of the enzyme when the pH is unchanging, the enzyme is added sample mixed and is tempered in optimal enzyme reaction temperature for 6 hours the pH is revised simultaneously if needed

Thermopressure preparation: 1) 10% solid fytomass material prepaired 2) in compressive vessel heated up to 180°C (0,6 MPa) for 30 minutes 3) release the valve for fast decompression Acid thermopressure preparation: 1) 10% solid fytomass material mixed with 300 ml H2O and (3, 6, 9, 12%) H2SO4 2) in compressive vessel heated up to 180°C (0,6 MPa) for 30 minutes 3) release the valve for fast decompression Alkalic thermopressure preparation: 1) 10% solid fytomass material mixed with 300 ml H2O and (3, 6, 9, 12%) NaOH 2) in compressive vessel heated up to 180°C (0,6 MPa) for 30 minutes 3) release the valve for fast decompression

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Viktor Antonov, Josef Marousek, Jan Marek et al. Procedure of glucose equivalent determination: 1) solution A = 388g of crystalline Na2CO3+ 50g C6H8O7+ 25g crystalline CuSO4+ H2O (till 1 000 ml) 2) 10g of 10% solid fytomass + 10ml solution A 3) 10 minutes boiling, that fast cooling 4) add 5ml 30% KJ and 5ml 25% H2SO4 5) difference of Na2S2O3 . 5 H2O titre used for blind sample and titre used for tested sample multiplied by 1,745 gives the mg amount of glucose-equipollents (sugars) from 1g of tested sample

4. RESULTS In the first test there were compared 2 cellulolytic (CLC, APN), 2 hemicellulolytic (HMC, HMP) and 1 amylolytic enzymes (ASD) and their effect to different types of biomass. All enzymes were dosed in the concentration of 1 ml of enzyme on 1 litre of biomass solution. All biomass samples had 10% of dry mass. Results are measured and shown (on the Y axis) in percentage growth of the content of glucose equipollent sugars towards the original content.

Figure 1. Only enzymatic treatment.

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Figure 2. Enzymatic treatment after thermopressure pretreatment.

Figure 3. Enzymatic treatment after acid thermopressure pre-treatment (6% sulphuric acid).

Figure 4. Enzymatic treatment after alkaline thermopressure pretreatment m(10% sodium hydroxide).

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It is obvious from the figures above that after the thermopressure pre-treatment the highest effect had both hemicellulases in comparison with enzymatic treatment without any pretreatment where also both cellulases played an important role. Results obtained from the first test lead to adding of several other enzymes to the experiment (APC, AO, BS, SLAD) and to the development of the new combined cellulolytic/hemicellulolytic product suitable for common pH and temperature conditions in biogass fermenters – Texazym BIO!TK. Table 2. Corn grains saccharification CORN GRAINS mg glucose equivalents / g dry mass Type of enzyme used – ASD AO HMP SLAD CLC enzymes only 80 46 84 186 74 enzymes after thermopressure 91 221 148 138 322 201 preparation

HMC APN BS TK APC 63 48 87 115 82 184

113

12 201 143 4

Figure 5. Corn grains saccharification.

Table 3. Comparison of different pretreatment of corn grains, i.e. different types of thermopressure process compared with the effect of Texazym BIO-TK CORN GRAINS BIO-TK thermopressure preparation BIO-TK after thermopressure preparation 3% acid thermopressure 6% acid thermopressure 9% acid thermopressure 12% acid thermopressure 3% alkal. thermopressure 6% alkal. Thermopressure 9% alkal. Thermopressure 12% alkal. thermopressure

mg glucose equivalents / g dry mass 115 91 201 131 137 141 164 115 117 121 133

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In the second test there were compared 3 cellulolytic enzymes (CLC, APN, APC), 2 hemicellulolytic enzymes (HMC, HMP), 1 combined enzyme (BIO!TK), 1 amylolytic enzyme from Inotex (ASD), 2 amylolytic enyzmes from Fluka (AO, BS) and 1 sample of malt representing a diastase enzyme (SLAD). All enzymes were dosed again in the concentration of 1 % from the dry biomass. Results are measured and shown (on the Y axis) as a weight (in mg) of glucose equipollent sugars in 1 g of dry biomass.

Figure 6. Comparison of different pretreatment of corn grains, i.e. different types of thermopressure process compared with the effect of Texazym BIO‐TK.

Table 4. Corn silage saccharification CORN SILAGE Type of enzyme used enzymes only enzymes after thermopressure preparation

mg glucose equivalents / 1 g dry mass no ASD AO HMP SLAD CLC 46 36 35 41 26

HMC APN BS 16 15 16

33

96

95

Figure 7. Corn silage saccharification.

114 125

118

137

87

TK 20

APC 14

117 136 127

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Table 5. Comparison of different pretreatment of corn silage, i.e. different types of thermopressure process compared with the effect of Texazym BIO-TK CORN SILAGE BIO-TK thermopressure preparation BIO-TK after thermopressure preparation 3% acid thermopressure 6% acid thermopressure 9% acid thermopressure 12% acid thermopressure 3% alkal. thermopressure 6% alkal. Thermopressure 9% alkal. Thermopressure 12% alkal. thermopressure

mg glucose equivalents / g dry mass 20 33 136 16 16 16 17 14 13 15 16

Figure 8. Comparison of different pretreatment of corn silage, i.e. different types of thermopressure process compared with the effect of Texazym BIO‐TK.

Table 6. Beet tuber saccharification BEET TUBER Type of enzyme used enzymes only enzymes after thermopressure preparation

mg glucose equivalents / 1 g dry mass no

184

ASD

AO

HMP SLAD CLC

HMC

APN

BS

164

175

295

332

265

201 386

180

202

346

215 246

277 277

410

299

TK

226 392

APC 271 295

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Figure 9. Beet Tuber saccharification.

Table 7. Comparison of different pretreatment of beet tuber, i.e. different types of thermopressure process compared with the effect of Texazym BIO-TK BEET TUBER BIO-TK thermopressure preparation BIO-TK after thermopressure preparation 3% acid thermopressure 6% acid thermopressure 9% acid thermopressure 12% acid thermopressure 3% alkal. thermopressure 6% alkal. Thermopressure 9% alkal. Thermopressure 12% alkal. thermopressure

mg glucose equivalents / g dry mass 386 184 392 254 278 281 291 185 189 199 204

Figure 10. Comparison of different pretreatment of beet tuber, i.e. different types of thermopressure process compared with the effect of Texazym BIO‐TK.

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CONCLUSION There were compared several types of enzymes for the saccharification of different biomass sources in these first 2 testings. Results have proved the ability of all enzymes used to saccharify different cellulosic and other sugar based biomass. The effect of each type of enzyme is dependent on type of biomass used. Enzymes with cellulolytic and hemicellulolytic activities are effective for beet tuber and grains saccharification whilst amylase together with hemicellulase enzymes are effective in case of corn silage and grain. When a thermopressure pretreatment is used the effect of enzymes is much higher and moving towards cellulase and other glucanase activities. Nowadays next tests are running with saccharification and biogas production in plant scale. The biomass consists mainly of corn silage and green grass. Texazym BIO!TK is tested in this test.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 14

SUSTAINABLE LOGISTICS CENTERS Hartmut Zadek1* and Robert Schulz†2 1

University Magdeburg ―Otto-von-Guericke‖, Chair of Logistics, Institute of Logistics and Material Handling Systems, 2 Universitaetsplatz 2, 39106 Magdeburg, Germany

ABSTRACT Today it is already possible to perform a company's intra-logistics with renewable energies and other innovative technologies. This paper will outline which technologies can already be used in logistics centers today and which concepts must still be developed in the future to improve the energy efficiency and make logistics centers more sustainable. A sample concept will show how a company can produce hydrogen (H2) with renewable energies, for example wind energy or photovoltaic (PV), and to use this H 2 for the powertrain of its forklifts.

Keywords: intra-logistics, logistics center, energy efficiency, sustainability, hydrogen powered forklifts, renewable energies, biomass micro power plant

1. INTRODUCTION Since the automotive industry conducts research in the use of H2 technologies as powertrain in cars especially the intra-logistics industry could use this technology for the reduction of CO2 emissions. Companies are not dependent on political decisions regarding the H2 infrastructure because they work in their own production or logistics system. The use of H2 as a fuel for the powertrain of forklifts is only one possibility to reduce CO2 emissions. However, there are other existing technologies like Ethylene Tetraflouroethylene (ETFE)

* †

E-mail: [email protected]. E-mail: [email protected].

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inflated foil cushions with integrated PV cells, bio-fuel plants or kinetic plates which generate electricity when vehicles pass over it. The combination of these various technologies was not realized in a logistics center yet. Either the electricity produced on-site is not used in the facility itself or the electricity required for the powertrains of intra-logistics equipment and other electric consumers such as lighting is not generated by renewable energies. Thus an integrated concept for logistics centers completely supplying itself with electric energy from renewable energies is still missing. Such a concept would make logistics centers really CO2 neutral buildings.

2. EXISTING TECHNOLOGIES AND CONCEPTS ―Green Logistics‖ becomes more and more relevant for logistics companies. A couple companies have started to accept the challenge of more sustainability of their logistics centers. For producers of intra-logistics equipment energy efficiency becomes a competitive advantage. Hence they focus on developing energy efficient products. Linde and STILL are two companies which have developed forklifts driven by H2 fuel cells. These H2 forklifts are for example tested by the BASF coatings AG in Muenster [1]. The advantages of H2 used for forklifts are the following: Compared with electric motors the charging time is reduced from several hours to a few minutes. The mileage is improved and the operating costs are reduced due to the long lifetime of the fuel cell system. Compared to diesel combustion engines no emissions occur and the forklifts can also be used inside of logistics or production halls. However, the use of H2 powered forklifts is only ecological when the H2 is produced with renewable energies. In the case of the BASF coatings AG Linde produces the H2 and supplies the H2 refueling station with transports on the road. The transport on the road is not ecological, too. Thus the only solution making sense is the H2 production with renewable energies at the facility itself. The Fronius International GmbH has installed such a system at its site in Sattledt, Austria. The system is called ―HyLOG‖ (Hydrogen Powered Logistics System). The electricity required for the production of H2 with an electrolyzer is generated by a 615 kWp photovoltaic power plant. The efficiency of the electrolyzer is approximately 50%. The H2 is then brought to the H2 refueling station which fills up exchangeable H2 tanks. Together with the energy cell the tanks are being installed at the tow truck (see figure 1). For the testing phase one Linde P30 tow truck was modified. Four other conventional tow trucks operate at the Sattledt site at the moment. With the installed electrolysis power of 1 Nm3 per hour 13 to 15 H2 tow trucks could be operated. Fronius has estimated that 30,646 kg CO2 could be saved per year compared to a conventional diesel forklift if the H2 is produced from solar energy [2]. Compared to a conventional electric forklift, powered with electricity from the public grid 16,021 kg CO2 could be saved per year. If the H2 is produced from natural gas still 21,166 kg CO2 (diesel forklift) and 6,541 kg CO2 (electric forklift) could be saved per year. As mentioned before, at the moment Fronius produces the hydrogen with solar energy. In the long-run this is not economical because the feed-in compensation will be omitted some day. Thus Fronius wants to produce the H2 with biogas. The process will be cheaper and has a higher efficiency (70-80%). According to Fronius the production of H2 with solar energy and

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an electrolyzer efficiency of 50% costs about 0.64 €/kWh. However, for being able to compete economically with conventional lead-acid battery solutions a price of less than 0.24 €/kWh is necessary. Fronius estimates prices of 0.04 €/kWh when the H2 is decentralized produced with natural gas or biogas.

Figure 1. The HyLOG project [2].

Besides the use of H2 as a fuel for forklifts or tow trucks, other technologies can be installed to reduce the dependence on fossil resources. Examples are combined heat and power plants operated with biomass or underground water basins. Combined with modern air conditioning technology these water basins allow to cool the buildings in summer and to heat them in winter [3]. Many companies have realized the need to reduce CO2 emissions and thus to reduce the energy consumption. One of the first firms which started to build energy efficient logistics centers is Gazeley. In 2009 its logistics center in Chatterley Valley, UK was finished. This center is considered as one of the world‘s first real sustainable logistics buildings. Actions were taken in the areas lighting, heating and cooling, the use of renewable energies and biomass [4]. In the intra-logistics sector about 15% of the energy is used for the buildings‘ lighting [5]. Consequently, Gazeley installed energy efficient lighting. The daylight can be controlled and dimmed. Motion detectors make sure that the light is only switched on when it is really necessary. The relatively new material Ethylene tetrafluoroethylene (ETFE) was used for daylight cushions covering 15% of the roof. At daytime the cushions inflate due to the solar radiation and deflate at night. Together with additional ribbons over the gates the need for artificial lighting is reduced. On the other hand the ETFE cushions reduce the light emissions at night. On the area of heating and cooling solar thermal panels were integrated into the south wall of the building. The panels save solar energy for the building‘s heating. An underfloor heating operated with biomass increases the building‘s efficiency. The advantage of such a heating system is that each building area can be heated separately. The disadvantage is the small flexibility when the warehouse is reorganized. The thermal energy loss is minimized by

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installing a strong isolation. The waste heat from biomass can be used for the heating of the offices, too. The energy production from renewable energies and biomass is an important factor for the energy efficient operation of this logistics center. Kinetic energy plates are an innovative technology. These plates are located in the entry area and generate energy while trucks or cars drive over. PV panels on the ETFE roof lights are installed to heat the water in the office rooms. The biomass micro power plant supplies the whole logistics center with electricity and heat. The biomass plant in Chatterley Valley produces more energy than needed for the operation of this logistics center. With this energy 3,100 local homes can be supplied with electricity and heat, too. Each mentioned action taken in Chatterley valley and the impacts on energy savings and CO2 reductions are shown in figure 2.

Figure 2. Energy savings of Chatterley Valley [4].

Additional actions for increasing the energy efficiency in the area of intra-logistics are for example the recuperation of storage and retrieval vehicles (SRVs), slow driving of SRVs, the use of efficient powertrains in conveyors, smart metering, or intelligent software for the control of SRVs. Viastore, for instance, found out that the energy consumption of a SRV is 14% lower when the engines work with half the speed [6]. With energy recuperation the movements of SRVs change. While one engine is recuperating the energy, another engine can use this energy at the same time. According to the ZVEI (central association for electrical and electronic industry) 40% savings potential could be realized with efficient powertrains (EFF2 and EFF1 engines) and other innovative powertrain concepts. This potential correlates to a payoff time of 12 to 18 months [7]. However, for all these technologies smart metering becomes more and more important. The company must monitor the energy consumption of each of its equipment to recognize the energy savings potentials.

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CONCLUSION AND OUTLOOK All of the presented technologies and possible actions are able to reduce the energy consumption and hence the CO2 emissions. However, none of the examples in this paper illustrates an integrated approach for a sustainable logistics center. Either the focus of the solution is put on the energy production and the building efficiency or on the intra-logistics technology. The reason is that the owner/ builder and the operator of a logistics real estate are often times not the same. In many cases the builder must decide what is best for the future operators. At the moment a lot of operators do not want to use energy efficient technologies such as a biomass micro power plant because they want to make a decision on their energy supply independently. On the other hand, the operator, usually a logistics service provider (LSP), decides on the used intra-logistics technology. Since sustainability more and more becomes a competitive advantage the clients of LSPs pay attention to the sustainability of the LSPs‘ operations and not only to costs. An integrated solution would combine many of the mentioned technologies. A logistics center such as Chatterley Valley with all the described features is the basis for a sustainable energy production. The operator should install innovative technologies such as H2 fueled forklifts and tow trucks. The H2 should be produced especially at off-peak times, hence when there is a low need for energy in the public grid. H2 is a good solution for the storage of (renewable) energy, too. The energy needed for the production of the H2 would then be generated by the biomass micro power plant located on the site of the logistics center. The requirement for an ecological operation of this biomass micro power plant is that the biomass is not transported over long distances. Ideally the biomass is grown on the site of the logistics center, too or next to it. A good example of energy crops is Miscanthus. It is a crop that needs little space, fertilizer and water, is perennial, fast growing, and delivers a high energy output. Because Miscanthus is a reed the processing into pallets or briquettes is necessary. To reduce the energy consumption of the logistics operations, such as storage and handling activities, the operator of the logistics center should install efficient powertrains in conveyors, use recuperation of SRVs and intelligent software for their control. However, a warehouse management system which uses strategies not exclusively based on time and process efficiency, but on energy efficiency as well, is still missing. For example such software could shift the order load over the day and thus reduce the peak load.

REFERENCES [1] [2]

[3]

Logistik fuer Unternehmen: Alternative Antriebstechnik im Praxistest. In: Logistik fuer Unternehmen, issue 1/2-2010, pp. 18-19. Springer-VDI-Verlag, Duesseldorf (2010). Wahlmüller, E.: HyLOG - Demonstration of a Fuel Cell Range Extender for Zero Emission Material Handling Application. Vienna (2008). http://www.a3ps. at/site/images/stories/a3ps_allgemein/A3PS_HEV_2008/session01/ 04_wahlmueller_hev_2008_10_21.pdf. Logistik fuer Unternehmen: Brennstoffzellen-Projekt demonstriert den CO2-freien Materialtransport. In: Logistik fuer Unternehmen, issue 3/4-2010, pp. 24-25. SpringerVDI-Verlag, Duesseldorf (2010).

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Hartmut Zadek and Robert Schulz Brexel, F.: Energieeffizienz und Nachhaltigkeit - Zukunft der modernen Logistikim mobilie. (2009). http://www.straubing-sand.de/fileserver/straubingsand/files/ 31.pdf. Kramm, M.: Der Energieausweis fuer Distributionszentren. In: Tagungsband Energieeffizienz im Lager , Cologne (2008). Müller, B.: Veraltete Lagertechnik kostet mehr Energie, als sich viele Firmen traeumen lassen. In: VDI nachrichten, issue 8/2010, p. 7. VDI-Verlag, Düsseldorf (2010). Scharf, A.: Klimaschutz erfordert Umdenken in der Antriebstechnik. In: VDI nachrichten, issue 45/2009, p. 9. VDI-Verlag, Düsseldorf (2009).

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 15

RELATION BETWEEN THE CELL-FREE DNA CONTENT AND THE LIPID PEROXIDATION IN THE BLOOD PLASMA OF MICE UNDER DAMAGING ACTION Lyudmila N. Shishkina *, Mikhail A. Klimovich, Mikhail V. Kozlov and Margarita A. Smotryaeva Department of the chemical and biological process kinetics, Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russia

ABSTRACT The detail analysis of interrelation between the cell-free DNA content and the LPO intensity in blood plasma of intact different species mice and under damaging factors (low-toxicity chemical agent, industrial toxicant and ionizing radiation at low doses) were done. It is shown that both the LPO intensity and cell-free DNA content have the high sensitivity to the action of chemical factors and ionizing radiation at the low doses. However, the scale of its changes is substantial dependence on the factor origin and also its concentrations and the dose rate radiation.

Keywords: mice, blood plasma, cell-free DNA, lipid peroxidation, X-rays, chemical agent, black liquor, low doses

1. INTRODUCTION As known, the most acute response to various damaging factors is produced by the hematopoietic system, which participates in maintaining the homeostasis on the level of *

Department of the chemical and biological process kinetics, Emanuel Institute of Biochemical Physics of Russian Academy of Sciences,119334 Moscow, Russia, Kosigin st.,4. E-mail: [email protected].

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organism. Changes in the hematopoietic system under the action of the chemical factors and ionizing radiation are of prime importance in forming both early and long-term biological consequences after the damaging actions. Among the most sensitive parameters to the radiation action especially at the low doses under the low dose rate there are parameters of the physicochemical regulatory system of the lipid peroxidation (LPO) which is maintained the steady-state of the LPO in the tissues of the intact laboratory animals [1]. At present the cellfree DNA content is widely suggested as the marker in the case of many diseases including pathologies in pregnancy [2-8]. A function and origin of the low-molecular cell-free DNA are extremely studied under different diseases and conditions, but a fact is turned one‘s attention that there are simultaneously changes of the LPO regulatory system parameters. Since the LPO initiators (hydroxyl radical, superoxide anion radical, peroxides) cause damages to not only lipids but also DNA bases [9], one might expect that there is a relationship between damages to the genetic structures and/or DNA biosynthesis and the LPO intensity. This assumption is in accordance with the data about the decreased oxidative DNA damage in human white blood cells after the antioxidant supplementation in diet [10, 11]. Moreover, in our investigations there were the reverse correlation between the rate constant for alkaline elution of the peripheral blood lymphocyte DNA and the oxidation products in blood plasma for the reference animals and irradiated mice at the dose of 15 cGy under the different dose rate and the direct correlation between the cell-free DNA content and the oxidation products in blood plasma of mice SHK whose drinking water was daily supplement with the industrial chemical toxicant at various concentrations [12]. However the linear regression coefficients of these correlations were distinguishable between the reference and experimental groups of mice. The aim of this work is the detail analysis of interrelation between the cell-free DNA content and the LPO intensity in blood plasma of intact mice and under damaging factors.

2. MATERIALS AND METHODS Experiments on Animals The X-irradiation at low doses and also the low-toxicity chemical agents and the industrial toxicant were used as damaging factors. The 130 white outbreed mice, 88 mice SHK and 45 Balb/c mice made a choice as the experimental animals because they characterize the various antioxidant status of tissues and different sensitivity to ionizing radiation [13, 14]. To modify the antioxidant status experiments were performed at the different seasons. Animals during experiments were in the special cages per 10 mice, a food and fresh drinking water mice received daily. As the low-toxicity chemical agents we used polyoxyethylensorbitanmonooleate (Tween 80) which is usually applied for the hydrophobic agent administration and the relative lowtoxicity chemical solvent acetone. The experiments were carried out on Balb/c mice (males, weight 16 – 20 g before the experiments, n = 13) during October and November. The mice were divided into two groups. The first group (8 mice) was used as the intact biological control. The Tween 80 (Ferak Berlin, FRG) at the dose of 30 mg/kg as 0.3% solution in 10% mixture of acetone in water was administration intraperitoneally at the second group of mice

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(5 mice). The solution of 0.3% Tween 80 in the 10% aqueous solution of acetone (special purity grade) was prepared immediately before the experiment. As the industrial toxicant we used the black liquor (a residue in cellulose pulping process), which were daily added in drinking water. The experiments were carried out on 60 mice SHK (females) during January and February. Mice were divided into 4 groups per 15 mice. Mice in experimental groups were received a drinking water which the black liquor was supplement at concentrations of the 0.005 (group I); 0.015 (group II) and 0.05% (group III), correspondingly. Mice of group IV were the intact age biological control and received a common drinking water without supplement. The single X-irradiation of white outbreed mice (females) at the doses less than 0.1 mGy under the decreased dose rate during exposure was performed by a Microwave electricElectron Cyclotron Resonance Accelerator which was developed and in detail described in Ref. [15, 16]. The absence of the microwave component during exposure was specially controlled. The experiments were carried out on 130 white outbreed mice (females) during November and December (experience 1) and September and October (experience 2). Mice per 10 in group were irradiated in special container where the each mouse was singly and could easily drive. The exposure time was about 18 minutes. Decapitation of mice was performed within 1 week after X-rays (30 mice, experience 1) and 1 month after damaging actions all another experiments. Decapitation of mice from the same animal group which served as the intact age biological control was simultaneously performed with the experimental groups. After decapitation of mice, blood was collected in test tubes treated by 5% solution of sodium citrate. The erythrocytes from the blood plasma were separated by centrifugation. All parameters of intact mice were determined for 1 or 2 animals. Mice in experimental groups were divided per 1 – 3 animals.

Biochemical Analyses The content of LPO products, which have reacted with 2-thiobarbituric acid (TBAreactive substances, TBA-RS) was determined spectrophometrically at the wavelength 532 nm with Beckman Du-50 (Austria) or KFK-3 (Russia) instruments by the method described in [17] with adding 10 l of the 0.01% 4-methyl-2,6–ditert.butylphenol (BHT) solution in ethanol. Protein was determined according to [18]. The content of cell-free DNA concentration in blood plasma was determined by fluorescence technique [19, 20] with the help of a Hitachi M 850 (Japan) spectrofluorimeter. Blood plasma (1 ml) was mixed with 1 ml of 4‘,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (0.4 μg/ml). The mixture was incubated for 30 min at 37 C since the whole binding of DNA and DAPI was within 15 – 30 min according to [21]. The fluorescence measurements were carried out on a spectrofluorimeter in a 0.02 M solution of disodium EDTA, pH 10. The wavelength of the exiting UV-radiation was 338 nm, the fluorescence spectra were measured at a wavelength of 457 nm, slit was 5 nm, the scanning rate was 60 nm/min. The amount of DNA (μg/ml) in the sample was determined by calibration curve, which was done by the native DNA of murine liver. The data were processed by a commonly used variational statistic method and by the KINS program given in [22]. The variability of indices was evaluated as ratio of mean square

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error of average mean to average mean for group expressed as a percentage. The significance of differences were evaluated by Student t- criterion.

3. RESULTS AND DISCUSSION As shown, the LPO is the common physiological process which is need for the functioning all types of biological membranes, cells and tissues [23, 24]. Earlier it was obtained that there are the substantial season differences both in the antioxidant activity of the spleen and liver lipids of mice SHK and Balb/c [13, 14] and the liver lipid composition and also the LPO products in the lipids and liver homogenate of mice Balb/c [25]. It is well known that LPO intensity is evaluated by the TBA-reactive substances content in a complex biological system [26]. Indeed, the existence of the reverse correlation between the TBAreactive substances content in the liver homogenate and the lipid antioxidant activity of mice SHK liver which is presented in [27] is a accordance with this conclusion. As the first step of our investigation, a possibility of the season changes of the TBA-RS content and cell-free DNA amount in the blood plasma of mice was studied. The data obtained are presented in Figure 1 for mice Balb/c and in Table 1 for the white outbreed mice. It is seen that studied parameters are for certain differed in dependence on season in which experiments were done. Table 1. The TBA-reactive substances amount and DNA content in the blood plasma of the white outbreed mice (females) at the different season Parameter [TBA-RS], nmol/mg of protein [DNA], μg/mg of protein

September

October (beginning)

October (the end of month)

November

December

0.076 ± 0.007 (n* = 4)

0.0765±0.004 (n = 2)

0.172± 0.005x (n = 2)

0.148±0.014x (n = 6)

0.082 ± 0.013 (n = 10)

0.037± 0.003 (n = 10)

0.0415 ± 0.0075 (n = 4)

0.060±0.009xxx (n = 6)

0.0615±0.008xx (n = 10)

* - Number of replicate measurements. Significant differences from the September data ( x p < 0.01; xx p < 0.02; xxx p < 0.03).

Figure 1. The TBA-reactive substances content (a) and the cell-free DNA amount (b) in the blood plasma of mice Balb/c (males) at the different season. Significant differences from the control group: * - p < 0.1, ** - p < 0.01.

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Thus, the TBA-reactive substances content in the blood plasma of mice Balb/c which are characterized a more low antioxidant status of tissues than the white outbreed mice [13, 14] may differ from 10.9 to 18.3 times in autumn and spring as compared with winter season. Besides the DNA amount in the blood plasma of these groups of mice is also substantially higher under performing experiments in winter. The most low levels both the LPO intensity and cell-free DNA content in the blood plasma of white outbreed mice reveal in September (Table 1). Earlier the high variability of the TBA-RS content was also found in the blood plasma of mice SHK (males) [28]. It is interesting to compare the average values of the cellfree DNA content and TBA-reactive substances amount in the blood plasma for groups of mice. This comparison is presented in Figure 2 for mice Balb/c (males). It is seen that this interrelation has the complex character. Moreover, the scale of interrelation between the cellfree DNA content and TBA-reactive substances amount in the blood plasma of the different species of mice is also unequal and substantially depends not only on the LPO intensity in the murine blood plasma. That assumption allow us to advance data which are presented in Figure 3. As could be seen, the increase of the LPO intensity in blood plasma doesn‘t always lead to the rise of the interconnection between the cell-free DNA and TBA-reactive substances amounts. Since the damaging factors cause disturbances in the LPO regulatory system functioning [1, 13, 14, 27] it might be expected that there are revealed changes both the level of the cellfree DNA content and the LPO intensity and the scale and direction of the relation between these indices in the blood plasma of mice after damaging actions. The obtained data which are presented in Table 2 and also in Figure 4 are confirmed this assumption.

Figure 2. The interrelation between the average values of the cell-free DNA content and TBA-reactive substances amount in the blood plasma for groups of intact mice Balb/c.

As seen, the administration of the low-toxicity chemical agents results to the reliable diminution of the cell-free DNA content, however the LPO intensity reveal the tendency to the rise (Table 2). The presence of the black liquor in the drinking water of mice causes the same dynamic changes of both studied parameters, but the effect depends on the toxicant concentration. The most effect is obtained by the black liquor concentration of 0.005 % (Table 2). There are only the tendency to the increase of both investigated indices after the Xray irradiation of mice at the low dose because of their high heterogeneity in the blood plasma of the experimental group of mice (Table 2). It is need to note that high heterogeneity of the system response is one of the distinctive peculiarities under a weak actions [12, 29].

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Moreover, the response of a complex biological system to weak influence can be substantial dependence on the initial values of its parameters. Data which are presented in Figure 4 are a accordance with this conclusion. Besides, the scale of the TBA-reactive substances content is dependent not only on its initial value in the blood plasma of mice in the control group, but the dose rate change during exposure (Figure 4 a and b).

Figure 3. Dependence of the correlation coefficient for the interrelation between the cell-free DNA content and LPO product amount on the LPO intensity in the blood plasma of the white outbreed mice (females) (1) and mice SHK (females) (2).

Figure 4. Influence of the X-rays dose on the TBA-reactive substances content and cell-free DNA amount under the irradiation of white outbreed mice in November (a) and September (b). Significant differences from the control group: * - p < 0.01, ** - p < 0.05.

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Table 2. The TBA-reactive substances amount and DNA content in the blood plasma of mice within 1 month after the damaging action Species of mice

Variant of experiment

Mice Balb/c (males)

The intact age control (n* = 4) Administration of 0.3% Tween 80 in 10% water acetone (n = 2)

Mice SHK (females)

The intact age control The black liquor in drinking water at the concentration of 0.005% 0.015 % 0.05 %

Mice SHK (females) [27]

The intact age control (n = 6) X-rays at the dose of 16 (n = 3)

[TBA-RS], nmol/mg of protein 0.021  0.006

[DNA], g/mg of protein 0.078  0.016

0.046  0.012 0.092  0.007 (n = 5)

0.0325  0.0055xx 0.102  0.008 (n = 9)

0.146  0.029 (n = 5) 0.143  0.029 (n = 5) 0.075  0.007 (n = 5) 0.1915  0.028 0.42  0.20

0.144 0.012 (n = 3)x 0.116  0.016 (n = 5) 0.096  0.025 (n = 5) 0.072  0.008 0.117  0.043

* Number of replicate measurements. x – Significant differences from the control mice (p < 0.02). Significant differences from the control mice (p < 0.06).

xx

-

CONCLUSION In this paper the season changes both the LPO product content and cell-free DNA amount in the blood plasma of the different species of mice are revealed.

Figure 5. Effect of damaging factors on values of the linear regression coefficients of the direct correlations between the cell-free DNA content and TBA-reactive substances amount in the blood plasma of mice with 1 month after action. a)– presence of the black liquor in the drinking water. b)– Xrays irradiation at the low doses under changing the dose rate. c) – X-rays irradiation at the dose of 16 cGy.

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It is found that both the LPO intensity and cell-free DNA content have the high sensitivity to the action of chemical factors and ionizing radiation at the low doses. However, the scale of its changes is substantial dependence on the factor origin and also on its concentrations and the radiation intensity. This can be seen in Figure 5 where are presented the effect of the different damaging factors on values of the linear regression coefficients for the direct correlations between the cell-free DNA content and TBA-reactive substances amount in the blood plasma of mice. Obviously, the reliable season changes of the cell-free DNA content and the LPO intensity in the murine blood plasma, its high and unequal sensitivity to the damaging factors and substantial dependence on the initial values in the control mice and also the intensity and/or concentration of factor would be expected to be also a reason for the existence of several hypotheses about the cell-free DNA origin.

ACKNOWLEDGMENTS This work was supported by the International Science and Technology Center Program, Projects No 547-98 and 1032, and also The Program of Fundamental Research of Presidium of the Russian Academy of Sciences ―Fundamental sciences – for Medicine‖ (2006 – 2008).

REFERENCES [1]

[2]

[3]

[4]

[5]

[6]

[7] [8]

L.N. Shishkina, E.V. Kushnireva, M.A. Smotryaeva, ―A new Approach to the Assessment of Biological Consequences of Exposure to Low-dose Radiation‖, Radiation Biology. Radioecology, vol. 44, no. 3, pp. 289-295, 2004 (in Russian). R. Gonzales, J.M. Siva, G. Domingues, et al., ―Microsatellite alternations and TP53 Mutations in Plasma DNA of Small Cell Lung Cancer Patients: Follow-up study and Prognostic Significance‖, Annual Oncology, vol. 11, pp. 1097-1104, 2000. V.G. Vladimirov, I.N. Vasil‘eva, L.A. Sharova, ―Extracellular DNA and Its Sign ificance for Current Medicine‖, Clinical Medicine and Physiopathology, no. 1-2, pp. 110-119, 1998 (in Russia). B. Burwinkel, M.W. Kilimann, ―Unequal Homologous Recombination Between Line-1 Elements as a Mutational Mechanism in Human Genetic Disease‖, Journal of Molecular Biology, vol. 277, pp. 513-517, 1998. Y.M.D. Lo, M.S. Tein, T.K. Lau, et al., ―Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis‖, American Journal of Human Genetics‖, vol. 62, pp. 768-775, 1998. L.Y. Chan, T.N. Leung, K.C. Chang, et al., ―Serial analysis of fetal DNA concentr ations in maternal plasma in late pregnancy‖, Clinical Chemistry, vol. 49, pp. 678 – 680, 2003. W.D. Bianchi, ―Circulating fetal DNA: its origin and diagnostic potential – a review‖, Placenta , vol. 18, pp. 93-101, 2004. S. Grill, C. Rusterholz, R. Zanetti-Dallenbach, et al., ―Potential markers of preeclampsia – a review‖, Reproductive Biology and Endocrinology, 7:70doi:1 0.1186/1477-7827-7-70, 2009.

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[10]

[11]

[12]

[13] [14]

[15]

[16] [17] [18] [19] [20]

[21]

[22] [23]

[24]

[25]

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A.R. Proteggente, T.G. England, C.A. Rise-Evans, B. Halliwell, ―Iron Supplementation and Oxidative Damage to DNA in Healthy Individuals with High Plasma Ascorbate‖, Biochemical and Biophysical Research Communications, vol. 288. pp. 245-251, 2002. M. Porrini, P. Riso, ―Lymphocyte Lycopene Concentration and DNA Protection from Oxidative Damage is Increased in Women after a Shot Period of Tomato Consumption‖. Journal of Nutrition, vol. 130, pp. 189-192, 2000. P. Moller, S. Loft, ―Oxidative DNA damage in human white blood cells in dietary antioxidants intervention studies‖, American Journal of Clinical Nutrition , vol. 76, pp. 303-310, 2002 L.N. Shishkina, M.A. Smotryaeva. ―Relationship of Membrane and DNA Damage with Lipid Peroxidation under Weak Influence‖, Biophysics, vol. 45, no. 5, pp. 818 – 826, 2000. E.B. Burlalova, A.V. Alesenko, E.M. Molochkina, et al., Bioantioxidants in Radiation Damage and Tumor Growth. Moscow, Nauka, 1975 (in Russian). L.N. Shishkina, E.B. Burlakova, ―The value of antioxidant properties of lipids in radiation damage and membrane repair‖, InŚ Chemical and Biological Kinetics. New horizons / Eds. E.B. Burlakova, S.D. Varfolomeev, Leden, Boston: VPS, pp. 334 – 364, 2005. K.F. Sergeichev, D.M. Karfidov, N.A. Lukina, ―Electron Cyclotron Resonance Acceleration of Electrons to Relativistic Energies by a Microwave Field in a Mirror Trap‖, Plasma Physics Reports, vol. 33, no. 6, pp. 455-473, 2007. D.M. Karfidov, K.F. Sergeichev, ―Characteristics of X-rays from ECR-discharge in a Mirror Trap‖, Applied Physics, no. 6, pp. 102-105, 2007 (in Russian). T. Asakawa, S. Matsushita, ―Coloring Conditions of Thiobarbituric Acid Test for Determination Lipid Hydroperoxides‖, Lipids, vol. 15, no. 3, pp. 137–140, 1980. R. Itzhaki, D.M. Gill, ―A micro-biuretic method for estimating proteins‖, Analytical Biochemistry, vol. 9, no. pp. 401 – 410, 1964. J. Kapuscinski, B. Skoczylas, ―Simple and rapid fluorimetric method for DNA microassay‖, Analytical Biochemistry, vol. 83, pp. 252-257, 1977. J. Kapuscinski, B. Skoczylas, ―Fluorescent complexes of DNA with 4‘,6-diamidino-2phenyl indole.2HCl or DCl 4‘,6-dicarboxyamide-2-phenyl indole‖, Nucleic Acids Research, vol. 5, no. 10, pp. 3775-3799, 1978. N.V. Blukhterova, M.A. Smotryaeva, K.E. Kruglyakova, ―Comparative Study of the Effect of 1-Methyl-1-Nitrosourea and 1,3-Dimethyl-1-Nitrosourea on DNA of Tumor Cells in vitro and in vivo by the Method of Alkali Elution‖, Biological Bulletin, no. 3, pp. 276-281, 1996 (in Russian). E.F. Brin, S.O. Travin., ―Modeling of chemical reaction mechanism‖, Chemical Physics Reports, vol. 10, no. 6, pp. 830-837, 1991 (in Russian). E.B. Burlakova, N.G. Khrapova, ―Membrane lipid peroxidation and the natural antioxidants‖, Achievements of Chemistry, vol. 54, no. 9, pp. 1540-1658, 1985 (in Russian). K. Hensley, K.A. Robinson, P. Gabbina, et al., ―Reactive Oxygen Species, Cell Signaling and Cell Injury‖, Free Radical Biology and Medicine, vol. 28,no. 10, pp. 1456-1462, 2000. M.V. Kozlov, V.V. Urnysheva, L.N. Shishkina, ―Interconnection of Parameters of Regulation System of Lipid Peroxidation and Morphophysiological Parameters of

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Lyudmila N. Shishkina, Mikhail A. Klimovich, Mikhail V. Kozlov et al. Mouse Liver‖, Journal of Evolutionary and Biochemical Physiology, vol. 44, no. 4, pp. 470-475, 2008. V.E. Kagan, O.N. Orlov, L.I. Prilipko, ―Problems of Analysis of Lipid Peroxidatoion Products‖. InŚ Itogi nauki I tekhniki. Moscow: VINITI Akad. Nauk SSSR. Ser. Biofizika, 1986 (in Russian). L.N. Shishkina, Ye.V. Kushnireva, M.A. Smotryaeva, ―The combined effect of surfactant and acute irradiation at low dose on the lipid peroxidation process in tissues and DNA content in blood plasma of mice‖, Oxidation Communications, vol. 24, no. 2, pp. 276– 286, 2001. L.N. Shishkina, Ye.V. Kushnireva, O.F. Bespal‘ko, N.V. Polyakova, ŚThe Role of Antioxidative Status in Development of the Consequences of Biological Action Under Low Doses and Low Intensity Irradiation‖, Radiation Biology. Radioecology, vol. 40, no. 2. pp. 162-7 (in Russian). E.B. Burlakova, ―Effect of ultra low radiation doses‖, Herald of Russian Academy of Sciences, vol. 64, pp. 425-431, 1994.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 16

MECHANISM OF STABLE RADICAL GENERATION IN LIGNIN UNDER THE ACTION OF NITROGEN DIOXIDE E.Ya.Davydov*, I.S. Gaponova, S.M. Lomakin, G.B. Pariiskii, T.V. Pokholok and G.E. Zaikov NM Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russia

ABSTRACT Lignin is the very sensitive to the nitrogen dioxide action as evidenced by reasonably high concentrations of stable radicals which can be accumulated in exposed samples (~2 x 1019 spins/g). The generation of nitrogen containing radicals is connected with oxidative electron-transfer reactions initiated by dimers of NO2 in phenol units and nonphenolic structures. The subsequent radical conversions include processes of degradation and modification (nitrosation and nitration) of a lignin. The formation of stable iminoxyl radicals in the presence of NO2 is the typical process for lignin.

Keywords: nitrogen dioxide, lignin, stable radicals, kinetics, mechanism, application

INTRODUCTION N.M. Emanuel Institute of Biochemical Physics (Russian Academy of Sciences) has several perfect installation of electron spin resonance (ESR). ESR-method is very effective and very sensitive for investigation of radical reaction in chemistry and biochemistry. This method was created in 1944 [1] by Prof. K.M. Zavoiskii (Academy of Sciences of USSR). V.V. Voevodskii, L.A. Blumenfeld, Ya.S. Lebedev, E.L. Frankevich, A.L. Buchavhenko,

*

NM Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, 4 Kosygin str., 119334 Moscow, Russia. [email protected].

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E.G. Rozantsev, A.M. Wasserman and A.L. Kovarskii did very much for application of this method for investigation of chemical and biochemical reactions [2-8].

AIM AND SCOPE The contributors of this paper published in 2009 the book [4] about interaction of polymers with polluted atmosphere (nitrogen oxides). Our new paper is devoted for the problems of interaction between nitrogen dioxide and lignin by ESR. Mankind accumulated tremendous amount of lignin and did not find real ways for applications of it. So, any kind of research about lignin (applications) is very important for looking for the fields of utilization.

METHODS OF EXPERIMENTS All information about application of ESR as well as spectroscopy for investigation of reaction between nitrogen dioxide were published recently [3].

EXPERIMENTAL RESULTS AND DISCUSSION Detection of specific stable radicals by ESR in plants can be considered as a sensitive method of air pollution monitoring. The stable radicals are associated with products of free radical reactions initiated by various air pollutants, in particular nitrogen oxides.

The nitrogen dioxide pre-treatment of wood pulp before oxygen bleaching leads to appreciably decreased lignin contents after a given duration of the oxygen bleaching (Figure 1). The impact of NO2 on pines growing in cities (Vilnius, Kaunas) has been revealed. The reduction in NO2 concentration in the atmosphere (1990 – 2006) determines an increase in pine radial increment. It is quite possible that this result is conditioned by interaction of NO2 with reactive groups of lignin (Figure 2).

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Figure 1. Dependence of kappa number against time for different conditions (2% NO2 and 4% NO2).

Figure 2. Dependence of ratio of radical increment on the year in Kaunas and Vilnius cities.

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Lignin is evidently reactive to nitrogen dioxide and capable of destroying in NO2 atmosphere. The purpose of the present research was to examine the mechanism of primary radical reactions determining subsequent conversions of lignin units (Figure 3).

Figure 3. Dependence of phenoxyl radical concentration on time in nitrogen dioxide atmosphere.

Decay of phenoxyl radicals is accompanied by appearance of iminoxyl radicals (Figure 4).

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Figure 4. Accumulation of aminoxyl radicals in time.

Change in the iminoxyl radical concentration in the course of thermolysis of lignin exposed to NO2 at room temperature. Figure 5 gives information about concentration of iminoxyl radicals in different temperatures.

Figure 5. Concentration of iminoxyl radicals for different temperatures.

Thermolysis shifts the equilibrium decay of iminoxyl radicals by recombination with NO2 to stable radicals.

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Rates of iminoxyl radical accumulation and phenoxyl radical decay are approximately equal at various NO2 concentrations (Figure 6).

Figure 6. Dependence iminoxyl radical accumulation and phenoxyl radical decay in time.

Mechanism of nitrosation of monomethoxyphenol groups of lignin with the formation of iminoxyl radicals. Initiators are dimers of NO2 in the form of nitrosyl nitrate.

In parallel with conversions of phenols, nitrosyl nitrate is capable of oxidising multitude of hydroxyl groups of nonphenolic structures linking aryl rings in lignin. As a result, aldehydes are formed.

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Changes of IR spectra on exposure of lignin to NO2 confirm the presented mechanism (Figure 7).

Figure 7. Infrared spectra of lignin before and after interaction with nitrogen oxides.

Aldehyde groups should be accumulated in the course of the lignin exposure to NO2. The band of hydroxyl groups in lignin (3300 cm1) appreciably reduces for two days of NO2 exposure. In addition, the decrease in intensity is observed for bands corresponding to stretching vibrations of C=C bonds of phenyl rings (1512 and 1450 cm1). Moreover, the appearance of new intense bands at 1558 and 1337 cm1 belonging to asymmetric and symmetric stretching vibrations of N=O bonds in nitro groups takes place. Aldehydes are precursors of acylaminoxyl radicals observed after prolonged exposure of lignin to NO2.

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Dependencies of the iminoxyl radical yield in lignin on NO2 concentrations is presented on the figures 8 and 9.

Figure 8. Dependence of accumulation rate of iminoxyl radicals on concentration of nitrogen dioxide.

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Figure 9. Dependence of the iminoxyl radical yield in lignin on concentration of nitrogen dioxide.

If to plot concentrations of Im  on steady-state levels ([Im ]st) on NO2 concentrations, the dependence reminding in form the Langmuir isotherm can be obtained.T the adsorption of nitrogen dioxide on the lignin surface is the rate-determining factor for the iminoxyl radical generation.

CONCLUSION 1. Lignin is the very sensitive to the nitrogen dioxide action as evidenced by reasonably high concentrations of stable radicals which can be accumulated in exposed samples (~2 x 1019 spins/g). 2. The generation of nitrogen containing radicals is connected with oxidative electrontransfer reactions initiated by dimers of NO2 in phenol units and nonphenolic structures. The subsequent radical conversions include processes of degradation and modification (nitrosation and nitration) of a lignin. 3. The formation of stable iminoxyl radicals in the presence of NO2 is the typical process for lignin.

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REFERENCES [1] [2]

[3]

[4]

[5] [6]

[7]

[8]

N.M. Emanuel, G.E. Zaikov, V.A. Kritsman ―Chemical kinetics and chain reactions. Historical aspects‖, Nova Science Publishers, New York, 1992, 625 pp. ―Chemical kinetics‖, Ed. by E.B. Burlakova, A.E. Shilov, S.D. Varfolomeev, G.E. Zaikov, VSP International Science Publishers, Leiden-Utrecht (The Netherlands), 2005, 682 pp. ―Biological kinetics‖, Ed. by E.B. Burlakova, A.E. Shilov, S.D. Varfolomeev, G.E. Zaikov, VSP International Science Publishers, Leiden-Utrecht (The Netherlands), 2005, 356 pp. G.E. Zaikov, E.Ya. Davydov, G.B. Pariiskii, I.S. Gaponova, T.V. Pokholok ―Interaction of polymers with polluted atmospheres. Nitrogen oxides‖, Smithers, Shawbury, Shrewsbury, Shropshire, UK, 2009, 270 pp. G.E. Zaikov ―Chemical and biochemical physics. New frontiers‖, Nova Science Publishers, New York, 2006, 272 pp. ―Resent advances in polymer nanocomposites: synthesis and classification‖, Ed. by S. Thomas, G.E. Zaikov, S.V. Valsaraj, A.P. Meera, Brill Academic Publishers, LeidenBoston (The Netherlands - USA), 2010, 436 pp. ―Resent advances in polymer nanocomposites‖, Ed. by Thomas, G.E. Zaikov, S.V. Valsaraj, VSP International Science Publishers, Leiden- Boston (The Netherlands USA), 2009, 528 pp. G.E. Zaikov, S.K. Rakovsky ―Ozonation of organic and polymeric compounds‖, Smithers, Shawbury, Shrewsbury, Shropshire, UK, 2009, 414 pp.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 17

SPRAY FORMATION OF ALTERNATIVE DIESEL FUELS UNDER ENGINE-LIKE CONDITIONS Dennis Backofen1, Michael Könnig 2, Helmut Tschöke 1 and Jürgen Schmidt2 1

Institute of Mobile Systems, Otto-von-Guericke University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany 2 Institute of Fluid Dynamics and Thermodynamics, Otto-von-Guericke University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany

ABSTRACT The need to reduce CO2 emissions of modern diesel powertrains is going to intensify in the near future, because of the current European legislation and the rising shortage of fossil energy sources. The using of alternative fuels, extracted from biomass, has a high potential to reduce CO2 emissions from their production to their combustion in the engine. Engine modifications, like the improving of the mixture formation and especially the increasing of the injection pressure can increase the efficiency and reduce the CO 2 emissions. The employment of alternative fuels with simultaneous optimizing the mixture formation presents an important mean to reduce the emissions of modern diesel engines. This paper presents results of the spray behaviour, which were measured in a pressure chamber with optical measuring devices at conditions like in the engine. The macroscopic structure of the spray (sprayangle, penetration) will be investigated at different injection pressures and for different alternative fuels. By variation of the chamber pressure and temperature, the fuel and the injection parameters like injection pressure and injection duration, tendencies about the behaviour of the mixture formation with alternative fuels under engine-like conditions can be derived. The paper presents the main results of the investigations and concludes with an outlook to ongoing research activities.

Keywords: Ultra-high pressure injection, diesel engine, CO 2 reduction, alternative fuels, optical measuring devices, spray formation

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1. INTRODUCTION, AIM AND BACKGROUND Development targets of modern engines for passenger cars are the reducing of the fuel consumption and the emissions. The reducing of emissions, like harmful carbon monoxides, unburned hydrocarbons as well as nitrogen oxides was objected during the last decades. Especially nitrogen oxides and carbon particulate matter are emissions which have to be reduced in the future because of the tighter limits of the European exhaust emission standards like EURO 6. In the last years the reducing of the fuel consumption of modern powertrains becomes more important because of the worldwide climate discussion and the increasing demand of fossil raw materials, especially of emerging markets like India and China. A promising approach to reduce the emissions and the specific fuel consumption (CO2emissions) of the Diesel engines simultaneously is the optimization of the mixture formation, especially by increasing the injection pressure. With the higher injection pressure, the spray velocity increases and influences the mixture formation: the increasing spray velocity generates smaller droplets, which leads to an optimization of the fuel atomization and an improved mixing of the fuel and the air in the combustion chamber. The optimization of the atomization and the increasing injected fuel mass lead to a higher cylinder pressure rate at the beginning of the combustion, which result in an increasing indicated mean effective pressure and a lower specific fuel consumption [4]. With the higher injection pressure and spray impulse, the Flame Lift Off (FLO), which describes the distance between the nozzle and the solid flame front near the nozzle, is increasing [5, 6]. The local fuel-air-ratio rises up with an increasing FLO, which leads to worse conditions inside the flame of the particulate matter formation and a faster process of the particulate matter oxidation along the external areas of the flame. Because of the faster oxidation, the particulate matter emissions decreases with the higher injection pressure. The maximum temperatures increase with a high injection pressure during the combustion, because of the accelerated energetic conversion of the fuel. This leads to an intensified formation of nitrogen oxides. This effect can be reduced very successfully by using an increased emission gas recirculation rate (EGR rate). The combination of a high injection pressure and a high EGR rate can help to reduce simultaneously the specific fuel consumption, respectively the CO2 emission and the nitrogen oxides and particulate matter emissions. The supply industry of injection systems works intensively on the increasing of the injection pressure of common rail systems. While actual injection systems for passenger cars are offered with an injection pressure up to pInjection = 2000 bar, the supply industry of injection systems for commercial vehicles offered systems with an injection pressure up to pInjection = 2500 bar. They are planning injection systems with pInjection = 3000 bar in the near future. In addition to the optimization of the mixture formation, more CO2 savings are realizable by using alternative fuels. For that, fuels made out of biomass, like bio fuels of the first and second generation are most suited. Particularly bio fuels of the second generation, called synthetic fuels, which are made out of the whole plant by using gasification and FischerTropsch synthetic processes, have the highest potential to reduce CO2 and present a high sustainability factor. Beside these biomass-to-liquid fuels (BTL), the use of blend fuels which

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consist of conventional diesel fuel and bio fuel of the first generation, like rapeseed methyl ester lead to lower particulate matter emissions at constant fuel consumption. The combination of optimization the mixture formation by increasing the injection pressure and the use of alternative diesel fuel presents an effective instrument to reduce the fuel consumption respectively the CO2-emissions, the particulate matter and nitrogen oxide emissions of diesel engines. Within the framework of the main research at the Otto-von-Guericke University called Automotive, an injection system was build up in the project ―Ultra high pressure injection of alternative diesel fuels‖, which can inject alternative diesel fuels with a high injection pressure up to pInjection = 3000 bar. At the beginning of this project the spray was investigated under ambient pressure and temperature conditions by using optical measurement devices [11]. After that the spray was investigated in a pressure chamber under engine-like conditions. In view of these results the injection system will be implemented at a single-cylinder engine to measure the influence on the combustion process of the extreme high pressure in combination with alternative fuels. Finally the potential to increase the power and to decrease the fuel consumption and the emissions of the high pressure injection of alternative fuels will be presented at a four-cylinder engine during the project. In this paper the development of a spray under engine-like conditions will be investigated in a pressure chamber. With the use of a high-speed camera the spray will be recorded by varying the injection pressure and by using rapeseed methyl ester in the fuel. The maximum pressure level of rapeseed methyl ester published in actual papers [7, 8, 9 and 10] is about pInjection = 1600 bar, like the standard pressure level of serial injection systems. In this paper the macroscopic structure of the spray of different shares of RME in the fuel will be investigated and analysed up to pInjection = 3000 bar. With that, questions about the influence of the pressure and the share of the rapeseed methyl ester in the fuel on the tip penetration, the spray angle, the maximum diameter of the spray and the spray volume have to be answered. Finally the influence on the engine operation can be described with these results.

2. EXPERIMENTAL SET-UP To generate a high pressure a special injection system was used, which injected the spray with an injection pressure up to pInjection = 3000 bar. In addition optical measurement devices and special software was used to capture the spray and to calculate characteristic values.

2.1. Generate the High Injection Pressure An injection system, which contains a CP3-common rail pump with a maximum pressure of pInjection = 2000 bar, a rail and a light-duty injector was used to generate the extreme high injection pressure. This experimental injector generates an injection pressure of pInjection = 3000 bar with an internal pressure intensifier. The used injection nozzle is a seven sac-hole nozzle with the following technical values:

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Dennis Backofen, Michael Könnig, Helmut Tschöke et al. Table 1. Specifications of the fuel injection nozzle Specification fuel injection nozzle Denotation sac-hole nozzle Number holes 1 (of 7) Diameter of the nozzle hole 0.151 mm (conical) Hydraulic discharge 425 – 444 cm³ / 30s at 100 bar Nozzle hole angle 158 °

For investigating only one spray jet and to minimize the wetting of the pressure chamber windows with fuel, six of seven nozzle holes were weld up by using laser welding at the Ottovon-Guericke University.

2.2. Pressure Chamber and Optical Measurement Devices Figure 1 shows the optical measurement setup and the integration of the injector in the pressure chamber. The injector was placed in that way, that the investigating jet was exactly parallel with the camera window. The window for PDA (Particle Dynamic Analysis) investigations, which is already turned for 45°, could be used to put the jet in the right direction. To get the largest view of the jet, the injector was placed in the window in that way, that the nozzle tip could be seen at the edge of the window. The light is transmitted in the pressure chamber of the upstream placed window. Halogen light with a power of 1000W was used as the light source. The chamber has a volume of about V = 0.013 m³ and is only for spray investigations. The pressure and the temperature can be increased up to pChamber = 60 and TChamber = 200 °C. In the next step of the project, the chamber will be operated with nitrogen gas, so that the temperature can be increased up to TChamber = 500 °C without igniting the air-fuel mixture. To capture the spray jet by varying the injection pressure, the fuel, the chamber pressure and chamber temperature, a high-speed video system Spraymaster (LaVision) was used. The camera ―Speed-Star-Camera 6‖ has a maximum resolution of 1024 x 1024 pixels with a sample rate of f = 5.4 kHz and a maximum frequency of f =150 kHz with the smallest resolution.

Figure 1. Pressure Chamber.

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The size of the pixel on the chip is 20 x 20 µm at a digital resolution of 12 Bit. For capturing the complete spray jet a resolution of 1024 x 192 pixels was used. With a frame rate of f = 20 kHz the complete dynamic spray development from the injector opening until the closing could capture with 60 frames. To synchronize the spray development the start pulse of the camera was used for triggering the injector.

2.3. Alternative Fuels In this paper an experimental series of the spray behaviour of different blended fuels, which contains different shares of conventional diesel fuel and rapeseed methyl ester, should be presented. Table 2 shows the important characteristic specifications of the used fuels. Table 2. Characteristic specification of the used fuels Characteristic Dimension Diesel fuel1 specifications Density (at 15°C) kg/m³ 834 Kinematic viscosity mm²/s 2.9 (at 40 °C) Surface tension (at 20 °C) N/m 0.028 (25 °C) Boiling point/range °C 176 °C – 364 °C Ester content % 0 1 = Shell Global Solutions GmbH, 2009. 2 = According to Werkszertifikat Bio-Ölwerk Magdeburg, 2009.

Rapeseed methyl ester (RME)2 883 3.50 – 5 not spec. 330 °C – 360 °C > 99,0

Before the investigations started the complete high pressure unit was rinsed for three times with the fuel. After each rinsing, the fuel was discharged and new fuel was filled in. After the last rinsing, the investigating fuel was filled in. Overall four different blended fuels were investigated. They are described in this paper as    

B0 (conventional diesel fuel) B10 (10% RME and 90% conventional diesel fuel) B50 (50% RME and 50% conventional diesel fuel) B100 (rapeseed methyl ester, RME).

3. ANALYTICAL METHOD The varying parameters beside the fuel and the injection pressure are the pressure and the temperature of the chamber. The injection pressure is specified for the pressure levels of pInjection = 1500 bar, 2000 bar, 2500 bar and 3000 bar. For a short time the injector can be used with an injection pressure of pInjection = 3500 bar. Because of safety reasons the pressure is increased only till pInjection = 3000 bar. For further investigations the pressure will be increased.

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Dennis Backofen, Michael Könnig, Helmut Tschöke et al. Table 3. Characteristic specification of the used fuels Setting Parameter Parameter Range Injection pressure pInjection [bar] 1500 bar, 2000 bar, 2500 bar, 3000 bar Chamber pressure pChamber [bar] 20 bar, 40 bar, 60 bar Chamber temperature TChamber [°C] 20 °C, 100 °C, 200 °C Fuel [-] B0, B10, B50, B100 Control duration [ms] - Constant fuel volume 12 mm³ 1.18 ms (1500 bar), 1.01 ms (2000 bar), 0.93 ms (2500 bar), 0.86 ms (3000 bar) - Constant control duration 1.5 ms

The optical parameters were defined with a frame rate of f = 20 kHz at 100 frames. Each experiment was repeated 9 times. To minimize influences on the light intensity of the spray, like different daylight, the images are neutralised with a background correction. For calculating characteristic values based on captured high-speed images the Software Davis 7.2 (LaVision) was used. With this software the penetration, the spray angle and the maximum diameter can be determined. To determine the spray volume and the surface a routine was programmed in MATLAB. Because of the increasing injected fuel mass by rising the injection pressure the control duration was adapt for each injection pressure. So the results of different injection pressures can be compared. Therefore different control durations were determined by measuring a constant injected fuel mass for different injection pressures, as seen in figure 2. The chosen injected fuel mass of 12 mm³ of the single hole nozzle corresponds to an injected fuel mass of an engine which operates under full load.

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For discussing the spray image, the penetration, the spray angle, the maximum diameter and the volume were selected for this paper:

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+ Volume

Penetration Figure 3. Spray values.

With these characteristic values conclusions about the spray behaviour at high injection pressures and different chamber pressures and –temperatures can be derived.

4. RESULTS This chapter presents the influences of an extreme high injection pressure of the spray behaviour of fuels with different shares of rape seed methyl ester fuel. In the chapter 4.1 the influence at constant chamber pressure and temperature will be discussed. The following chapters show the influences by varying the chamber pressure and the chamber temperature at a constant injection pressure.

4.1. Constant Chamber Pressure and Temperature Figure 4 and figure 5 shows the influence of the injection pressure and the different fuels on the spray values. To get a clear overview, only the lowest and the highest injection pressures of these investigations are shown in the following figures. The injection pressure of pInjection = 1500 bar are comparable with an actual injection system of a modern diesel engine. Because of the high chamber pressure compared to [11], the time between start of injection and break up is getting shorter. So a linear progress of the penetration after starting the injection is not been clearly shown in figure 4 for the penetration.

Figure 4. Penetration and spray angle of the fuels by varying the injection pressure.

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Like shown in many papers [1, 2, 3, 9, 11 and 12] also in this investigation the increasing of the penetration by increasing the injection pressure could be measured for each fuel. The reasons for that effect are the higher kinetic energy and the higher velocity of the spray at the exit of the nozzle hole by increasing the injection pressure. At the start of the injection phase there are higher gradients of the penetration with an increasing injection pressure. This leads to a strong acceleration of the spray front, because of the increasing kinetic energy by the use of high injection pressures. Regarding the engine operation the larger penetration leads to high hydro-carbon-emissions because of the long jet lengths which hit the cold cylinder wall and piston bowl. A nozzle will be optimized by CFD simulations during this project to prevent this effect. Compared with the investigations at ambient pressures [11] the differences between the fuels regarding the penetration are not detectable, which is shown in the left picture in figure 4. The spray angle is detected a little bit earlier with an increasing injection pressure because of the higher kinetic energy of the fuel, which leads to an earlier open of the needle. For each fuel that is injected with the high pressure the spray angle is narrower compared to the fuel, which is injected with the low pressure. So the spray is more focused with the high injection pressure than with the low pressure for all fuels, because of the higher kinetic energy at the orifices of the nozzle holes at pInjection = 3000 bar. Only at low injection pressure there are differences between the fuel with 100% RME compared to the fuels with lower shares of RME. B100 has lower spray angles at the end of the injection than the other fuels. At pInjection = 3000 bar the differences of the spray angles between the fuels are negligible. So it seems that a high injection pressure increases more the turbulence of the spray with pure RME than for the other fuels. lso the maximum diameter increases for a high injection pressure, as seen in figure 5.

Figure 5. Maximum diameter and spray volume of the fuels by varying the injection pressure.

The main reason is the increasing level of turbulence because of the higher kinetic energy. Compared to the progress of the penetration the development of the maximum diameter of the spray is going asymptotic at the end of the curve. Only for the fuel with pure RME there are differences between the maximum diameter: corresponding to the spray angle, B100 has lower maximum diameters after t = 2.8 ms than the other fuels at low injection pressures. At high injection pressure, this difference is not

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detectable. So it also seems that the turbulence of a spray jet with pure RME-fuel becomes higher with an increasing injection pressure. Because of the high kinetic energy at pInjection = 3000 bar, the spray volumes of all fuels are increased heavily compared to the spray volume at low injection pressure, as seen in the right picture of figure 5. Remarkable in this figure is the low spray volume for B100 compared to the other fuels at low injection pressure and after t = 2.6 ms. Corresponding to the spray angle and the maximum diameter, this effect is decreasing with a high injection pressure, which is shown in figure 6 for different injection pressures.

12000 11000 Sprayvolume [mm³]

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Figure 6. Sprayvolume of B0 and B100 by varying the injection pressure.

Figure 6 also shows the lower spray volume of B100 compared to B0 for all injection pressures. A possible reason for the bad atomization of B100 compared to B0 can be the higher diameters of the droplets of RME, expressed as Sauter Mean Diameter (SMD). Because of a possible higher surface tension of RME, the ―internal forces‖ in a droplet are higher and it needs more energy to break them of [10]. Bigger droplets lead to a higher inertial mass and the droplets might go further. So the spray angle and the maximum diameter decreases and affects the general shape of the spray and leads to a smaller spray volume. The decreasing differences between the spray volumes of B0 and B100 with increasing injection pressures in figure 6 shows the potential of the extreme high injection pressure in combination with RME: the high injection pressure leads to an intensive atomization of RME compared to conventional diesel fuel. This effect is very important to get a high homogenization of the air-fuel mixture, which leads to lower particulate matter emissions and a low specific fuel consumption.

4.2. Variation of the Chamber Pressure Figure 7 shows the influence of the chamber pressure at high injection pressures and for different fuels on the penetration and the spray angle.

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Figure 7. Penetration and spray angle of the fuels by varying the chamber pressure.

A high chamber pressure means an increasing charge pressure of the engine at constant compression ratio or the retardation of the injection in the direction of top dead center (TDC). Expectedly the penetration is decreased with an increasing chamber pressure. Because of the higher density of the air in the chamber, the droplets of the spray are retarded. The friction forces on the droplets also increase with the higher chamber pressure and support the retard of the droplets. So many fast droplets catch up the retarded ones and break them in smaller droplets. For the engine operation this effect is preferable, because too long jet lengths lead to unburned hydrocarbon oxides when they hit the cold cylinder walls respectively the piston bowl especially during the warm-up operation of the engine. So it is important to increase the chamber pressure while the injection pressure is increased to limit the jet lengths. Therefore investigations with a high injection pressure and high charging pressures will be carry out at a single-cylinder engine in this project in the near future. The influences on the penetration and the maximum diameter of the chamber pressure progress of the different fuels at constant injection pressure are negligible. With the higher injection pressure the spray angle increases because of a more intensive retard of many droplets closer to the exit of the nozzle. Many droplets move in radial direction of the spray axis and increase the spray angle near the nozzle exit. The comparison of the different spray angle progresses between the fuels doesn`t reflect a clear statement. The slightly low spray angle of B50 at pInjection = 1500 bar is not explainable regarding the progress of the other fuels. The left picture of figure 8 shows lower maximum diameters of the sprays, which are injected with a high injection pressure. Even though higher spray angles are detected with an increasing chamber pressure, a higher density of the air leads to lower maximum diameters. The consequence in combination with the lower penetration is a narrower outline of the spray and a smaller spray volume, as seen in the right picture of figure 8. Compared with the investigations at ambient chamber pressure [11] the spray volume decreased about 50%. The comparison with the progress of the spray volume with an injection pressure of pInjection = 1500 bar shows the advantage of a high injection pressure at high chamber pressures: the spray volume increases impressively from pInjection = 1500 to pInjection = 3000 bar at high chamber pressures. This means a better homogenization of the air/fuel mixture in the combustion chamber and leads to the advantages which are described above.

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Figure 8. Maximum diameter and spray volume of the fuels by varying the chamber pressure.

Remarkable is the increasing difference between the progress of RME and pure diesel for high chamber pressures. It could be found out, that the difference of the spray volume between diesel and RME for high chamber pressures increases with high injection pressures. A possible assumption for that effect could not be found until finishing this paper. But further investigations are planned to explain this effect.

4.3. Variation of the Chamber Temperature The different influences on the spray values at low and high chamber temperatures are presented in figure 9 and 10.

Figure 9. Penetration and sprayangle of the fuels by varying the chamber temperature.

At high chamber temperatures a slightly higher penetration is detected for all fuels. The reason could be the lower density of the air in which the fuel is injected, so that the droplets of the spray can move a little bit longer from the nozzle. Also a lower spray angle is detected with the high chamber temperature at the beginning of the injection phase, as seen in the right picture of figure 9. A possible reason for that effect could be the early vaporization of droplets along the boundary of the spray with the result of

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decreasing spray angles. The spray also shows a low share of turbulence. This leads to the assumption, that many small droplets in the spray vaporized during the injection so that fewer droplets collide with each other compared to the diesel spray with a low temperature. More information especially about the droplet size are necessary to confirm these assumptions. Therefore investigations with PDA will be run in the near future. During the further progress of the project the chamber will be operate with nitrogen gas, so that the chamber temperature can be increased up to TChamber = 500°C.

Figure 10. Maximum diameter and sprayvolume of the fuels by varying the chamber temperature.

In the left picture of figure 10 the maximum diameter is shown for the different fuels with a low and a high chamber temperature. It seems, that for fuels with a high content of RME differences between the progresses of the maximum diameter are more detectable at late times after start of control than for fuels with a low content. For that, at high temperatures and with a high content of RME in the fuel larger maximum diameters are detectable. A possible reason could be the higher boiling point of the fuel with a high content of RME, which leads to more droplets in the spray compared to a fuel with a low content of RME, where many droplets are vaporized. The high number of droplets may lead to an intensive turbulence in the spray and to bigger maximum diameters. Because of the higher penetration and the slightly higher maximum diameters the volume of all sprays are rising with an increasing chamber temperature, as seen in the right picture in figure 10. A possible reason can be the low density of the air with the high chamber temperature. The forces that affect the spray are lower because of the low density, so that the droplets are less retarded. It can also be noticed that the differences in the spray volume for a low and a high temperature depends on the content of RME in the fuel: for a high content the difference is bigger than for a low content, respectively the conventional diesel fuel B0. The reason can be the higher boiling point of RME compared to the diesel fuel, see table 2. It is clear that the gradient of the spray volume curve of B0 becomes smaller after a short time at high chamber temperatures, because many droplets are vaporized at TChamber = 200°C. While the progress of B0 decreases dramatically after t = 3.2 ms, the gradient of B100 is not so small. The right picture of figure 10 shows the bad vaporization characteristic of RME at low temperatures. This leads to bad cold start ability of RME at engine operation.

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The difference between B0 and B100 will potentially be reduced if the chamber temperature increases. In the near future the chamber will operate with nitrogen gas so that the chamber temperature can be increased up to TChamber = 500°C.

CONCLUSION The influence of a high injection pressure on fuels with a different content of RME was investigated in this paper. Furthermore the ambient pressure and temperature of a close chamber in which the injector injected was varied up to pChamber = 60 bar and TChamber = 200 °C for this investigation. By using an internal pressure intensified injector, the injection pressure could be increased from pInjection = 1500 to 3000 bar. On the basis of high-speed images, the characteristic spray values, like the penetration, spray angle and maximum diameter could be calculated. With a MATLAB code the spray volume could be estimated. Compared with many papers it could be shown, that the penetration and the spray volume increases with higher injection pressures. While the longer jet lengths has disadvantages regarding the engine operation, the larger spray volume means a better atomization and homogenization of the air/fuel ratio in the combustion chamber, because more air can be captured by the spray. By increasing the chamber pressure, the large jet lengths can be reduced however the spray volume decreases. For investigations at the single-cylinder engine, it will be important to find a compromise between reducing the spray lengths of extreme high pressure injected fuels by increasing the charging pressure and a large spray volume to homogenize the air/fuel mixture as much as possible. A high injection pressure can help to increase the spray volume also at high charging pressures. With an increasing chamber temperature the penetration, the maximum diameters and the spray volume is increasing for all fuels because of the low density of the air in the chamber and the lower retardation of the droplets. A low spray angle at high temperatures leads to the assumption that the boundaries of the spray vaporized at first. It could be shown that with an increasing injection pressure the differences between the spray volumes of pure diesel and RME decreases impressively. With a high injection pressure the atomization of fuel with a high content of RME can be increased up to the level of diesel fuel. The better atomization leads to an intensive homogenization of the fuel in a diesel engine, which can improve the specific fuel consumption and the particulate matter emissions. Only the progress of the spray volume changed for the different fuels at different chamber pressures. Further investigations are necessary to explain this effect. The investigations at high chamber temperature show the bad ability to vaporize fuels with a high content of RME, because of the high boiling range. So the measured results are difficult to discuss because of the different boiling ranges of the fuels. It is important for the next investigations to increase the chamber temperature up to a level, at which pure RME vaporized. This will be achieved by using nitrogen gas, to increase the chamber temperature up to TChamber = 500 °C. Furthermore two more fuels will be investigates in the near future. Also measurement on microscopic level with the help of Particle Dynamic Analysis (PDA), to determine the

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velocity and the diameter of the droplets will complete the investigation of the extreme high pressure injection of alternative diesel fuels in a diesel engine for passenger cars.

REFERENCES [1]

Jeong, D. Y.; Lee, J. T.: Analysis of the Suitable Injection Pressure for Diesel Injection with High Pressure, International Journal of Automotive Technology, volume 6, No. 2, page 87-93, 2005 [2] Lee, S. H.; Jeong, D. Y.; Lee, J. T.; Ryou, H. S.; Hong, K.: Investigation on Spray Charactersitics under Ultra-High Injection Pressure Conditions; International Journal of Automotive Technology, volume 6, No. 2, page 125-131, 2005 [3] Nishida, K.; Zhang, W.; Manabe, T.: Effects of Micro-Hole and Ultra-High Injection Pressure on Mixture Properties of D. I. Diesel Spray, SAE-Paper Nr. 2007-01-1890, 2007, page 1353-1361 [4] Seebode, J.; Stegemann, J.; Sommer, A.; Stölting, E.; Buschmann, G.: Höchstdruckeinspritzung und Einspritzverlaufsformung am Nfz-Einzylindermotor, 15. Aachener Kolloquium Fahrzeug- und Motorentechnik 2006, Aachen [5] Marohn, R.; Rakowski, S.; Brauer, M.; Seebode, J.: Einspritzdruckbedarf für zukünftige dieselmotorische PKW und NKW Brennverfahren, Tagung Diesel- und Benzindirekteinspritzung V in Berlin, expert-Verlag, 2006 [6] Fischer, S.: Untersuchung des Effekts einer Höchstdruckeinspritzung auf die Ruß/NOxEmissionen bei hoher Last in einem PKW-Dieselmotor, Innovative Automobiltechnik, Tschöke, H.; Krahl J.; Munack, A., 1. Wissenschaftssymposium Automobiltechnik (WISAU) Magdeburg, 26th – 27th june 2009, page 46-60. [7] Tschöke, H.; Backofen, D.: CO2-Reduktionspotenzial im Automobilbereich, 8. Magdeburger Maschinenbautage and 7. MAHREG Innovationsforum „AUTOMOTIVE – Impulse für den Maschinenbau―, 10th -11th october 2007, Magdeburg. [8] Tschöke, H.; Backofen, D.: Zielkonflikte Alternativer Kraftstoffe, 11. Symposium Automobiltechnik, Technische Akademie Esslingen, 5th – 6th june 2008, Ostfildern. [9] Park, S. H.; Kim, H. J.; Suh, H. K.; Lee, C. S.: Experimental and numerical analysis of spray-atomization characteristics of biodiesel fuel in various and ambient temperature conditions; Elsevier; 2009. [10] Desantes, J. M.; Payri, R.; Garcia, A.; Manin, J.: Experimental Study of Biodiesel Blends` Effects on Diesel Injection Process; Energy and Fuels 2009, volume 23, page 3227-3235; 2009. [11] Backofen, D.; Könnig, M.; Geike, G.; Tschöke, H., Schmidt, J.: Spraycharakterisierung alternativer Kraftstoffe; 9. Magdeburger Maschinenbautage; 30th september -1st october 2009, Magdeburg. [12] Alfuso, S.;Allocca, L.; Auriemma, M.; Caputo, G.; Concione, F.E.; Montanaro, A.;Valention, G.: Analysis of a High Pressare Diesel Spray at High Pressure and Temperature Environment Conditions; SAE technical paper 2005-01-1239; 2005.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 18

DNA FINGERPRINTING AND CHARACTERISATION OF GENETIC VARIATION OF DIFFERENT CLONES OF URTICA DIOICA L. VIA RAPD AND RAPD-DERIVED SCAR MARKERS Bettina Biskupek-Korell1, Sabrina Becker1, Jasmin Dufrenne1, Patricia Rauscher1 and Carolin Schneider2 1

FH Hannover, Fakultät 2, Abteilung Bioverfahrenstechnik, Heisterbergallee 12, D-30453 Hannover 2 Institut für Pflanzenkultur e.K., Solkau 2, D-29465 Schnega

INTRODUCTION Urtica dioica L. (stinging nettle) is a so called low-input crop which is used for the production of textile and technical fibres. The nettle offers several advantages compared to common fibre crops like flax or hemp, but the fibre contents are still too low and/or production costs too high. So the aim of this research project is to develop new varieties with improved fibre contents and efficient propagation systems. The exact identification of different clones, cultivars and varieties is of special interest for the breeding, registration and protection of plant breeder‘s rights in general, and for vegetatively propagated species such as Urtica dioica in particular. In this context, the aim of this investigation was to develop specific genetic fingerprints of some agronomically important clones of Urtica dioica . The main focus was to achieve a reliable and preferably simple method to finally assign a specific, PCR based, fingerprint for each clone. Genetic diversity within plant species can be studied using either phenotypic traits or molecular markers. Random amplified polymorphic DNA (RAPD) markers represent a powerful tool for the investigation of genetic diversity (Williams et al., 1990). The RAPD procedure works with short oligonucleotids as PCR primers to produce anonymous genomic markers, the method requires only small amounts of template DNA, and is less laborious than other DNA markers (Caetano-Anolles et al., 1991). So the RAPD method is a good tool with

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many advantages such as low cost, easy operation, and high resolution, particularly when little genomic information of the species is available, as in the case of Urtica dioica (Wu et al., 2009). For this reason, in the research here presented, a set of 20 RAPD primers were tested to look at their ability to produce polymorphic bands within the investigated clones. Due to the common disadvantages of the RAPD method, with problems in terms of stability and reproducibility (Basha and Sujatha, 2007), the resulting polymorphic DNA fragments should be cloned and sequenced in order to obtain specific PCR primers for the precise classification of Urtica dioica individuals to their respective clone. These SCAR (= sequence amplified regions; Paran and Michelmore, 1993) markers will allow the unique genetic fingerprinting of each of the investigated clones. Advantages of the SCAR procedure, in comparison to RAPD, are higher specificity and improved repeatability (Guo et al., 2003).

MATERIALS AND METHODS Plant Material All the investigated clones of Urtica dioica were derived from the Institut für Pflanzenkultur, Schnega, Germany. At the beginning of the experiments, currently most important clones B1, B2, B12, B13, Z6 and Z10 were employed, whereas the further clones B4, B18, Z3 and Z5 were included in the second step to verify the results (Wartenberg, 2009). Leaves of field grown as well as of in vitro propagated plants of each clone were used for isolating total genomic DNA. The samples were stored at -28° C until further processing.

DNA Extraction Procedure 100–150 mg frozen leaf material from each sample was pulverised with a frozen mortar and pestle, and genomic DNA extracted using the DNeasy® Plant Mini kit according to the manufacturer's protocol. DNA was eluted with warm TE buffer in a final volume of 150 μl, quantified on a Nanodrop ND-1000 (Thermo Scientific), diluted to a final concentration of 5ng/μl, and stored at -20° C until used in the PCR experiments. Because of the inhibition of the PCR reactions of a couple of the investigated DNA templates (probably due to the abundance of a high concentration of polyphenolic substances, which are known to repress DNA polymerases) an improved method for isolating genomic DNA with the application of 10% (w/v) Polyvinylpyrrolidone (PVP) or 5% (w/v) Polyvinylpolypyrrolidone (PVPP) was carried out.

General PCR and Electrophoresis Conditions All PCRs were carried out in 25 μl reaction volumes, and amplifications were performed in a GeneAmp® PCR System 9700 (PE Applied Biosystems) with cycling conditions according to the respective PCR protocol.

DNA Fingerprinting and Characterisation of Genetic Variation of Different Clones… 183 Due to the trouble with the amplification of some of the templates, in addition to carrying out DNA isolation with PVP or PVPP, a couple of PCR enhancers were also tested. QSolution (1x), DMSO (2,5%), PVP (1%) or PVPP (1%) were added to the different master mixes. Following PCR, the samples were separated on 2% agarose, 1x TBE gels, stained with ethidium bromide, viewed and photographed under ultraviolet light.

PCR with Control Primers In order to prove the general amplification ability of all DNA templates prior to genotyping, PCRs with a well established control primer pair (EU+ and EU-) (Unterausschuß Methodenentwicklung des LAG, 2002) were carried out. This has turned out to be necessary because of the failure of the templates of a couple of clones to be amplified via PCR with the RAPD and SCAR primers. The PCR reactions were performed in a reaction volume of 25 µl containing 1x Taq polymerase buffer with 1.5 mM MgCl2, 0.2 mM of each dNTP, 5 pmol of each primer, 5 ng genomic DNA and 0.25 units Taq DNA polymerase. Temperature profile was according to Unterausschuß Methodenentwicklung des LAG (2002).

RAPD-PCR A set of 20 RAPD primers was tested concerning their ability to produce polymorphic bands within the investigated clones; two of them proved to be suitable to differentiate between the established Urtica dioica clones (Table 1). Table 1. Sequences and melting temperatures (Tm) of the RAPD primers KB-8657d and KB-8659d Primer

Sequence (5` 3`)

KB-8657d GGT GAC GCA G KB-8659d TGG GGG ACT C Tm = Melting temperature [4(G+C) + 2(A+T)].

Tm °C 34 34

Table 2. Temperature profile for RAPD PCR with Urtica dioica genomic DNA; primers KB-8657d and KB-8659d, 45 cycles Step Initial denaturisation Denaturisation Annealing Extension Final extension Storing

Time 2:00 min 30 sec 45 sec 1:30 sec 10:00 min ∞

Temperature °C 95 95 35 72 72 4

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All RAPD PCR reactions were conducted in a reaction volume of 25 µl with 1x Taq polymerase buffer, 3 mM MgCl2, 0.4 mM of each dNTP, 25 pmol of each primer, 20 ng genomic DNA and 1 unit Taq DNA polymerase. The respective temperature profile is given in Table 2.

Cloning and Sequencing of PCR Products Due to the common disadvantages of the RAPD method, with problems concerning stability and reproducibility, the resulting polymorphic DNA fragments were cloned and sequenced in order to obtain specific PCR primers for the precise classification of Urtica dioica individuals to their respective clone. After electrophoresis, the target DNA bands were cut out with a sterile cutter under ultraviolet light. DNA was recovered from agarose gels with the Double Pure Kit (BioBudget Technologies GmbH) and cloned with the T/A Cloning Kit (Genaxxon BioScience) as described by the manufacturers. Plasmid DNA was recovered with the Wizard Plus SV Minipreps DNA Purification (Promega) according to the manufacturer‘s protocol. DNA concentration was quantified via NanoDrop (Thermo Scientific) and the solutions were sent to a commercial DNA sequencing service.

Design and Application of Specific Primers The sequence information was then used to design specific PCR primers applying the software Primer 3 (http://frodo.wi.mit.edu/). All SCAR PCR reactions were carried out in a reaction volume of 25 µl with 1x Taq polymerase buffer, 3 mM MgCl2, 0.4 mM of each dNTP, 25 pmol of each primer, 20 ng genomic DNA and 1 unit Taq DNA polymerase. Table 3 gives the temperature profile for amplification. Table 3. Temperature profile for SCAR PCR with Urtica dioica genomic DNA, 35 cycles Step

Time

Temperature °C

Initial denaturisation

1:30 min

94

Denaturisation

30 sec

94

Annealing

45 sec

53

Extension

1:30 sec

72

Final extension

10:00 min

72

Storing

∞

4

DNA Fingerprinting and Characterisation of Genetic Variation of Different Clones… 185

RESULTS AND DISCUSSION PCR with Control Primer Pair EU+/EUThe first attempts at genotyping several Urtica dioica clones pointed out that in some cases, it was not possible to amplify the DNA templates via PCR. For this reason, troubleshooting was necessary before proceeding with fingerprinting. Due to the fact that Urtica dioica tissues can be rich in phenolic compounds (Bharmauria et al., 2009), which are known to inhibit PCR (Koonjul et al., 1999), two different approaches were tested. In the first case, a DNA isolation procedure was conducted with 5 % (w/v) Polyvinylpolypyrrolidone (PVPP) in the extraction buffer AP1. This molecule is known to absorb polyphenols during DNA and RNA purification (Santamaria et al., 2010). Additionally, a few common PCR enhancers were added to the respective master mixes: To overcome the inhibitory effects of polyphenols in the PCR reactions, PVP as well as PVPP was applied (Koonjul et al., 1999). Another fact that may limit the output of PCR reactions is that some DNA sequences are poorly amplifiable or not amplifiable under standard reaction conditions, either because of a high GC-content or due to their special ability to form secondary structures (Ralser et al., 2006). To overcome these problems, DMSO or Q-Solution was pipetted into the master mixes. It is well-known that DMSO is able to destabilise DNA in solution and affects the thermal stability of the primers, which leads to a higher specificity of amplification (Rådström et al., 2004). Q-Solution is a PCR additive which facilitates amplification of difficult templates by modifying the melting behaviour of DNA (Qiagen, 2010). In a first step, DNA templates isolated from field-grown leaves and from in vitro derived plant material were compared in PCRs with the control primer pair (Figure 1).The outcomes of different PCRs with the control primer pair clearly demonstrate that amplification of DNA from field-grown nettle plants can be insufficient, even if PVPP is added to the extraction buffer (Figure 1), whereas DNA from in vitro propagated individuals shows a much better performance, even if PVPP is not applied. The poor results for some of the field derived DNA templates coincide with unsatisfying DNA yields and low OD260/OD280 ratios in these samples.

Figure 1. PCR amplification patterns (including 1x Q-Solution) of 10 Urtica dioica clones (B1 –Z5) with primer pair EU+/EU-, product length 136 bp; A) DNA from field-grown leaves, DNA isolation with PVPP (5% w/v in buffer AP1), B) DNA from in vitro propagated individuals; DNA isolation with PVPP (5% w/v in buffer AP1) only for clones B4, B13, Z3, Z5; M = Marker, NTC = non template control.

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Moreover, the addition of Q-Solution to the PCR master mixes produced the best results compared to PVP, PVPP or DMSO (data not shown). To validate the indicated amplification characteristics of the DNA samples, PCRs with another control primer pair, A1/A2 (Unterausschuß Methodenentwicklung des LAG, 2002), were performed and led to identical findings (data not shown). As a result, all subsequent fingerprinting with RAPD and SCAR primers was carried out with DNA templates from in vitro propagated plants, isolated with PVPP added to the extraction buffer, and using Q-Solution as the PCR enhancer.

Genotyping via RAPD Markers In order to differentiate between individuals with close taxonomic relationships, RAPD has established itself as a good tool with many advantages, particularly when little genomic information about the species is available (Wu et al., 2009). For Urtica dioica , very little work has been done so far to investigate its molecular genetics. Bharmauria et al. (2009) successfully applied eight RAPD primers to differentiate between nettle plants from lower altitudes and samples collected from higher altitudes in the Himachal Pradesh region in the Himalayas, but had also some problems with DNA isolation and the amplification abilities of several genotypes. For other fiber crops like cotton (Chaudhary et al., 2010) or hemp (Mandolino and Carboni, 2004), the RAPD method was used to evaluate the genetic diversity or sex of the individuals and support marker assisted selections for low THC content. The motive behind the present investigation was to develop a reliable method enabling Urtica dioica individuals to be assigned to one of six currently agronomically important clones of this species.As Figure 2 shows, on the basis of two RAPD primers (KB-8657d and KB-8659d), it is possible to distinguish properly between the clones B1, B12, B13, Z6 and Z10.

Figure 2. RAPD PCR amplification patterns of 6 Urtica dioica clones (B1–Z10) with primers KB8657d (A) and KB-8659d (B); M = Marker, NTC = non template control.

With the help of four different PCR products generated by RAPD marker KB-8657d (1900 bp, 1200bp, 750 bp, 580 bp), the four clones B1, B12, B13 and Z6 can now be

DNA Fingerprinting and Characterisation of Genetic Variation of Different Clones… 187 identified directly, whereas to differentiate between clones B28 and Z10, a product of a second RAPD marker (KB-8659d, 950 bp) proved to be necessary (Table 4). Table 4. RAPD markers for genotyping of Urtica dioica clones; x = band present; - = band absent

RAPD marker KB-8657d

KB-8659d

clone Product bp 1900 1200 750 580 950

B1

B12

B13

B28

Z6

Z10

x -

x x -

-

x x

x -

x -

Genotyping via SCAR Markers The innate restrictions of RAPD fingerprinting, as well as problems with limited interlaboratory reproducibility of many RAPD markers, often led to their enhancements in terms of SCAR markers, amplifying only a single sequence linked to the trait of interest (Mandolino and Carboni, 2004) or for the assessment of the genetic diversity of several species (Basha and Sujatha, 2007).

Figure 3. SCAR PCR amplification patterns of 6 Urtica dioica clones (B1–Z10) with primers B12KB8657d-1200-1.0-l+1.1-r (A), B12-KB8657d-1200-1.0-l+2.0-r (B), B1-KB8657d-390-3.0-l+3.4-r (C), B28-KB8659d-950-6.0-l+6.1-r (D); M = Marker, NTC = non template control.

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For this reason, our next approach was to excise some of the polymorphic RAPD bands out of the gels. Selection criteria were their length (< 1200bp), to enable uncomplicated cloning and sequencing, their intensity, and not being too close to another band. Subsequently, they were purified, cloned and sequenced. On the basis of a couple of successfully isolated bands and sequenced clones, several specific primer pairs were designed and tested. This finally led to the establishment of four different SCAR markers (Figure 3, Table 5) for genotyping Urtica dioica clones B1, B12, B13, B28, Z6 and Z10. Table 5. SCAR markers for genotyping of Urtica dioica clones; x = band present; - = band absent SCAR marker B12-KB8657d-1200-1.0-l +1.1-r B12-KB8657d-1200-1.0-l +2.0-r B1-KB8657d-390-3.0-l +3.4-r B28-KB8659d-950-6.0-l + 6.1-r

clone Product bp

B1

B12

B13

B28

Z6

Z10

350 950 750 1350 910

x -

x x x

x -

x x x x

x x -

x x x x x

RAPD and SCAR PCR Results of Clones B4, B18, Z3 And Z5 Following the development of RAPD and SCAR markers for genotyping the established Urtica dioica clones, both methods should now also be tested with the further clones B4, B18, Z3 and Z5. Although the problems with the initially observed failure of the DNA templates of these clones in PCRs with plant specific control primers could be solved (Figure 1), amplification patterns with RAPD and SCAR primers still remained poor (Figure 4).

Figure 4. SCAR PCR amplification patterns of 10 Urtica dioica clones (B1–Z5) with primers B1KB8657d-390-3.0-l+3.4-r (A), B28-KB8659d-950-6.0-l+6.1-r (B); M = Marker, NTC = non template control.

This phenomenon is currently beyond final explanation, but corresponds closely to the findings of Bharmauria et al. (2009) and will be studied in detail during the following months. There is some evidence that a large concentration of polyphenols in plant tissues can strongly

DNA Fingerprinting and Characterisation of Genetic Variation of Different Clones… 189 influence PCR results (Knoll, 2002; Bharmauria et al., 2009), so this will be the starting point for our future investigations.

CONCLUSIONS The current status of our investigations verifies the conclusion that RAPD and SCAR techniques can be regarded as useful and reliable molecular tools in the identification individuals of different Urtica dioica clones. A couple of polymorphic RAPD bands could be converted to robust SCAR markers. On the other hand, it has to be noted that there are still some open questions concerning the unstable performance of some DNA templates during PCR amplification. A probable reason could be that the qualities of the Urtica dioica genomic DNA used to make templates in PCR strongly depend on the physiological state of the respective plant material as well as the DNA isolation protocol. So our next steps will comprise the further optimisation of the DNA extraction procedure, involving other plant tissues like stems or roots instead of leaves, in order to gain more knowledge about the reasons for the observed inconsistent results, particularly for the new clones.

REFERENCES Basha, S.D.; Sujatha, M., 2007: Inter and intra-population variability of Jatropha curcas (L.) characterized by RAPD and ISSR markers and development of population-specific SCAR markers. Euphytica , 156, 375–386. Bharmauria, V.; Narang, N.; Verma, V.; Sharma, S., 2009: Genetic variation and polymorphism in the Himalayan nettle plant Urtica dioica based on RAPD markers. Journal of Medicinal Plants Research, 3,166-170. Caetano-Anolles, G.; Bassam, B.J.; Gresshoff, P.M., 1991: DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. Biotech., 9, 553–556. Chaudhary, L.; Sindhu,A.; Kumar, M.; Kumar, R.; Saini, M., 2010: Estimation of genetic divergence among some cotton varieties by RAPD analysis. Journal of Plant Breeding and Crop Science, 2, 039-043. Guo W.; Zhang T.; Shen X.; Yu J. Z.; Kohel R. J., 2003: Development of SCAR Marker Linked to a Major QTL for High Fiber Strength and Its Usage in Molecular Marker Assisted Selection in Upland Cotton. Crop Sci., 43, 2252-2256. Knoll, A., 2002: Entwicklung schneller Verfahren zur DNA-gestützten Detektion von Fusarien und Analyse ihrer Mykotoxinbildung. Diss. TU München, 121 S. Koonjul, K.; Brandt, W.; Farrant, J.; Lindsey, G., 1999: Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA. Nucleic Acids Res., 27, 915–916. Mandolino, G.; Carboni, A., 2004: Potential of marker-assisted selection in hemp genetic improvement. Euphytica, 140, 107–120

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Paran, I.; Michelmore, R.W., 1993: Development of reliable PCR based markers linked to downy mildew resistance genes in lettuce. Theor. Appl. Genet, 85, 985–993. Qiagen,2010: ww.1qiagen.com/Products/PCR/TaqSystem/TaqDnaPolymerase.aspx#Tabs=t1; status: 20/01/2010. Rådström, R.; Knutsson, R.; Wolffs, P.; Lövenklev, M.; Löfström, C, 2004: Pre-PCR Processing, Strategies to Generate PCR-Compatible Samples. Molecular Biotechnology, 26, 133-146. Ralser, M.; Querfurth, R.; Warnatz,H.-J.; Lehrach,H.; Yaspo, M.-L.; Krobitsch, S., 2006: An efficient and economic enhancer mix for PCR. Biochemical and Biophysical Research Communications, 347, 747–751. Santamaría, E.; Toorop, P.E.; Rodríguez, R.; Cañal, J., 2010: Dormant and non-dormant Castanea sativa Mill. buds require different polyvinylpyrrolidone concentrations for optimal RNA isolation. Plant Science, 178, 55-60. Unterauschuß Methodenentwicklung des LAG, 2002: Methodensammlung des LAG: PCRNachweis der spezifischen gentechnischen Veränderung in Glyphosate-resistenten transgenen Pflanzen. www.lag-gentechnik.de/, soproundup.pdf; status 06.05.2010 Wartenberg, Sven, 2009: Personal communication, Institut für Pflanzenkultur, Schnega. Williams, J.G.K.; Kubelik, A. R.; Livak, K.J.; Rafaleski, J.A.; Tingey, S.V., 1990: DNA Polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18, 6531–6535. Wu, Y.; Zhang, Z.; Chen, Y.; Wang, B., Yang, G.; Yang, W., 2009: Authentication of Thailand jasmine rice using RAPD and SCAR methods. Eur. Food Res. Technol. , 229, 515– 521.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 19

IFT PERFORMANCE OF MES SURFACTANT FROM PALM OLEIN FOR EOR APPLICATION *

Erliza Hambali1, Mira Rivai1, Putu Suarsana2, Sugiharjo 3, Edi Zulchaidir4 and Hermansyah Handoko 5 1

Surfactant and Bioenergy Research Center (SBRC) - Bogor Agricultural University, Kampus IPB Baranangsiang, Jl. Raya Pajajaran No. 1 Bogor 2 PT Pertamina EP, Standard Charter Building, 25th Floor, Jl Prof Dr. Satrio 164 Jakarta 3 Lemigas-DESDM, Jl. Ciledug Raya Kav. 109, Kebayoran-Lama, Jakarta Selatan 12230 4 PT Findeco Jaya, Jl. Raya Bekasi km. 21, Pulogadung, Jakarta 13920 5 PT Mahkota Indonesia, Jl. Raya Bekasi Km. 21, Pulogadung, Jakarta 14250

ABSTRACT The application of EOR technology by using surfactants in oil industries in Indonesia is not well developed as most of formation water has high salinity and hardness level. One of the most potential surfactant from palm olein that can be applied for oil industries to obtain a higher oil recovery is MES surfactant. This research was aimed at assessing the performance of MES surfactant for oil industries. Results showed that refined MES surfactant was found to posses 10-2 - 10-3 dyne/cm of IFT values at salinity of formation water of 15,000 and 30,000 ppm by concentration of MES surfactant in solution only 0.3% to 1%.

Keywords: methyl ester sulfonate, MES surfactant, sulfonation process, interfacial tension enhanced oil recovery, EOR

*

Presented on 16th International Conference for Renewable Resources and Plant Biotechnology Magdeburg, Germany at 7-8 Juni 2010. Funded by Directorate General of Higher Education, Indonesia.

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INTRODUCTION Surfactant plays an important role in Enhanced Oil Recovery process by reducing interfacial tension (IFT) and altering wettability to facilitate the process of oil jetting from reservoir to production well. For surfactant application in EOR technology, oil industries require certain characteristics of surfactant, such as resistance to salinity (not coagulate) and relatively stable detergency in water with high hardness level, resistance at high temperature (100 – 115oC), and a range of IFT values within 10-3 to 10-6 dyne/cm. Today, the application of EOR technology by using surfactant in oil industries in Indonesia is not well developed. This is due to most of formation water has high salinity level ((5,000-30,000 ppm), high hardness level (100-500 ppm) and high temperature (80-150oC). One of the most potential surfactant from palm olein that can be applied for oil industries to obtain a higher oil recovery is MES surfactant. Refined bleached deodorized palm oil (RBDPL) consists of two fractions, namely solid fraction known as refined bleached deodorized palm stearin (RBDPS) and liquid fraction known as refined bleached deodorized palm olein (RBDPO). The composition of olein fatty acid is dominated by palmitic (C16:0) about 37.9 – 41.7 percent and oleic (C18 :1) about 40.7 – 43.9 percent. Table 1 below shows the composition of olein fatty acid. Table 1. The composition of olein fatty acid [1] Fatty acid Lauric (C12:0) Myristic (C14:0) Palmitic (C16:0) Palmitoleic (C16:1) Stearic (18:0) Oleic (18:1) Linoleic (C18:2) Linolenic (C18:3) Arachydate (C20:0)

Percentage (%) 0.1 – 0.5 0.9 – 1.4 37.9 – 41.7 0.1 – 0.4 4.0 – 4.8 40.7 – 43.9 10.4 – 13.4 0.1 – 0.6 0.2 – 0.5

As shown in Table 1, the dominant types of fatty acid in olein are C16 and C18. These types have a good detergency character that could be processed into a surfactant [2]. One of the potential surfactant that can be developed from palm olein is MES surfactant. Beside it can be utilized as cleaning agent, MES surfactant also has a prospect to be utilized for enhanced oil recovery (EOR) to increase oil recovery in mature oil fields.

AIM AND BACKGROUND A. Aim This research was aimed at assessing the performance of MES surfactant for oil industries. The MES surfactant produced by using palm olein as raw material and SO3 gasses as reactant in sulphonation process.

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B. Background The main and most important criteria of the surfactant for EOR is that the surfactant owns an IFT value lower than 10-2 dyne/cm. IFT alters wettability of core from oil wet to water wet. If the IFT of an applied surfactant could reach 10-3 dyne/cm, it could be predicted that the increasing oil recovery could reach up to 10-20 percent [3]. The important criteria of surfactant to be compatible for EOR are that it must have specific characteristics including ultra low interfacial tension, compatibility at water formation and core at reservoir, and the effectiveness of increasing oil mobilization at low concentration with low cost [4]. MES has been shown to have good dispersion characteristic, good detergency characteristic especially in high level of hard water and the nonappearance of phosphate, ester fatty acid of C14, C16 and C18 provide the best level of detergency, and good biodegradability characteristic. Compared to petroleum sulphonate, MES surfactant showed more advantages, including lower concentration of MES having the same detergency ability with petroleum sulfonate, having capability to maintain a better enzyme activity in detergent formula, and more tolerant to the presence of calcium, and having lower disalt content [5]. The process production of MES surfactant was performed by reacting methyl ester with a sulfonation agent. The reactants that can be used on the sulfonation process are H2SO4, oleum, SO3, NH2SO3H, and ClSO3H [6,7]. In this research, SO3 is used as a reactant, using Singletube Film Sulfonation Reactor (STFR) for the sulfonation process.

EXPERIMENTAL PART Materials used in this study were palm olein, SO3 gas, NaOH, methanol, H2O2 50%, NaCl, aquades, xylene, methylene blue, ethanol 95%, HCl, iodine, amylum, phenolphthalein, BaCl2, isobuthanol, KOH, BF3, Na2SO4, bromide, starch, tetrachloride, n-hexane, isoprophanol, potassium hydrogen ptalate, cyclohexane, acetic acid glacial 96%, potassium iodide, Na2S2O3, K2Cr2O7, Wijs concentrate, toluene, diethyl ether, aluminum foil, periodate acid, chloroform, H2SO4 95%, filter paper Whatman 41, petroleum ether, methylene blue indicator, phenol red indicator, N cetylpyridinium chloride, amidos sulfonic acid, bromthymol blue, dedocyl sulfate sodium salt, cetyltrimethylammonium bromide (CTAB), H2O2, water formation, crude oil and other chemical material for others analysis. Equipment used in this study included transesterification reactor, STFR reactor, purification reactor, spinning drop tensiometer, microscope with camera system (karl fischer), centrifuge and tube, pH-meter, mixer vortexer , analytic balance, stopwatch, hotplate stirrer, glass breaker and other equipment for analysis. The stages of the research activity included analyzing of physicochemical characteristics of olein, producing of methyl ester and analyzing the characteristics of methyl ester, and producing MES surfactant by sulfonation process. In the process production of methyl ester by transesterification process, methanol was added as much as 15% (v/v) from the total of palm olein that was going to be processed and mixed with KOH 1% to form metoxide. Then the palm olein and the concentrate of metoxide were mixed into transesterification reactor. The process of transesterification lasted for an hour with a temperature of 600C during the blending. The next step was settling process to separate crude methyl ester and glycerol that

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were produced from the process. After that, the next activity was the process of cleaning crude methyl ester with water to remove impurities contained in the crude methyl ester such as the residual soap, catalyst, glycerol, and other pollutant. The sulfonation process was performed by reacting methyl ester olein with SO3 gas as reactant using the Singletube Film Sulfonation Reactor (STFR). After the temperature of methyl ester has reached at 80-100 oC, the SO3 gas was added. The flow rate of methyl ester into the reactor was 100 ml/minutes. A hot steam was utilized to maintain the stability of sulfonation temperature at 800C. The steam would protect the viscosity of methyl ester in the reactor so the formation of falling film would run smoothly. The sulfonation process of methyl ester produced an intermediate compound called MESA (Methyl Ester Sulfonic Acid) that has a dark color and a low degree of acidity (pH). The purification process including bleaching and neutralization was carried out to improve MESA characteristic. In the bleaching process, MESA was heated at temperature 750C and then methanol 31% and H2O2 4% were added and the mixture was stirred constantly for 1.5 hour. The MES neutralization was carried out by adding NaOH 50% until the pH reached 6-7. The test of Interfacial Tension (IFT) was conducted by using a Spinning Drop Tensiometer at water formation 15,000 ppm and variety of MES concentration of 0.1%, 0.3%, 0.5% and 1%.

RESULTS AND DISCUSSION Surfactant plays an important role in the process of Enhanced Oil Recovery (EOR) by lowering the interfacial tension, altering the wettability, behaving as emulsifer, reducing viscosity, and stabilizing the dispersion so that jetting crude oil from reservoir to producing well is easier to do. The main criteria a surfactant has to meet in order to improve oil recovery included low IFT level (minimum 10-3 dynes/cm), resistance to high salinity, stability at high temperature (80-1500C), and having low price [8]. The result of physico chemical characteristic analysis of palm olein are as follow: Free fatty acid content of 0.19%, acid value 0.41 mg KOH/g, iodine number of 61.33 mg Iodine/g, saponification number of 208.40 mg KOH/g, density of 0.906 g/L, viscocity 61.5 (29oC), water content 0.103 %, cloud point 15oC, pour point 9oC, and unsafonification fraction 0.38%. According to the result analysis of methyl ester olein noted that its characteristic has acid value 0.94 mg KOH/g, iodin value 63.74 mg Iod/g, safonification value 27.63 mg KOH/g, glycerol total 0.06 %, ester content 95.55 %, water content 0.13% and unsafonification fraction 0.14%. Interfacial Tension (IFT) is a measure of cohesive energy at the interfacial that arises from the imbalance forces among the interface molecules (gases/liquid, liquid/liquid, gases/solid, liquid/solid). When the two different phases (gases/liquid, liquid/liquid, gases/solid, or liquid/solid) made contact with each other, thus the molecules at interface will experience an imbalance force that might cause accumulation of free energy at interface. The excess energy is called surface free energy. It can be measured as energy measure, which is the energy required to increase the surface area interface/contact. This situation also illustrates the formation of line tension or interfacial tension (IFT), which is calculated as a force. This force tends to minimize the surface area, thus explaining the phenomenon of the

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formation of liquid droplets and round shape air bubble. Common unit for the interfacial tension is dyne/cm or mN/m. This unit has an equal value. The spinning drop method is one of the methods used to measure interfacial tension. Basically, the measurement of IFT is carried out in a horizontal rotating tube (cylinder) filled with fluid. Another drop of fluid is placed in that solution. The rotation from the horizontal tube will create a centrifugal force toward the tube wall, the liquid droplet will elongate and this extension will stop when the tension of inter face and centrifugal force are balanced. This value is obtained at the equilibrium point used to estimate the tension of a specific liquid surface using an appropriate correlation. A device called ―spinning drop tensiometer― is commonly used for this purpose. Spinning drop method is usually preferred for accurate measurement having the IFT value lower than 10-2 mN/m. The working tool principle used can be seen in Figure 1.

Figure 1. The working tool principle of spinning drop tensiometer.

where Vonnegut equation used to measure IFT

Description: w : angular velocity AP: the difference of density between two fluids R: radius or radius of droplet Results of IFT value measurement of MES surfactant as sulfonation of methyl ester olein using SO3 gas at STFR with variation of steam flow 0%, 50%, and 75%, are presented in Table 2 below. Measurement of IFT values of MES surfactant was obtained at the water formation and crude oil from an oil well in Indonesia with salinity level of 15,000 ppm. Droplet appearances of crude oil and IFT value measurement using various MES surfactant concentration are seen in Table 3. Table 2. The IFT value of MES surfactant

No

Sample

Steam

Input Temperature (oC)

1 2 3 4 5 6

EH 1 EH 2 EH 3 EH 4 EH 5 EH 6

0% 0% 0% 75% 75% 75%

80 90 100 80 90 100

IFT at different MES concentration (dyne/cm) 0.1% 0.3% 0.5% 1.0% 0.0327 0.0269 0.0236 0.0201 0.0222 0.0172 0.0188 0.0067 0.0620 0.0356 0.0307 0.0276 0.0248 0.0467 0.0036 0.0053 0.0453 0.0046 0.0063 0.0043 0.0224 0.0048 0.0078 0.0458

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The data showed that the value of IFT which generated from MES olein is within average range 10-2 – 10-3 dynes/cm. When two fluids that are immicibles made a contact, these two fluids will form a border in between. The interfacial tension measures of how much work is needed to increase the area of the interfacial. The increasing area of interfacial will produce dispersion of a liquid phase on the other liquid in a small droplets. A lower interfacial tension will emulsify one liquid phase on the other liquid phase so that a low tension on interfacial correlated with displacement efficiency [9]. Steam condition is used to maintain the temperature of sulfonation process at 800C. The reaction of sulfonation occurred along the reactor. There are three interactions occured within the reactor: 1) The contact between gas and liquid phases, 2) The absorption of SO3 gas from the gas phase, and 3) the reaction in liquid phase. Table 3. Droplet appearance of crude oil and IFT value of various MES surfactant concentration Sample

0.1%

0.3%

0.5%

1%

3.27 x 10-2

2.69 x 10-2

2.36 x 10-2

2.01 x 10-2

2.22 x 10-2

1.72 x 10-2

1.88 x 10-2

6.7 x 10-2

6.20 x 10-2

3.56 x 10-2

3.07 x 10-2

2.76 x 10-2

2.48 x 10-2

4.67 x 10-2

3.6 x 10-3

5.3 x 10-3

4.53 x 10-2

4.6 x 10-3

6.3 x 10-3

4.3 x 10-3

2.24 x 10-2

4.8 x 10-3

7.8 x 10-3

4.58 x 10-2

EH 1

EH 2

EH 3

EH 4

EH 5

EH 6

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The decreasing in the interfacial tension of oil-water is caused by the amphyphylic structure of surfactant which consisting of two groups with different degrees of polarity, namely hydrophilic (like water) and hydrophobic (like oil). Surfactant with a chemical formula RSO3H will be separated in the water into ions RSO3- and H+. Ions RSO3- will intersect with the surface of oil bubbles and wrap it through a layer of the water film forming emulsion particles. Simultaneously, there is also the intersection between the surfactant and the surface of core particles. This interaction will affect the adhesive force among the oil bubbles with core of oil reservoir, as a result the bond among the oil bubbles will be greater while the adhesive force between oil and the rocks will be smaller and therefore the pressure from capillary performing in the area where the pores are tight can be reduced. There are still some conditions need improvements in order to lower the IFT value of MES from palm olein so it can produce an IFT value smaller than 10-3 dyne/cm. One of the possible way to reduce the IFT value of MES to get lower IFT value than 10-3 dyne/cm is through a separation process of methyl ester C-16 from methyl ester C-12, C-14, C-18 and then continue with the sulfonation process, aging, bleaching and neutralization. Another important issue noted from the sulfonation process is the concentration of SO3 gas, the flowrate of organic feed/compound and SO3, this is related to the molar ratio between SO3 and methyl ester to achieve maximum conversion from methyl ester to methyl ester sulfonate. In the contact phase of methyl ester with SO3, SO3 is aborbed by methyl ester to form an intermediate product, and the mol ratio of SO3-ME should not be smaller than 1.2 because it will fail to achieve a full conversion of methyl ester to methyl ester sulfonate [10]. The visualization of IFT value measurement of MES is presented in Figure 2.

Figure 2. Graphic of IFT values based on the use of steam at sulfonation process at some MES concentrations.

The IFT value presented in Figure 2 showed the measurement of IFT performance of MES olein on the variations of several surfactant concentrations towards the water formation.

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This test is intended to obtain an optimum concentration of surfactant to produce a maximum reduction of interfacial tension so the oil recovery from reservoir is higher. From Table 1 showed that the concentration of surfactant from 0.3% to 1% can reduce the IFT down to 10-3 dynes/cm. Thus, concentration of 0.3% is the most efficient concentrate to use. One thing to be considered in the EOR process is the process efficiency and the economic value where the use of a small concentration of surfactant can reduce the interfacial tension so that the oil recovery will be higher while the cost is low. The surfactant performance to reduce the interfacial tension between oil-water also changes the wettability through the interaction of surfactant with the pores from the rocks formation is largely determined by the surfactant concentration. Basically, the use of surfactant in EOR technology not only can reduce the interfacial tension oil-water but also plays an important role in changing the wettability. The characteristic of interfacial, wettability (contact angle), and interfacial tension also the characteristic of fluid flow (cover velocity and viscosity) are correlated with the petroleum recovery through the characteristic of Capillary Number (Nc),

where v and u are the speed (velocity) and thickness (viscosity) of displacing phase, while θ is the contact angle between the interfacial fluids with a solid surface, and σ is pressure of interfacial tension among fluid phase. The greater the capillary number, the lower the oil residual oil saturation so that it will enhance oil recovery. The combination of a decrease from IFT and a decrease of contact angle (θ) by surfactant will produce an optimum increase in oil production. Petroleum-based surfactant with a great performance of IFT reduction has a price that is relatively expensive [11]. Therefore, if MES can combine the reduction of IFT and good wettability, this opportunity can be used on EOR to improve the oil production. On average, only one third of the OOIP can be recovered through primary and secondary recovery, while most of the remaining trapped in the reservoir pores. This oil can be recovered by reducing the capillary force that hold the oil to flow at the core pores at the reservoir going towards production wells. Surfactant with high molecular weight will be easily absorbed by the reservoir core surface compared with the surfactant with low weight molecular, although the surfactant with high BM is important to reduce IFT, the decrease absorption of surfactant will reduce the ability to lift the oil residue in the reservoir. The molecular weight of Surfactant MES is approximately 420 g/mol.

CONCLUSION One of the most potential surfactant from palm olein that can be applied for oil industries to obtain a higher oil recovery is MES surfactant. MES surfactant produced has a range of IFT value approximately at 10-2 – 10-3 dynes/cm. Result showed that the MES surfactant has a great opportunity to be applied at the EOR technology.

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As recommendation, to reduce the IFT value of MES from methyl ester olein until reaching below 10-3 dyne/cm, a repairing process through separation of methyl ester C-16 from C-12, C-14 and C-18 using multilevel distillation technique is needed. Furthermore, the methyl ester C-16 which is already separated is to be sulfonated, aged, bleached and neutralized utilizing NaOH 50%.

REFERENCES Basiron, Y. 1996. Bailey‘s Industrial oil and Fat Products. Fifth Edition, Volume 2. Hui, Y.H. (Ed.). John Wiley and Sons, Inc., New York. [2] Watkins, C. 2001. All Eyes are on Texas. Inform 12 : 1152-1159. [3] Akzo Nobel Surfactants. 2006. Enhanced Oil Recovery (EOR) Chemicals and Formulations.Akzo Nobel Surface Chemistry LLC. www.surfactants. [4] Lee, C and P. Berger, 2010. Surfactant Injection Projects-Field Cases. Oil Chem Technologies, Inc.www.oil-chem.com. [5] Matheson, K. L. 1996. Surfactant Raw Materials : Classification, Synthesis, and Uses. In : Soap and Detergents : A Theoretical and Practical Review. Spitz, L. (Ed). AOCS Press, Champaign, Illinois. [6] Bernardini, E. 1983. Vegetable Oils and Fats Processing . Volume II. Interstampa, Rome. [7] Pore, J. 1976. Oil and Fats Manual. Intercept Ltd, Andover, New York. [8] Pithapurwala, Y.K., A.K. Sharma, and D.O. Shah. 1986. Effect of salinity and alcohol partitioning on phase behavior and oil displacement efficiency in surfactant-polymer flooding. JAOCS 63 (6) : 804-813. [9] Borchardt, J.K., 2010. Using Dynamic Interfacial Tension to Screen Surfactant Canditates. Tomah Products. [10] Roberts, D.W., L. Giusti dan A. Forcella. 2008. Chemistry of Methyl Ester Sulfonates. Biorenewable Resources 5 : 2-19. [11] Xu, Wei, Ayirala, S.C. and Rao, D.N., 2005. Experimental Investigation of Oil Compositional and Surfactant Effects on Wettability at Reservoir Conditions . Louisiana State University. International Symposium of Society of Core Analyst, held in Toronto, Canada, 21-25 August 2005. [1]

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 20

THE DEVELOPMENT PROCESS OF JATROPHA METHYL ESTER SULFONIC ACID (MESA) TO ENHANCE OIL RECOVERY *

Siti Mujdalipah 1, Mira Rivai1, Erliza Hambali1, Ani Suryani1, Hermansyah Handoko2 and Edi Zulchaidir3 1

Surfactant and Bioenergy Research Center (SBRC) - Bogor Agricultural University, Kampus IPB Baranangsiang, Jl. Raya Pajajaran No. 1 Bogor 2 PT Mahkota Indonesia, Jl. Raya Bekasi Km. 21, Pulogadung, Jakarta 14250 3 PT Findeco Jaya, Jl. Raya Bekasi km. 21, Pulogadung, Jakarta 13920

ABSTRACT Enhancement of jatropha oil‘s potential through the development of its value added by using it as feedstock for MES surfactant production is possible as C16 and C18 fatty acids contained in oleic, linoleic, and stearic acids have excellent detergency property. It shown MES surfactant produced can be appropriately used as stimulation agent for fossil fuel recovery, but it needed improvement of sulphonation process to gain IFT value range of 10-3 dyne/cm.

Keywords: jatropha curcas, MESA, MES surfactant, IFT, enhanced oil recovery, EOR

INTRODUCTION There are several vegetable oil that we can used as raw material to produce surfactant such as plam oil, coconut oil, soybean oil, and jatropha curcas oil. Fatty acid composition of jatropha curcas consists of 22.7% saturated and 77.3% unsaturated fatty acids made it can be *

Presented on 16th International Conference for Renewable Resources and Plant Biotechnology Magdeburg, Germany at 7-8 Juni 2010. Funded by Ministry of Research and Technology, Indonesia.

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appropriately used as feedstock for MES surfactant production. As it is biodegradable, MES surfactant has been utilized in personal care product, soap, and detergent industries to produce environmentally friendly products. Utilization of MES surfactant in oil industry is a potential application. One of the most important criteria of the surfactant for EOR is IFT. IFT alters wettability of core from oil wet to water wet. If the IFT of an applied surfactant could reach 10-3 dyne/cm, it could be predicted that the increasing oil recovery could reach up to 10-20 percent [1]. The important criteria of surfactant to be compatible for EOR are that it must have specific characteristics including ultra low interfacial tension, compatibility at water formation and core at reservoir, and the effectiveness of increasing oil mobilization at low concentration with low cost [2].

AIM AND BACKGROUND A. Aim This study was aimed at obtaining the process conditions of jatropha oil-based MES surfactant production by using SO3 gas reactant for enhanced oil recovery in oil industry.

B. Background Fatty acid composition of jatropha curcas consists of 22.7% saturated and 77.3% unsaturated fatty acids including linoleic (40.2%), oleic (37.1%), palmitic (17.0%), and stearic (5.7%) acids. These C16 and C18 fatty acid contents have so good detergency property that they can be appropriately used as feedstock for MES surfactant production. MES has been shown to have good dispersion characteristic, good detergency characteristic especially in high level of hard water and the nonappearance of phosphate, ester fatty acid of C14, C16 and C18 provide the best level of detergency, and good biodegradability characteristic. Compared to petroleum sulphonate, MES surfactant showed more advantages, including lower concentration of MES having the same detergency ability with petroleum sulfonate, having capability to maintain a better enzyme activity in detergent formula, and more tolerant to the presence of calcium, and having lower disalt content [3]. MES surfactant applications are so far limited to detergent and cleaning material formulations. Utilization of MES surfactant in oil industry is a potential application. Application of MES surfactant as oil well stimulation agent has been developed by Hambali et al. (2005) by using PKO methyl ester containing C12 dominant fatty acid and NaHSO3 reactant. In the study, oil well stimulation agent was developed in a formula consisting of 70% MES of PKO methyl ester, 20% solvent, 7% non-ionic surfactant, and 3% co-solvent. It was shown that at the stimulation agent rates of 0.5 and 1% with salinity levels of 10,000; 20,000; and 30,000 ppm, the IFT value reached 10-3 dyne/cm. In a test by using core lab, total oil recovery at agent stimulation rate of 0.5%v/v ranged from 88 to 94% [4]. These findings indicated that jatropha oil is also potential to be used as MES feedstock as C16 and C18 are the predominant fatty acids in this oil.

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EXPERIMENTAL PART Materials used in this study included jatropha oil, H2SO4, NaOH, methanol, H2O2 50%, NaCl, SO3 gasses, distilled water, xylene, methylene blue, ethanol 95%, HCl, iodine, amylum, phenolphthalein, BaCl2, isobuthanol, KOH, BF3, Na2SO4, bromide, starch, tetrachloride, n-hexane, KOH, isopropanol, potassium hydrogen ptalate, cyclohexane, glacial acetic acid 96%, potassium iodide, Na2S2O3, K2Cr2O7, Wijs solution, toluene, diethyl ether, aluminum foil, periodic acid, chloroform, HCl, methanol, H2SO4 95%, Whatman 41 paper, petroleum ether, methylene blue indicator, phenol red indicator, N cetylpiridinium chloride, amidos sulfonic acid, brome thymol blue, dedocyl sulphate sodium salt, cetyltrimethylammonium Bromide (CTAB), H2O2, and other chemicals fo other analyses. Equipment used included jatropha oil pressing device, esterification reactor, transesterification reactor, STFR reactor, purification reactor, spinning drop tensiometer, microscope with camera system, karl fischer, centrifuge and its tubes, pH-meter, mixer vortexer, dropper, screwed tube, analytical balance, stopwatch, graduated glass, grind cover, hotplate stirrer, burette, and glass equipment for other analysis. The study was conducted in stages including proximate analysis, jatropha seed pressing, jatropha oil physicochemical analysis, esterification-transesterificsation processes to produce jatropha methyl ester, sulphonation process to produce MESA, purification to produce MES. Jatropha seed pressing was done by using a screw pressing device. Esterification process was done at 55oC for 1 hour by using methanol of 225% FFA and sulphuric acid catalyst of 5% FFA. Transesterification was done for 1 hour at 60oC with stirring followed by warm water washing 30% (V/V), and drying. Sulphonation process was done by using a Singletube Film Sulfonation Reactor (STFR) developed by Hambali et al. (2009). Parameters assessed in sulphonation process included flow rate of SO3 gas as sulphonation agent, reaction temperatures (80, 100, 120oC), flow rate of jatropha-based methyl ester, and sampling times in 30, 45, 60, 75, and 90 minutes. Purification of resulted MESA was done by using 31-32% methanol solvents and H2O2 50% which were further neutralized by using NaOH 50%.

RESULTS AND DISCUSSION 1. Proximate Analysis and Jatropha Seed Pressing Jatropha seeds used in this study were obtained from PT Rajawali Nusantara Indonesia (Nusindo) in Cirebon, Indonesia. The seeds were packed in 30 kg sacks. Results of proximate analysis of the seeds are given in Table 1. Table 1. Results of Proximate Analysis of Jatropha Seeds Proximate Analysis Component Water content (%) Ash content (%) Oil content (%)

Value 8.90 4.62 39.87

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The oil content of 39.87% was the oil content of the seeds measured at laboratory level. However, when the oil was extracted by using extraction equipment such as a screw press, the oil content could be lower as during the processing some oil was trapped in the cake. The existence of water in the seed also affected the quality of oil produced. High water content in the seed makes the oil content easy to get hydrolyzed.

2. Analysis of Jatropha Oil Physicochemical Properties Jatropha seeds were then air dried and pressed by using a screw press. The resulted jatropha oil was collected and precipitated to separate it from solid impurities. Smaller sizedimpurities were filtered by using a filter press. The oil coming out from the screw press was black/dark in color as it contained impurities from the seed skin and other chemical compound including alkaloids, phosphatides, carotenoids, chlorophyll, and other particles. To get rid of sap/mucous containing phosphatides, protein, carbohydrate, residue, water, and resin, a degumming process was conducted. Water contained in the oil simulates hydrolysis reaction and oxidation process making the oil become rancid easily. A good storage to reduce the effect of oxidation and hydrolysis is required. Results of the degumming process showed a clear color difference (clear yellow) from the original one. Results of analysis of jatropha oil physicochemical properties are presented in Table 2. Results of FFA test with the value of higher than 2% indicated that an esterification reaction is needed in methyl ester production process before it was continued with a transesterification process. Table 2. Results of analysis of jatropha oil physicochemical properties Analysis component Water content Ash content FFA Acid number Iodine number Saponification number Density

Unit % % % mg KOH/g fat mg iodine/g fat mg KOH/g fat g/cm3

Value 0.25 0.042 10.98 20.94 99.34 197.6 0.91

3. Esterification-Transesterification Process of Jatropha Oil Jatropha methyl ester was produced in two reaction stages. The first stage was esterification reaction followed by a transesterification reaction. In esterification reaction, results of the analysis showed the value of 10.98% so that 24.71% methanol and 0.55% H2SO4 were added. The esterification reaction was done for 1 hour in a stirring rate of 300500 rpm at 50-60 oC. A transesterification was then conducted by reacting jatropha oil with 15% methanol and the inclusion of 1% KOH catalyst. The esterification/ transesterification of jatropha oil into methyl ester was done in a production scale of 100 L/batch. Results of the analysis of jatropha-based methyl ester physicochemical properties are shown in Table 3.

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Table 3. Results of the analysis of jatropha-based methyl ester physicochemical properties No 1 2 3 4 5 6

Property Water content Acid value Iodine number Total glycerol Unsaponified value Ester content

Unit % mg KOH/g ME mg Iodine/g ME %-(w/w) % %, counted

Value 1.33 – 2.29 0.16 – 0.41 98.33 0.12 – 0.27 0.39 98.9

4. Sulphonation Process Sulphonation is a process of attaching a sulphonate group to an organic compound. Sulphonation reaction occurred in a Single Tube Falling Film Reactor. Three stages occurred in MES surfactant production process included a contact between gas and liquid phases, SO3 gasses adsorption from the gasses phase, and reaction in a liquid phase. Characterization of sulphonation products (methyl ester, sulphonic acid, MESA) was conducted towards acid number, active ingredient, IFT value, pH, and iodine number. It was found that the resulted MESA had an average acid number of 12.4 mg NaOH/g MES. The lowest acidic number of 4.0 mg NaOH/g MESA was obtained at 80oC and 30 minutes sampling time. The highest acidic number of 20.64 mg NaOH/g MESA was obtained at 100oC and 90 minute sampling time. The average MESA active ingredient content was 17.10%. The lowest active ingredient content of 1.2% was obtained at 80oC and 30 minute sampling time while the highest of 31% was obtained at 100oC and 75 minute sampling time. Active ingredient content was increasing until 75 minute sampling time but it started to decrease in 90 minute sampling time. For IFT values, the average value was 1.96 dyne/cm. The lowest IFT value of 0.697 dyne/cm was obtained at 100oC and 75 minute sampling time and the highest value of 2.73 dyne/cm was obtained at 120oC and 45 minute sampling time. Table 4. Physicochemical properties of jatropha MESA No 1 2 3 4 5 6

Parameter Acid number Active ingredient IFT Value pH Iodine number Surface tension

Unit mg KOH/g MESA % dyne/cm Mg iodine/g MESA dyne/cm

Value (average) 19.81 30.41 0.73 1.15 33.53 32.38

The average MESA pH was found to be 1.35. The lowest pH of 1.12 was obtained at 100oC and 90 minute sampling time and the highest pH of 1.66 was obtained at 80oC and 30 minute sampling time. Meanwhile, the average MESA iodine number was 48.66 mg Iodine/g MESA. The lowest iodine number of 33.27 mg iodine/g MESA was obtained at 100oC and 75 minute sampling time and the highest iodine number of 73.84 mg Iod/g MESA was obtained at 100oC and 30 minute sampling time. Based on its IFT value, the best MESA was

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obtained in treatment conditions of reaction temperature of 100oC, 75 minute sampling time, 100 ml methyl ester flow rate, and 0.7 kg/hour gas flow rate. MESA physicochemical properties in these reaction conditions are given in Table 4. After the best conditions were obtained, MESA was then produced and purified. Purification process was done through bleaching and neutralization.

5. Purification Process Purification process was done to reduce or eliminate impurities found in products to result in better quality products. MES produced from jatropha oil was found to have dark color (pitch black) and low pH. In order to overcome these problems, bleaching and neutralization processes were conducted. It was found that MES had active ingredient content of 29%. Results of analysis of MES physicochemical properties are given in Table 5. Table 5. Results of analysis of MES physicochemical properties No 1 2 3 4

Physicochemical properties Acid number Active ingedient IFT value (1% MES in water formation) Iodine number

Unit mg KOH/g ME % dyne/cm Mg iodine/g MES

Value (average) 1.34 29 8.08 x 10-2 56.91

It was found from IFT analysis that MES was more effective in reducing interfacial tension at salinity levels of 15,000 ppm compare to 30,000 ppm. This was seen from the comparison of IFT values at the same rate. Results of IFT values measurement are given in Table 6. Table 6. Results of IFT values measurement in Formation Water at salinity levels of 15,000 and 30,000 ppm IFT value (dyne/cm) 15,000 ppm 8.76 x 10-01 8.14 x 10-02 8.67 x 10-02 8.08 x 10-02

Surfactant rate (%) 0.1 0.3 0.5 1

a)

b)

30,000 ppm 1.67 x 10-00 1.45 x 10-01 1.14 x 10-01 1.40 x 10-01

c)

d)

Figure 1. Fossil fuel droplet appearances when IFT measurement was done at salinity levels of 15,000 ppm and surfactant rates of 0.1 (a), 0.3 (b), 0.5 (c), and 1% (d).

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Fossil fuel droplet appearances when IFT measurement was done at surfactant rates of 0.1, 0.3, 0.5, and 1% and salinity levels of 15,000 ppm were shown in Figure 1 and at salinity level of 30,000 ppm in Figure 2.

a)

b)

c)

d)

Figure 2. Fossil fuel droplet appearances when IFT measurement was done at salinity levels of 30,000 ppm and surfactant rates of 0.1 (a), 0.3 (b), 0.5 (c), and 1% (d).

CONCLUSION Jatropha-based MESA resulted from the sulphonation process had the following physicochemical properties: acid number 19.81 mg KOH/g MESA, active ingredient content 30.41%, IFT value 0.73 dyne/cm, pH 1.15, iodine number 33.53 mg iodine/g MESA, and surface tension 32.38 dyne/cm. Bleaching and neutralization processes were found to maintain products with 29% active ingredient content. Assessed in formation water with salinity level of 15,000 ppm, IFT values were found to be in the range of 1,45 x 10-01 – 1,67 x 10-0 dyne/cm. This indicated that improvement in sulphonation process was needed in order to obtain an IFT value range of 10-3 dyne/cm because this range could improve EOR by up to 10-20%. It was concluded that jatropha oil was potential to be utilized as an oil well stimulation agent in fossil fuel recovery process.

REFERENCES [1] [2] [3]

[4]

Akzo Nobel Surfactants. 2006. Enhanced Oil Recovery (EOR) Chemicals and Formulations.Akzo Nobel Surface Chemistry LLC. www.surfactants. Lee, C and P. Berger, 2010. Surfactant Injection Projects-Field Cases. Oil Chem. Technologies, Inc.www.oil-chem.com. Matheson, K. L. 1996. Surfactant Raw Materials : Classification, Synthesis, and Uses. In : Soap and Detergents : A Theoretical and Practical Review. Spitz, L. (Ed). AOCS Press, Champaign, Illinois. Hambali, E., P. Permadi, A. Pratomo, A. Suryani, dan R. Maria. 2008. Palm oil-based methyl ester sulphonate as an oil well stimulation agent. J. Oil Palm Research, (special issue- October 2008) : 8-11.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 21

RAPESEED PROTEINS – RECENT RESULTS ON EXTRACTION AND APPLICATION Frank Pudel Pilot Pflanzenöltechnologie Magdeburg e.V.Berliner Chaussee 66, D-39114 Magdeburg

ABSTRACT The added value of rapeseed processing can be improved by extraction and use of the contained proteins. This would also contribute to close the growing gap between worldwide plant protein demand and supply. Because of their nutritional and functional properties, rapeseed proteins are valuable raw materials for new applications in animal feeding, in the non food sector and in human nutrition as well. But, there are some reasons why rapeseed proteins are not used by now, except of cattle feeding. On the one hand rapeseed proteins are so called ―novel food‖ according to the EU regulations, on the other hand it is much more difficult to extract them from cake or meal in comparison to e.g. soy. The presentation will discuss these challenges and will describe a modular concept for rapeseed processing in order to get high value rapeseed protein products which is based on the conventional oil mill technology. The most crucial step is desolventizing because it decides about protein extractability from the meal and yield. A new fluidized bed desolventizing system will be described which ensures gentle desolventizing with low PDI decrease. Finally some ongoing rapeseed protein application projects will be presented.

Keywords: rapeseed, protein, processing, desolventizing

1. INTRODUCTION There is a worldwide growing demand on plant proteins. Large amounts of plant proteins are needed for the production of animal proteins, like meat, fish, eggs or milk, taking into account that 8 kg oilseed meal are needed to produce 1 kg meat. Particularly, there is a

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rapidly increasing shortage of proteins which are prospectively needed for aquaculture, because fishmeal production is not more to expand. Additionally, due to their nutritional and/or functional advantages it is to expect that animal proteins more and more will be partially or completely replaced by proteins of vegetable origin in both certain human nutrition and industrial applications. In the most cases meals as by-products of the conventional oil mill process are not able to meet the quality requirements. Protein products of high purity, like pure protein fractions, protein isolates or concentrates are needed. Rapeseed is a potential source for such products because it is the major crop in Europe and one of the most important oil seeds worldwide and contains between 20 to 25% proteins. In 2008 an amount of about 8 m t rapeseed oil was produced in Europe, almost twice as much in 2004 [1]. The only reason for this development is the predominant use of rapeseed oil for biodiesel production. Taking into account that rapeseed contains up to 45 % oil, there is an amount of about almost 10 m t rapeseed meal (or cake) available in Europe, which is used for animal, particularly cattle feeding, having a comparably low price. Rapeseed proteins possess besides their high nutritional value a distinct functional potential enabling stabilization of emulsions and foams as well as formation of gel-like [2] and other structured systems with high water binding capacity. Therefore a lot of new value added applications in human nutrition, animal feeding (like the use of rapeseed protein concentrates in aquaculture) and for different technical purposes may be expected. Technologies for processing and application of rapeseed proteins are manifold described in the literature. However, no single commercial plant has been installed so far. The presentation will describe the main reasons for that and will show prospects for the next future.

2. THE POTENTIAL OF RAPESEED PROTEINS Like other oilseeds too, rapeseed contains not only oil, but also considerable amounts of proteins, polysaccharides, fibers and secondary plant substances. These compounds are to a different content located in the various seed compartments (Figure 1). After conventional oilseed processing, most of them are be enriched in the cake or meal. Comparing the amino acid composition of rapeseed proteins with other sources and with the requirements of FAO it can be seen rapeseed proteins are of high nutritional value (Figure 2).

Figure 1. Composition of rapeseed, hull and cotyledon [3].

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Figure 2. Comparison of FAO/WHO/UNO suggested pattern of amino acid requirements with the composition of various protein sources [4].

On the other hand, proteins possess very interesting functional properties. Due to their specific structure with both hydrophilic and hydrophobic properties they can stabilize interfaces and form films. By physical, enzymatic or chemical modification the subunits can be dissociated and the polypeptide chains can be unfolded which improves the interface stabilizing properties. And last but not least they can form networks to build bio-plastics (Figure 3).

Figure 3. Correlation between structure change and surface functionality of proteins [4].

Based on their manifold functionalities, proteins can be used both in various technical and nutritional applications. Texturized proteins can be used as be used as meat extenders and replacers as well as fibers for textiles. Protein stabilized emulsions and foams can be used in food dressings as well as asphalt emulsions or fire control foams. Protein based films and coatings can be used for fruit moisture control as well as for packaging purposes. Most important technical application possibilities are [4]:    

fillers and binders for chipboards, binders for papers and cupboards, label glues and adhesives, solubilizers, dispersion agents and emulsifiers,

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surface coatings for papers and paper boards, packaging films, encapsulation of pharmaceuticals, flavoring agents and vitamins, detergents and cosmetics, xerogels and thermoplastics.

Currently, the main problem consists in rapeseed proteins classification as ―novel food‖ according to the EU regulations, which not allows its use as food or food additive before passing time and cost consuming admission procedures. But, besides the ―novel food‖ hurdle, there are also other reasons why rapeseed protein is not produced in industrial scale up to now. Particularly, there are some processing difficulties in comparison with, for instance, soybean protein.

3. WHY IS RAPESEED PROTEIN EXTRACTION MORE DIFFICULT? 3.1. Storage Proteins Rapeseed contains two major storage proteins: the 2 S albumin napin with a molar weight of 12 – 17 kDa and the 11/12 S globulin cruciferin with a molar weight of about 300 kDa. The napin cruciferin ratio depends on the rapeseed variety and is for 00 quality about 1.1 – 1.3. This is significantly different to soybean, which contains about 90% globulin [3]. To be economic, both protein fractions have to be extracted, and this requires the adjustment of different extraction parameters in a multi step process.

3.2. Secondary Plant Substances Furthermore, rapeseed contains different specific secondary plant substances. The best known are the glucosinolates. If a rapeseed cell is damaged the enzyme system myrosinase begins immediately to decompose the gluco-sinolates. It´s break down products have mostly negative nutritional effects. Mainly isothiocyanates are formed being very reactive substances which react already at mild conditions with some functional groups of the proteins changing their solubility, isoelectric point (IP) and ratio of hydrophilic/ hydrophobic properties as well as their molar weight. Polyphenols, particularly trans sinapic acid, create dark color and bitter taste and react also with proteins, in a similar way like glucosinolates break down products. Finally phytic acid forms complexes both with trace metals, which lowers their bioavailability, and with globulins too, which change their IP to low pH values [5]. Therefore these secondary plant substances should be removed before or during protein extraction in order to secure high yield and quality of rapeseed proteins. In the literature a lot of different technological options are proposed for detoxification procedures.

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4. HOW TO DESIGN VALUE ADDED RAPESEED PROCESSING? There are a lot of different possibilities to design a process which produces both rapeseed oil and proteins. Because each single process step is able to influence yield and quality of oil and proteins as well the final process design strongly depends on the requirements of the targeted applications, particularly of the proteins due to their wide range of functionality. There is no rapeseed processing technology suitable for all purposes. Therefore a modular process design concept was developed which can fulfill various requirements. This concept consists of four processing steps: a) de-oiling process leading to cake or meal, b) protein concentrate process based on cake, c) protein isolate process based on meal and d) protein fractionation process in order to get high purified rapeseed protein fractions for special applications.

4.1. De-Oiling Process To design a whole rapeseed oil and protein extraction process at first it is to decide, whether oil and protein are to extract separately (conventional oil mill process added by protein extraction) or simultaneously. For a longer period a lot of work has been done to develop simultaneously aqueous (or aqueous alcoholic) extraction of oil and protein from specific pre-treated seeds. An overview is given by Natsch [3]. What we know now is both oil and protein yield due to not sufficient cell disruption [6], emulsification and interactions between the secondary plant substances and proteins are in maximum of about 80% and therefore too low to be economic [3]. The conventional oil mill process is optimized to high oil yield. There are oil mills extracting the oil only mechanically by one or two pressing steps. Other oil mills use hexane extraction after pre-pressing. Only pressing is cheaper, but the oil yield is lower and the cake contains 7% oil or more. This residual oil in the cake causes emulsion forming during the following aqueous protein extraction. Pressing followed by hexane extraction leads to the highest oil yield. The meal contains only about 1% residual oil. After solvent extraction the used hexane has to be removed from the meal which is done in desolventizer toaster (DT) systems. During this process step the proteins within in the meal are partially damaged leading to losses of functionality [2], [3], [4]. In soybean processing this is quantified by PDI (protein dispersibility index). Large PDI values stand for good solubility (in water) and high functionality. Measuring rapeseed products PDI only covers the globulin portion of the proteins because PDI determination is carried out in aqueous solutions using distilled water at neutral pH [7]. Nevertheless PDI can also be useful for a first estimation of the influence of rapeseed processing steps on the protein quality. In our modular process design concept first step is conventional de-oiling, improved by proper conditioning in order to inactivate the myrosinase, gentle desolventizing which keeps a high PDI in the meal and final milling (Figure 4).

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Further improvements of this basic de-oiling process could be reached by additional options. The use of yellow seed as well as seed dehulling could additionally lower the content of secondary plant substances, particularly phytic acid and phenolics, but yellow (winter rape) seed is not available, and dehulling is not be used in oil mills by now. A new crushing alternative is pressing with CO2, proposed as HIPLEX® by Crown and Harburg-Freudenberger. This shall lead to higher oil yield and better oil and protein quality due to the lower temperature stress in the press [8]. Finally, some detoxification and re-functionalization steps could be arranged between desolventizing and milling.

Figure 4. De-oiling process.

4.2. Protein Concentrate Process In order to get protein concentrates cake can be used as raw material. After (aqueous) counter current extraction and thermocoagulation the proteins can be separated. The dried matter contains about 60 to 70% proteins. The separated by-product can be used as fiber concentrate in animal feeding or as a feedstock for fermentation (Figure 5).

Figure 5. Protein concentrate process.

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Figure 6. Protein isolate process.

4.3. Protein Isolate Process If the target is protein isolate with protein contents higher than 90%, defatted rapeseed meal as feedstock is required. The process is similar to the concentrate one. The protein separation is done after counter current extraction and membrane filtration (Figure 6).

4.4. Protein Fractionation Process High purified rapeseed protein fractions are able to get by ion exchange chromatography. The process is comparable with the protein isolate process. A protein rich solution recovered by counter current extraction of a gentle deodorized meal is given into an ion exchange chromatography column and fractionated (Figure 7). This process is very simple, feasible in industrial scale, has high yield and leads to products with more than 95% purity. Figure 8 shows SDS-PAGE blots. On the left side the protein solution after extraction, on the right side the fractionated proteins, pure cruciferin and pure napin. The cruciferin fraction has good emulsifying, film and gel formation properties, whereas the napin fraction is characterized by a high solubility and foam stabilization properties.

Figure 7. Protein fractionation process.

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Figure 8. SDS-PAGE before and after fractionation.

5. HOW TO GET BEST RAW MATERIAL FOR PROTEIN EXTRACTION? As already mentioned meal desolventizing is the most critical step regarding to the protein quality. Figure 9 shows the conventional oilmill process. After oil extraction the oil content in the meal is decreased to about 1%. The hexane content is about 30% and has to be removed up to lower than 300 ppm. This is made in desolventizer toaster (DT) systems. But there is not only a reduction of the solvent, but also an undesired reduction of the PDI. In Figure 10 a scheme of a conventional desolventizer is showed. Especially during the first desolventising and toasting/stripping steps temperatures of more than 100°C exist. The treatment takes between 1 - 2 hours. Under these conditions the proteins will be damaged and the PDI will be reduced.

Figure 9. Conventional oil mill process.

To avoid PDI decrease during desolventizing in soybean processing so called flash desolventizers (FDS) are used to produce white flakes. Flaked meal is given in a pipe, in which superheated hexane at about 85°C is circulating with high velocity evaporating of most of the solvent from the flakes.

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Figure 10. Conventional DT system [9].

This treatment is finished after some seconds. Subsequently residual solvent is removed from the mael by stripping with superheated steam in a flake stripper. In this way PDI up to 85% can be reached depending on the used raw material. To fulfill other requirements on PDI of the flakes it can be adjusted by a final flake cooking system. Cooking with addition of water, desuperheated or saturated steam leads to PDI values between 15 to 85% [10]. Flash desolventizing is possible due to a very high heat and mass transfer surface of the flakes. Vavlitis and Milligan described that in the case of 0.23 mm thick flakes there is an active surface in the tube of about 6.700 m² available. But, if flakes are only a little bit thicker, this active surface will be drastic lower. Finally, spherical particles lead to a very low active surface of about 1.500 m². Hexane wetted rapeseed meal has rather a spherical shape than that of flat blanks. Therefore a flash desolventizer system seems to be not suitable for gentle desolventizing of rapeseed meal [11].

Figure 11. Scheme of FDS system (12).

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Figure 12. FDS system [12].

A process alternative to realize very high heat and mass transfer is the application of the fluidized bed technology. In a joint project PPM Pilot Pflanzenöltechnologie Magdeburg e.V., Dr. Weigel Anlagenbau Magdeburg and Otto-von-Guericke-University of Magdeburg developed a new batch fluidized bed desolventizer system. Figure 13 shows the principle, Figure 14 the small pilot scale equipment.

Figure 13. Scheme of fluidized bed desolventizer system.

A fluidized bed is a quantity of solid particles which are placed by a fluid under such conditions that the solid/fluid mixture behaves as a fluid. In our case, the fluid (superheated hexane) is fed up from the bottom and distributed by a perforated plate. It leaves the separation chamber on top. The meal is fed in from top and fluidized by the fluid. After treatment the distributor plate is turned and the desolventized meal can be removed from the equipment. After filtration the fluid is partially condensed, hexane and water are separated and led back into the system.

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Figure 14. Small pilot scale fluidized bed desolventizer.

The velocity of the fluid must be higher than the minimum fluidized bed velocity; below that a fixed bed occurs. At the upper end, the velocity has to be lower than the fluctuation velocity; above that pneumatic transport begins. Minimum fluidized bed velocity and fluctuation velocity depend on particle size. Therefore, the operating range of a stable fluidized bed is defined by the minimum fluidized bed velocity of the largest particles and the fluctuation velocity of the smallest particles. In our case particles with a size less than 0.4 mm would begin to leave the apparatus if the fluid velocity is just high enough that particles of about 5 mm can be fluidized. PDI %

Glucosinolates µmol/g

Phytic Acid g/kg

Sinapic Acid g/kg

AOCS Ba 10b-09

EG 1864/90L (LUFA)

SAA A004 (ÖHMI)

LC/MS (LUFA)

Commercial Press Cake (Oil mill)

23,7

18,2

16,9

11,5

Commercial Meal (Oil mill)

13,6

7,0

16,7

8,0

Gentle Desolventized Meal (Fluidized Bed, 75° C)

33,4

20,3

Gentle Desolventized Meal (Fluidized Bed, 95° C)

32,0

18,6

14,4

13,6

“Air Desolventized” Meal

30,4

20,5

16,8

13,7

13,9

Figure 15. Results of fluidized bed desolventizing.

Figure 15 shows the main results. It is compared PDI and contents of secondary plant substances of different materials: a commercial press cake from an oil mill, a commercial meal made from this press cake in the same oil mill, two at 75°C and 95°C fluidized bed desolventized meals, produced by extraction of the commercial press cake in our own small pilot scale extraction facility as well as a meal ―desolventized‖ by drying at air under ambient conditions. It can be seen, that the meals desolventized in the fluidized bed desolventizer have the highest PDI. On the other side, there is no effect of the fluidized bed desolventizing on secondary plant substances.

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Figure 16. Economic figures of commercial batch fluidized bed desolventizing plants.

Figure 16 shows the main economic figures for commercial batch fluidized bed desolventising plants. The 1 m diameter plant has a max capacity of 1.200 t per year, the 2 m plant of 6.000 t per year. Now we are going to develop a continuous one. It will consist of three parts: a predesolventizing step followed by the desolventizing step, realized as a fluidization channel, and a gas exchange step.

6. ONGOING RAPESEED PROTEIN APPLICATION PROJECTS As described before there are a lot of interesting possible applications for rapeseed proteins. At PPM there are actually three projects in development dealing with   

use of concentrates, isolates or pure protein fractions as aquafeed for rainbow trout and turbot feeding, use of rapeseed proteins as additives for plastic films in order to improve vapor barrier and oxygen permeation properties and use of rapeseed proteins as a paper additive in order to improve water retention and coating hold out as well as printability at reduced costs.

Additionally, there is a project running investigating the possibilities to use the byproducts from rapeseed processing (after protein extraction) as fermentation substrates.

CONCLUSIONS Because of the characteristic rapeseed composition containing two different major storage proteins with quiet different properties and some specific secondary plant substances which can react with the proteins under certain conditions rapeseed protein extraction is

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difficult. Each single processing step influences possible protein yield and quality. Therefore the process design strongly depends on the targeted application of the extracted proteins. A modular concept for value added rapeseed processing was developed based on conventional de-oiling done by oil mills. The use of a new fluidized bed desolventizer system allows gentle solvent removal without PDI decreasing. Other technology steps, like dehulling, pressing under CO2 atmosphere or detoxification / re-functionalization steps may lead to further improvements. After de-oiling rapeseed proteins can be extracted and purified in order to get concentrates, isolates or pure albumin and cruciferin fractions which are suitable for a wide range of applications in human and animal nutrition as well as for chemical and technical purposes. For the next future it is necessary to identify high value and high volume applications for rapeseed proteins in order to initiate first process installations.

REFERENCES [1] [2] [3]

FEDIOL. [Online] 2010. http://www.fediol.be/6/index.php. Becker, K. W. Current trends in meal desolventizing. JAOCS. 2 1983, pp. 216-219. Natsch, Axel. Untersuchung der Herstellbarkeit von Rapsproteinprodukten auf der Grundlage verschiedener Entölungsverfahren. Berlin : Dissertation, TU Berlin, 2006. [4] Krause, Jens-Peter, Kroll, Jürgen and Rawel, Harshad M. Verarbeitung von Rapssaat Eigenschaften und Gewinnung von Proteinen. [book auth.] UFOP-Schriften Heft 32. Rapsprotein in der Humanernährung. Berlin : UFOP, 2007. [5] Kroll, Jürgen, Krause, Jens-Peter and Rawel, Harshadrai M. Native sekundäre Inhaltsstoffe in Rapssamen - Eigenschaften und Wechselwirkungen mit Proteinen. Deutsche Lebensmitel-Rundschau. 4 2007, pp. 149-153. [6] Heckelmann, A., et al. Entwicklung eines Hochspannungsimpuls-unterstützten Verfahrens zur Verdrängungsextraktion von Ölen und funktionellen Proteinen aus Ölsaaten am Beispiel von Raps (Abschlussbericht AiF 15241 BG). Bonn : Forsch-ungskreis der Ernährungsindustrie e.V., 2010. [7] AOCS Standard Procedure Ba 10b-09. [8] Crown. http://www.crowniron.com/userimages/Crown_HIPLEX.pdf. [Online]. [9] Desolventizing and meal quality. De Kock, J. Leipzig, Germany : s.n., 2007. New Trends in Oilseed Crushing, DGF Symposium . [10] Milligan, E. D. and Suriano, J. F. System for production of high and low protein dispersibility index edible extracted soybean flakes. 4 1974. [11] Flash desolventizing. Vavlitis, Andreas and Milligan, Edward D. s.l. : AOCS Press, Champaign, Illinois, USA, 1993. Proceedings of the World Conference on Oilseed Technology and Utilization. pp. 286-289. [12] Harburg-Freudenberger. http://www.harburg-freudenberger.com/files/prospekt_extrak tion.pdf. [Online].

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 22

PCR-BASED DETECTION OF DIFFERENT ENDOPHYTIC BACTERIA APPEARING IN IN VITRO CULTURES OF DIFFERENT CLONES OF U RTICA D IOICA L. USING SPECIFIC PCR-PRIMERS, DERIVED FROM BACTERIAL 16S RDNA SEQUENCES Carolin Schneider*1, Sven Wartenberg1, Jasmin Dufrenne2 and Bettina Biskupek-Korell2 1

2

Institut für Pflanzenkultur e.K., Solkau 2, 29465 Schnega, Germany FH Hannover, Faculty 2, Dpt. Bioprocess Engineering, Heisterbergallee 12, 30453 Hannover, Germany

Keywords: fibre plant, stinging nettle, micropropagation, somatic embryogenesis, contamination

INTRODUCTION Under natural conditions, bacterial endophytes are known to be beneficial or neutral regarding plant health, nutrient uptake and other ecological functions (Ryan et al. 2008); in contrast, if endophytes occur during in vitro culture, they may cause a lot of problems (Ulrich et al. 2008). Especially in callus cultures they often appear and may finally lead to necrosis of calli (Leifert and Cassels 2001). But, for many plant species particularly callus tissue is an important prerequisite to induce somatic embryogenesis in vitro. Within the framework of a research project, we currently develop an efficient propagation system for fibre nettle (Urtica dioica L.) via somatic embryos. Induction of callus itself proved to be quite uncomplicated, but after some time of cultivation, in several of the investigated clones different, apparently clone related, bacterial endophytes had to be *

Corresponding author: [email protected].

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noticed. In order to establish contamination-free callus cultures, a laboratory method to detect bacterial free explants for establishing new callus cultures was necessary. A well known molecular approach is to amplify conserved regions of 16S rDNA with bacterial specific PCR primers (Chelius and Triplett 2001). In this investigation we tested, if this method can be used to verify bacterial contaminations in Urtica dioica clones. After cloning and sequencing the respective PCR products, strain specific PCR primers should be designed and tried out. With those primer pairs, contaminations with endophytic bacteria in Urtica dioica tissues should be detectable and it should be possible to select explants without contamination to establish callus cultures for somatic embryogenesis in this species.

MATERIALS AND METHODS Isolation: Leaves from in vitro plants were harvested free of non-endophytic bacterial contamination and/or surface disinfected with sodium hypochlorite and were stored at -26 °C. Extraction of plant DNA was done with ―DNeasy Plant Mini Kit‖ (Qiagen). Bacterial DNA was isolated from colonies on agar plates under aseptic conditions with the kit ―DNeasy Blood and Tissue Kit‖ (Qiagen). DNA concentrations and qualities were measured with a spectral photometer ―NanoDrop‖ (Thermo Scientific). PCR for 16S rDNA analysis and PCR with specific primers: PCR with primers 799f and 14492r was performed after Chelius and Triplett (2001) with plant (mitochondrial DNA 1090 bp) as well bacterial DNA (735 bp, bacterial DNA of Escherichia coli, Staphylococcus aureus, S. xylosus and Pseudomonas spp. as a control). The same protocol was used for PCR with specific primers, the annealing temperature was lowered from 55 °C (Chelius and Triplett 2001) to 53 °C, the number of cycles was maximal 25. Gel electrophoresis: Via PCR amplified DNA fragments were separated with agarose gel electrophoresis (2 % Agarose), stained with Ethidium Bromide and analysed under UV light. Sequencing of DNAŚ Specific bacterial DNA bands (‗white bacterial mucus‘ of Urtica dioica clone B30, ‗yellow bacterial mucus‘ of Urtica dioica clone Z6) from gel electrophoresis were excised out of the gel and purified with ―Double Pure Kit‖ (Bio Budget Technologies). Purified DNA was cloned in vector PJET1.2 (―CloneJET™PCR Cloning Kit‖, Fermentas), plasmid preparation was done with ―GeneJET™ Plasmid Miniprep Kits‖ (Fermentas). The respective inserts in the purified plasmid DNA were sequenced by SRD, Bad Homburg. Detection and production of specific primers: With the software Primer 3 (http://fro do.wi.mit.edu/). The obtained sequences were used for designing of specific primers, which were purchased by biomers.net GmbH, Ulm.

RESULTS AND DISCUSSION The transfer of the protocol of Chelius and Triplett (2001) to a wide range of different plant species (Brassica oleracea, Cannabis sativa , Helianthus annuus, Lepidium sativum, Secale cereale) was possible (figure 1). No detection of the bacterial band of 735 bp support the conclusion that the tested Helianthus annuus (S1 and S2) and Cannabis sativa (H1-3) are

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free of bacterial endophytes. In the targeted plant species Urtica dioica, the detection of endophytic bacteria was principally possible, too: Via microbiological methods as ―contaminated‖ classified clones/explants were correctly detected as ―contaminated‖ with PCR of 16S rDNA analysis such as clones Z6 and B30 (figure 1), resp. correctly as ―not contaminated‖ (clone B9).

Figure 1. Electrophoresis of PCR 16S rDNA analysis with primers 799f and 1492: Different plant species. M = AppliChem DNA Ladder Mix 100-5000, NTC = Non template control, Z6, B30, B9 = Urtica dioica clones, RO = Secale cereale, H = Cannabis sativa , K = Lepidium sativum, S = Helianthus annuus, RD = Brassica oleracea „Dakkar‟, RR = Brassica oleracea „Ramsch‟.

Figure 2. Electrophoresis of PCR 16S rDNA analysis with primers 799f and 1492: Urtica dioica clones Z6, B30 and B9 and resp. endophytic bacteria B-Z6 and B-B30. M = AppliChem DNA Ladder Mix 100-5000, NTC = Non template control, A = Staphylococcus aureus , P = Pseudomonas spp., RR = Brassica oleracea .

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The expected mitochondrial band with a length of 1090 bp could not be found in Urtica clones (figure 2), nor in meristematic root tissue, surface sterilized material or via amendment of additives (results not shown), it was detected in Brassica oleracea (RR, figure 2) and as expected in all tested bacteria (figure 2). Two unexpected bands are detected in plant material of Urtica dioica clones Z6 and B9 with a size of 2500 bp (figure 2). Comparison of sequences of endophytic bacteria of Urtica clone Z6 with the data base NCBI (www.ncbi.nlm.nih.gov, Basic Local Alignment Search Tool BLAST with 1.200 bacterial sequences in archive) revealed similarity with DNA sequences of bacterial genera Plantibacter , Leifsonia , Microbacterium and Curtobacterium as well as additional non-culturable bacteria of the classes Alphaproteobacteria and Actinobacteria . Microbacterium and Curtobacterium are well known endophytic bacteria in maize, cucumber and several woody plant species (Schulz et al. 2006; Ulrich et al. 2008; Scherling et al. 2009). In Urtica clone B30, the data base alignment resulted in similarity with DNA sequences of bacterial genera Agrobacterium and Rhizobium, which belong to the class Alphaproteobacteria , too, and other non-culturable bacteria.

Figure 3. Electrophoresis of PCR with specific primer pairs BacZ6-1-Prod.228 L+R and BacB301Prod.165 L+R. M = GeneCraft 100 bp DNA Ladder, NTC = Non template control, 1 = Urtica dioica clone B24-1 callus, 2 = Urtica dioica clone B24-2 callus, 3 = Urtica dioica clone Z6, 4 = Urtica dioica clone B30-1, 5 = Urtica dioica clone B30-2, 6 = Urtica dioica clone B9-1, Urtica dioica clone B9-2.

Of the designed primer pairs, two primer systems (BacZ6-1-Prod.228 L+R and BacB301-Prod.165 L+R) provided a specific and reliable detection of each of the two bacterial contaminations. Whereas the three specific primer pairs BacZ6-2-Prod.220 L+R, BacZ6-3Prod.198 L+R and BacB30-2-Prod.250 L+R amplified the expected products in several unwanted samples (results not shown), BacZ6-1-Prod.228 L+R detected exclusively the ―yellow bacterial mucus‖ of Urtica clone Z6, and primers BacB30-1-Prod.165 L+R solely the ―white bacterial mucus‖ of Urtica clone B30 (figure 3). With these two primer systems the expected product was never detected in non-endophytic bacteria (results not shown).

CONCLUSIONS AND SUMMARY With the developed primer pairs and PCR protocols, contaminations with endophytic bacteria in Urtica dioca tissues can now be reliably detected and thus the method enables to

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select explants to establish non-contaminated callus cultures for somatic embryogenesis in this species.

REFERENCES Chelius, M. K.; Triplett, E. W. 2001: The Diversity Of Archaea And Bacteria In Association With The Roots Of Zea Mays L. Department Of Agronomy, University Of WisconsinMadison, Madison, Usa, Microbial Ecology (2001) 41:252–263, Springer-Verlag New York Inc. Leifert, C.; Cassels, A. 2001: Microbial Hazards In Plant Tissue And Cell Cultures. In Vitro Cell Dev. Biol. Plant. 37: 133-138. Ulrich, K.; Ulrich, A.; Ewald, D. 2008: Diversity Of Endophytic Bacterial Communities In Poplar Grown Underfield Conditions. Bundesforschungsanstalt Für Forst- Und Holzwirtschaft, Institut Für Forstgenetik- Und Forstpflanzenzüchtung, Waldsieversdorf, Und Leibniz-Zentrum Für Agrarlandschaftsforschung (Zalf), Institut Für Landschaftsstoffdynamik, Müncheberg, Fems Microbiol. Ecol. 63 169–180. Ryan, R.; Germaine, K.; Franks, A.; Ryan, D., Dowling, D. 2008: Bacterial Endophytes: Recent Developments And Applications. Fems Microbiol Letters 278: 1-9. Scherling, C.; Ulrich, K.; Ewald, D.; Weckwerth, W. 2009: Kleine Bakterien-Große Wirkung? Endophytische Bakterien Fördern Das Wachstum Von Bäumen. Aus: Internationale Vernetzung, Forschungsreport 2/2009, Seite 40-42. Schulz, B.; Boyle, C.; Sieber, T. 2006: Microbial. Root Endophytes. Springer-Verlag Berlin Heidelberg.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 23

SURVEY OF JATROPHA CURCAS ENERGY POTENTIAL FOR AN EFFICIENT PRODUCTION OF BIODIESEL AS RENEWABLE ENERGY Sékou Traoré1, Amadou Diarra1, Macki Traoré2 and Sékou Magassouba3 1

2

Polytechnic Institute, University of Conakry, Conakry; Guinea FSE; Department of Economics, University of Sonfonia, Conakry, Guinea 3 Institute of medical plants,Dubreka; Guinea

ABSTRACT Security of fuel supply, reduction of pollution and social development are worldwide acknowledged as the most important drivers for Biofuel. Vegetable oil from the physic nut (Jatropha curcas L.) can be used for mobility (vehicles), electricity (generator), lighting and cooking. Jatropha distinguishes from many other Biofuel crops because of benefits it can offer. There is however a lot of uncertainty on the yield optimization in terms of oil quantity and quality, the improvement of oil recovery in seeds processing. Physical, mechanical and chemical properties of seed and kernel are needed for the design of equipment to handle, transport, process, store and assessing the product quality. Chemical quality and composition of the oil are investigated with regard to its behavior in the motor. In order to provide a tool to decision makers, plant growers, oil processing sector, engine manufacture and to the end users themselves, a wider approach is necessary. Therefore an exhaustive investigation of Jatropha energy resource should take into account agricultural techniques, physical, mechanical and chemical properties of the seeds and kernel, the physico-chemical properties and composition of crude oil and the derivated Biofuel. It should include also the genotype/environment dimension.

INTRODUCTION As fossil fuel costs soar and pollution increases, the need to switch the so called petrocentric society to alternative energy resources becomes ever more apparent. The extraction of

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and demand for crude oil increased exponentially as new uses for oil were discovered. It is astonishing how fast humans learned to make and use fuel from fossils. Plants transform solar energy into chemical energy in the form of hydrocarbons. A promising local renewable energy source is Jatropha Curcas L.; a plant that grows oil yielding seeds. What distinguishes Jatropha from many other bio fuel crops are the benefits it can offer. It can be utilized for various purposes of which application as transport fuel is probably the most interesting one from both an economical and ecological point of view. The most interesting application for the Jatropha oil is to use it as a fuel. It can be used for mobility (vehicles), electricity (generator), lighting and cooking. The oil content of the seeds varies with origin and growing conditions and is between 30-40 weight %, which makes it a high oil content seed. However attempts to cultivate the plant are limited because of uncertainty about how to optimize seed quality, oil quantity, oil recovery, etc. Many research works on the topic investigate physical, mechanical and chemical properties of the capsule, seed or kernel. In order to provide a tool to decision makers, plant growers, oil processing sector, engine manufacture and to the end users them-selves, a wider approach is necessary. Therefore an exhaustive investigation of Jatropha energy resource should take into account agricultural techniques, oil extraction technologies based physical, mechanical and chemical properties of the seeds and kernel and oil processing for uses based on the investigation of the physicochemical properties and composition of crude oil and the resulting Biofuel.

RENEWABILITY OF BIOMASS AND ENERGY In view of the energy problems which mankind faces, the issue of biomass and solar energy has attracted recently more and more attention. The renewability of biomass energy lies in the cyclic nature of their manu-facture. Generally natural processes on the earth are cyclic. The circulation of water between oceans, atmosphere and continents is a familiar example. Another is the transformation and movement of carbon-containing compounds for which the obvious elements are the photosynthetic generation by plants of carbohydrates from carbon dioxide and the consumption of carbohydrates by herbivores that regenerate carbon dioxide through respiration. The complete carbon cycle involves a number of additional processes and is often referred to as metabolic, biogeochemical, global cycles, etc. The terms are most commonly used to refer to cycles of the organogenic elements C, O, N, S, and P; but its use is extended as well to regional cycles and to other elements or components. The study of metabolic cycles is the study of the transformation and transport of substances in the Earth's systems involving atmosphere, oceans and earth itself. Such a cycle can be represented as follows (Figure I). The symbol M (with units of mass or moles) stands for matter; Ma: matter in atmosphere; Mt: in earth; Mo: in oceans. F (the exchange rates or flows F's have units of mass or moles per unit of time) stands for flux; Fta: flux from earth to atmosphere; Fat: that of atmosphere to earth; Foa: flux from ocean to atmosphere; and Fao: that of atmosphere to oceans. A quantitative description would give numerical values of the amounts and fluxes would give expressions for the F's in terms of the M's.

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Figure I. Schema of a metabolic cycle of organic matter.

The transformation and movement of carbon-containing compounds for which the immediately obvious elements are the photosynthetic generation by plants of carbohydrates from carbon dioxide and the consumption of carbo-hydrates by herbivores who regenerate carbon dioxide through respiration. Photosynthesizing plants are an immense organic factory and a giant energy transformer station. Photosynthetic organisms, transform visible light in the 400-700 nm part of the spectrum - called photosynthetically active radiation (PAR) - into the chemical energy of carboncontaining compounds. Photosynthetically active radiation varies with latitude, seasonality and geographical factors. The energy - in the form of biomass - that can be obtained via photosynthesis thus depends on the level of PAR and the efficiency of the conversion process Q. E biomass = PAR x Q Photosynthesis in plants is superimposed on the reverse process-respiration that is, slow combustion of organic matter to water and carbon dioxide. An excess of photosynthesis over respiration is what permits growth of plants and storage of starch, fats, oils in them. The total production of organic matter on earth can be used to calculate the total energy storage by photosynthesis. A simple ratio exists between the chemical turnover and the storage of energy, determined by the amount of chemical energy stored in a unit mass of synthesized organic matter [1]. Organic matter varies considerably in composition and energy content. How-ever, its average composition is close to that of carbohydrate Cn(H2O)m. All carbohydrates have approximately the same energy content of about 112 Kcal per gram atom (12g) of carbon contained in them. One calorie is the amount of heat needed to heat 1g water by 1deg. Centigrade.

As for Jatropha curcas oil the organic structure is as follows:

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Figure II. The Jatropha curcas oil molecule.

The formula (Figure II) shows that the synthesis of a mol of J. curcas oil requires 57 atoms of carbon (12g/mol) from CO2; 107 H (1g/mol) from water and 6 atoms of O (16g/mol). Hence Jatropha curcas mmol weighs 0.888g. This means whenever this stechiometry between CO2 and H2O is available in presence of sunlight and green leafs a corresponding amount of Jatropha oil will be photo chemically produced [2]. The gross energy content of Jatropha oil varies between 30.1 – 45.1 MJ/kg. That the oil resulting from the pressing is not pure since the expected value for pure vegetable oil is 45 MJ/kg. After pressing up to 35% of small impurities remain in the oil which should be filtered in order to increase the gross energy content. For a rational setting of biodiesel production from Jatropha seeds it is necessary to design a scale between the phases and operations involved.

I. Agricultural Techniques Several investigations have determined a geographical belt suitable for Jatropha growth. Tropical regions of the earth offer good conditions for the plant cultivation [3]. The photosynthesis active radiation as key parameter for the crude oil production depends on environment factors. Therefore identification of land area and the applied agricultural techniques are important steps. Soil properties and preparation, climate, application of diverse inputs, dis-serve a particular attention in the production of purging nut as energy source. As for soil quality, Jatropha curcas demand is low. Jatropha curcas is a monoecious shrub or small tree with staminate (male) flowers and pistillate (female) flowers on the same inflorescence. Male flowers are more numerous (8090%). Flowering is one of the most important crop phonological stages for Jatropha curcas oil production, as the number of female flowers and their fertilization determines how many fruits and seeds eventually will develop. Surely high oil content of the seeds is an important crop characteristic, but if size, the number of seeds or the number of fruits per tree (or per square meter) is not accurately accounted for, oil yields per ha are easily overestimated. The oil has a very good quality for burning. Cetane number of Jatropha curcas oil (23-40) is close to cottonseed and better than rapeseed (30-36) and sunflower (29-37). [4]

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Oil yield is the most important part in Jatropha production. As the maximum number of seeds per capsule is limited and the agronomic factor of planting density does not offer much flexibility for increasing yields, selection should focus on the other yield components. Production of Jatropha seeds and kernel should include a deep analysis of the relationships between the 1000 seed-weight, the oil quantity and quality on one side and the climate parameters (temperature, precipitations) and the altitude on the other side. Genetic and environment factors have significant impact on oil yield. However a report on genetic resource indicates that in some cases environmental factors were predominant over genetic ones [5], environ-ment exerts more effect on Jatropha than the genetic resources. Heavy rainfalls about 4000 m yearly on the Coast region in Guinea affect negatively the 1000 seed-weight as well as oil quantity as shows [6] (Figure 2). The graph exhibits a strong correlation between rainfalls 1000 seed-weight with a correlation coefficient r2=0.7256 in linear form. Precipitations exert a similar influence on oil content in Jatropha seed. As for altitude of the area a correlation exists in a less extend as shows figure 2: r2= 0.2997 and 0.257 for the polynomial and linear form respectively.

Figure 2. Effecr of precipitations on Jatropha oil content.

Figure 3. Effect of rainfalls and altitude on oil content.

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II. Oil Extraction Technologies Oil extraction and recovery efficiency are based on the knowledge of physical and mechanical properties of seeds and kernel aimed to the design of equipment for oil optimal oil recovery and conservation. Once optimal yields of capsules, seeds and kernels containing oil are obtained, the next step consists of recovering as much as possible the oil contained in seed or kernel. For Jatropha curcas oil extraction different processes are conducted before: dehulling, separating hull from nut, deshelling, separating shell from kernel, drying and finally oil extraction. In order to improve oil recovery i.e. to get the highest possible amount of crude oil physical, mechanical and chemical properties of seed and kernel are needed for the design of machines and equipment to handle, transport, process, store and assessing the product quality [7]. The mechanical properties of Jatropha seeds comprise: rupture force, deformation at rupture point, hardness and energy used for rupture on three different position loads (horizontal, transversal and vertical). Restrictions on how to store the seeds are linked to these properties. Hardness values (Table I) indicate that Jatropha seeds are relatively soft compared to for example rapeseed (> 52.6 N/mm) [8] and sunflower (35.3-65.3 depending on seed orientation). The seeds weigh about 1 ton/m3. Mechanical properties provide insights on how to adapt the pressing process to Jatropha seeds. Table I. Physical and mechanical properties of Jatropha seed [11] Physical properties

Nut

Kernel

Length [mm]

21.02 ± 1.03

15.45 ± 0.54

Equatorial width perpendicular to length [mm]

9.58 ± 0.28

7.41 ± 0.33

Breadth perpendicular to length and width [mm]

11.97 ± 0.30

10.25 ± 0.36

Solid density [kg/m³]

1040

1020

Bulk density [kg/m³]

1040

420

Mechanical properties

Nut

Kernel

Rupture force [N]

146.63 ± 14.82

67.72 ± 19.03

Hardness [N/mm]

69.98 ± 6.22

38.52 ± 5.59

Energy used for rupture [Nmm]

124.44 ± 19.95

51.61 ± 26.84

Mechanical pressing and solvent extraction are the most commonly used methods for commercial oil extraction. Screw pressing is used for oil recovery up to 90-95%, while solvent extraction is capable of extracting 99% [9]. In spite of its slightly lower yield, screw pressing is the most popular oil extraction method as the process is simple, continues, flexible and safe. The oil content of the seeds varies with origin and growing conditions and is between 3040 wt.%, which makes it a high oil content seed.

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Table 3 shows that seeds from Guinea possess relative high oil crude oil content: 38.699% in average. Seeds were collected from life fences without any specific instructions or methods. But they quite representative of Jatropha landscape in Guinea. Table II. Jatropha oil content in seeds from different provinces in Guinea Provenance

Seed quantity (g)

Oil (g)

Oil (%)

Dabola

76,40

24,50

32,06

Dinguiraye

87,70

39,55

45,09

Faranah

81,90

33,75

40,13

Kankan

53,50

35,35

42,33

Mali

84,10

35,19

41,84

Conakry/M

76,60

28,45

37,14

Conakry/H

97,72

37,89

38,77

Conakry/S

56,70

19,99

35,25

Siguiri/K

217,31

81,15

37,34

Siguiri/C

104,57

38,74

37,04

Figure 4. Oil (%) content in Jatropha seeds from different provenances.

III. Oil Processing for Use This step requires the determination of the oil composition and its physico-chemical properties to meet motor operating regime.

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III.1. Physico-Chemical Properties Much research has been conducted on the composition and properties of Jatropha seeds. These studies also provide insight in the possibilities of using Jatropha oil for fuel purposes. [10]. Crude vegetable oil that has not been treated apart from filtering is often referred to as Pure Plant oil. Purging nut oil belongs to a wide variety of pure plant oils. The main reason to use pure plant oil is the costs related to the transesterification process. Compared to conventional diesel the use of crude oil in a diesel engine reduces the emission of sulfur oxides, carbon monoxides, polyaromatic hydrocarbons, smoke, particle matter and noise [11]. The main disadvantage of vegetable crude oil on the other hand is its high viscosity that leads to unsuitable pumping and fuel spray characteristics. The high viscosity of Jatropha oil is a result of the presence of the saturated and unsaturated fatty acids. The acids consist of relatively long carbon chains (C14:0-C20:0) when compared to conventional diesel (C12:0C15:0) [12]. The acid composition also hints an acidic nature of Jatropha oil, which is harmful to rubber engine components. Poor atomization, low volatility and insufficient fuelair mixing can lead to combustion chamber deposits, gum formation and unburned fuel.

III.1.2. Biodiesel Biodiesel is a fuel type that has already been proven suitable for use in diesel engines. Biodiesel consists of fatty acid methyl esters of seed oil and fats and is produced through transesterification. The triglyceride esters of oil are changed into methanol monoesters (methyl esters), each with single fatty acid chains causing the lower viscosity of biodiesel. Fatty acid methyl esters are environmentally safe, non-toxic and biodegradable making them a suitable transport fuel. The biggest advantage of biodiesel over pure plant oil is its lower viscosity. One of the important properties for selection of fatty acids methylester for biodiesel is the Cetane number. This is the ability of a fuel to ignite quickly after injection. Higher values indicate better ignition quality. Some of the most widely used biodiesel standards ASTM D 675, DIN 51606 and EN 14214 set Cetane umber at 47, 49 and 51 respectively. The CN for Jatropha biodiesel is on the high end being 52.31 [13]. Another key property for biodiesel quality is the iodine value, which indicates the degree of unsaturation (amount of double bonds). Some level of unsaturated fatty acid components is necessary to prevent fatty acid methyl esters from solidification. High temperatures occurring in internal combustion engines tend to accelerate this process. III.1.2.1.Transesterification Transesterification is most commonly used and important method to reduce the viscosity of vegetable oils. In this process triglyceride reacts with three molecules of alcohol in the presence of a catalyst producing a mixture of fatty acids, alkyl ester and glycerol. The process of removal of all the glycerol and the fatty acids from the vegetable oil in the presence of a catalyst is called transesterification. Biodiesel results from transesterification. Biodiesel properties are similar to diesel fuel. It is renewable, non-toxic, bio-degradable and environment friendly transportation fuel. After transesterification of the vegetable oil its density, viscosity, Cetane number, calorific value, atomization and vaporization rate, molecular weight, and fuel spray penetration distance are improved more.

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Transesterification reaction equation [11]:

Physical and chemical properties are more improved in transesterified vegetable oil because transesterified vegetable oil contains more Cetane number than diesel fuel. These parameters induce good combustion characteristics in vegetable oil esters. So unburnt hydrocarbon level is decreased in the exhaust. It results in lower generation of hydrocarbon and carbon monoxide in the exhaust than diesel fuel. The vegetable oil esters contain more oxygen and lower calorific value than diesel. So, it enhances the combustion process and generates lower nitric oxide formation in the exhaust than diesel fuel. Table III. Compared Fatty acids composition of Jatropha methylester Sample

Fatty acids (%) C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

C20:0

Guniea

16,22

1,05

8,97

47,25

26,23

0,17

0,11

Typ I *

14,60

0,85

7,15

46,27

30,80

0,20

0,21

Typ II *

13,45

0,72

7,46

34,30

43,12

0,20

0,21

Typ III *

15,20

0,90

6,70

42,60

33,90

0,20

0,20

(*) Data from literature.

CONCLUSION Photosynthesizing plants are an immense organic factory and a giant energy transformer station. The renewability of biomass energy lies in the cyclic nature of their manufacture. Generally natural processes on the earth are cyclic. The transformation and movement of carbon-containing compounds for which the immediately obvious elements are the photosynthetic generation by plants of carbohydrates from carbon dioxide and the consumption of carbohydrates by herbivores that regenerate carbon dioxide through respiration. Agricultural techniques in conjunction with environment and genetic potential determine the yield parameters of Jatropha curcas. Tropical regions of the earth offer good conditions for the plant cultivation.

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Oil extraction and recovery methods based on mechanical, physical properties define the oil amount and quality with this regard moisture content has the strongest effect on oil recovery. Optimal oil recovery is expected at a moisture content of 2-4%. Restriction size and rotational speed of the screw are other influential parameters. Jatropha oil content is relatively high in Guinea. Oil processing technologies based on the knowledge of chemical properties of the oil and its composition allow an efficient use of the oil and provide safety of motors and equipment. With regard to fatty acid composition the Jatropha oil originating from Guinea seems to have a good stability since it has a very low amount of fatty acids with double bounds.

REFERENCES Yamamoto, H.K., Yamaji, K. and Fujino, J.ś 1999Ś ‗Evaluation of bioenergy resources with a global land use and energy model formulated with SD technique‘, Applied Energy 63(2), 101–113. [2] Jongschaap, R.E.; Corré, W. I., Bindraban, P. S.; Brandeburg, W. A. 2007: Claims and facts on Jatropha curcas L., PRI, Report 158; Wageningen, pp.6-21. [3] Heller, J. Physic nut. Jatropha curcas L. Promoting the conservation and use of underutilized and neglected crops.: Institute of Plant Genetics and Crop Plant Research, Gatersleben/International Plant Genetic Resources Institute, Rome, 1996. [4] Guebitz, G. M.; Mittelbach,M.; Trabi, M.: Biofuels and industrial products from Jatropha curcas. Dbv-Verlag fuer die TU Graz, Graz. Austria ; pp. 98-109. [5] Kaushik, N.; Kumar, K.; Kumar, S. 2007: Genetic variability and divergence studies in seed traits and oil content of Jatopha (Jatopha curcas L.) accessions, Biomass and and Bioenergy 31, 497-502. [6] Traoré, S.;Traoré, M; Becker, K.: Naossa 14.International Conference on Renewable resources and plant Biotechology ; Magdeburg 2008: Profiles of Jatropha curcas oil as sustainable source of energy and raw material in Guinea. [7] P. Sirisomboon, P. Kitchaiya, T. Pholpho, and W. Mahuttanyavanitch, "Physical and mechanical properties of Jatropha curcas L. fruits, nuts and kernels," Biosystems Engineering, vol. 97, pp. 201-207, 2007. [8] Faborode, M. O. and Favier, J. F., 1996, Identification and Significance of the Oil-point in Seed-oil Expression: Journal of Agricultural Engineering Research, v. 65, p. 335345. [9] Shahidi, F., 2005, Bailey's Industrial Oil and Fat Products volume 5: "Edible oil and fat products: Processing Technologies": New Jersey, John Wiley and Sons, Inc.. [10] Openshaw, K., 2000, A review of Jatropha curcas: an oil plant of unfulfilled promise: Biomass and Bioenergy, v. 19, p. 1-19. [11] Berens, P. 2007: Screw-pressing of Jatropha seeds for fuelling purposes in less developed countries, M.Sc. Thesis; TU Eindhoven; Department of Sustainable Energy Technology, Eindhoven, pp.13-19 [1]

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[12] Akintayo, E. T., 2004, Characteristics and composition of Parkia biglobbossa and Jatropha curcas oils and cakes: Bioresource Technology, v. 92, p. 307-310. [13] Mohibbe Azam, M., Waris, A., and Nahar, N. M., 2005, Prospects and potential of fatty acid methyl esters of some non-traditional seed oils for use as biodiesel in India.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 24

GENETIC RESOURCES OF CANNABIS SATIVA L. IN THE COLLECTION OF THE GENE BANK AT INFANDMP IN POZNAN Magdalena Chudy* and Grażyna Mańkowska

Institute of Natural Fibres and Medicinal Plants, Poznań, Poland

ABSTRACT The hemp gene collection at the Institute of Natural Fibres and Medicinal Plants is unique in Poland in terms of diversity and the number of gathered accessions. It holds about 150 accessions from various regions of the world. It includes mono- and dioecious forms representing different degree of adjustment to climate-soil conditions and also local ecotypes and breeding lines of stable genotype, distinctive because of a single treat e.g. 9THC and cellulose content, yielding, fibre quality and fatty acid composition. Each accession is characterized with the morphological and biological features and use value. The INFandMP collection makes a valuable source for breeders when selecting useful parental components and also secures breeders‘ achievements.

Keywords: gene bank; genetic resources; monoecious hemp; dioecious hemp; seed

INTRODUCTION Species – sowing hemp (Cannabis sativa L.) belongs to the family Cannabaceae, the type of Cannabis. Hemp plants are considered to be easily adapts to changing conditions for plant growth. Within the species there are many types, forms and varieties of significant biological

*

Institute of Natural Fibres and Medicinal Plants, ul. Wojska Polskiego 71b, 60-630 Poznań, Poland. tel.:+48 61 84 55 844 (832), fахŚ +48 61 84 17 830, E-mail: [email protected].

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differences - and different morphological economic value [Białousowa i in., 1958, Bocsa i Karus, 1997] In terms of different environmental conditions to which had spread to adapt in different climatic zones, hemp can be divided into 3 groups:   

Northern hemp. Plants are low, their height does not exceed 80 cm, thin stem, little branched with a short growth duration 60 to 75 days. Give a little fat and fiber divisible. Hemp south. Characterized by high stem (300-400cm) and a long growing season (140-160 days). Under favorable conditions, produce a high yield of good quality fiber. Mid-European (intermediate) Occupy the middle zone of Europe between 1951 and 58 latitude [Bytnerowicz and al., 1968]. This group includes Polish varieties of hemp. Characterized by a relatively short growing season 80-120 days and plant height of 200-300 cm.

There is a distinction between hash and hemp fiber. Hemp fiber according to Polish law (Act of 29 July 2005. On Counteracting Drug Addiction - Journal Laws No. 179, position1485) and EU law, the hemp containing less than 0.2% 9 tetrahydrocannabinol (9 THC a chemical compound decide on the hallucinogenic effect of hemp) and tetrahydrokannabinolic acid (delta-9-THC-2-carboxylic acid) Sownig hemp is an annual plant, dioecious or monoecious. Monoecious hemp produces male and female flowers on one plant. At the dioecious hemp male and female plants are separate plants subject to different laws of development. Male plants stop the growing period of about four weeks earlier, which causes several complications, both in harvesting and processing straw. In order to eliminate these problems have been grown monoecious hemp which at the same time maturing and giving a uniform raw material and higher seed yield [Jaranowska 1962; Van der Werf, 1994, ]. Cultivating hemp in Poland has a long tradition. In 1928 it was cultivated on area of 29 300 hectares. First agronomic research was conducted in the period between the two World War. Breeding work with hemp began in 1946 under the direction of J. Jagmin. The starting material for breeding was local populations and Schurig form, in which the content fiber in the straw was only 14.3%. As the result of many years of breeding work the contents of this fiber grew to 25 - 30%. [Grabowska L. i in. 2009]

AIM AND BACKGROUND The Gene Bank of Cannabis sativa L. has collected hemp from different regions of the world (Figure 1). These include varieties, local ecotypes and breeding lines of stable genotype, distinctive because of a single treat e.g. Δ9THC and cellulose content, yielding, fibre quality and fatty acid composition in oil obtained from seed.

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Collecting of varieties and ecotypes of hemp is done not only to provide a differentatied source material for breeding and protection of many achievements of breeders, it is also done for maintaining of biodiversity. In the case of hemp this is particularly important element, because until recently the cultivation of hemp because of the risks drug addiction, was banned in many countries. Not only material for breeding, but also the local populations destroyed. 2 2

24

2

11

2 27

14

2 3

24 2

15 25

9

2 1 11

Bulgaria Yugoslavia Russia Cuba

China Poland Ukraine Turkey

Finland Slovakia Thailand Breeding lines

France Netherland Hungary

India Romania Italy

Figure 1. Structure of INF and MP Cannabis collection according to origin.

35%

65%

monoecious

dioecous

Figure 2. The share of mono- and dioecious hemp accessions.

The starting material for creative breeding are variety and breeding lines collected in the Gene Bank INFandMP. Collection of Cannabis sativa L. holds of approximately 150 objects. The hemp collection at INFandMP is kept as a part of the national long-term program: „The plant improvement for sustainable Agri-Eco-Systems, high quality of food and plant production for non-food applications‖. The works comprise: „Collecting, protecting, evaluating, maintaining and providing access to the genetic resources of crop plants and their pathogens, in the scope of flax and hemp ecotypes and the sowing material of protected medicinal plants‖. The program is financed by the Ministry of Agriculture and Rural Development (Poland) and coordinated by the Institute of Breeding and Plant Acclimatization in Radzikow. The following topics are included: 1. Collecting and long-term storing in genetic purity and live state of crop plants varieties and ecotypes of flax and hemp and of sowing material of protected medicinal plants.

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2. Keeping inventory, evaluating and characterizing of the collected ex situ and in situ plant genetic resources - varieties and ecotypes of flax and hemp and of sowing material of protected medicinal plants. 3. Documenting and providing access to the information and the collected accessions for scientific and breeding purposes, and realizing pro-ecological policy of the government. Hemp seed, due to its structure and chemical composition, strongly react to adverse storage conditions. The proper conditions for storing hemp seeds are:  

Low seed moisture at about 7 - 8% Relative air humidity in the storing rooms not higher than 55% [Ziemnicki, Wierzchowiecka, 1984].

Photo 1. The core collection.

Photo 2. The reserve bank.

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245

In 2000, the institutes collections of hemp genetic resources were moved to new premises to ensure perfect storage conditions. So-called air-dry seed (ok.7% absolute humidity) are packed in heat sealable aluminum foil and sent to:  

the core collection – cooling room with temperature at 4C (Photo 1) the reserve bank – cooling room with temperature at - 17C (for long-term storing) (Photo 2)

At the moment we are at the stage of transfering seed from bags into jars (Photo 3, 4). Since they are more practical.

Photo 3. Bags and jars for storing the seeds.

Photo 4. Bags and jars for storing the seeds.

EXPERIMENTAL PART Propagation and restoring sowing value of the collected accessions is a very meticulous process. Space isolation is required because of hemp dioeciousness, allogamy and anemophily. Depending on amount of material we use different isolating methods. Small amount is sown into isolators in the pots inside the vegetation hall (Photo 5,6), and higher amounts into isolators in isolated chamber in the greenhouse and the isolators on fields (Photo 7-10). When monoecious forms are propagated it is necessary to remove male plants during flowering. The irregurality in maturation of seeds and their predisposition to shedding forces harvesting at optimal time and the need to secure panicles before shedding the seed and against birds feeding on the seed.

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Photo 5. The isolators in the pots inside the vegetation hall.

Photo 6. The isolators in the pots inside the vegetation hall.

Photo 7. The isolated chamber in the greenhouse.

Photo 8. The isolated chamber in the greenhouse.

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Photo 9. The isolators on fields.

Photo 10. The isolators on fields.

New directions for the use of hemp have set new tasks before the breeders and therefore an important issue is to determine the economic value of the materials held in the collection. Comparative experiments are carried out where the reference variety is the Polish cultivar Białobrzeskie. During vegetation period the developmental phases are monitored, also lodging and pest infestation. During flowering the samples of panicles are taken to test for THC (the case of dioecious varieties only the samples from female plants are taken). After harvesting morphological measurements are taken, yields, weight of 1000 seeds, fibre, cellulose and chemical composition of oil are determined.

RESULTS AND DISCUSSION The Jermakowska variety origates from Russia and is distinctive because of very valuable dietetically ratio of omega – 3 and omega – 6 fatty acids (3:1) (Mańkowska i in, 2007) and very high fiber qualityś Białobrzeskie a polish variety, shows the highest fiber content at about 26% (table 2); and are varieties producing seed yield over than 1,0 ha. The Ukrainian varieties Juso 11, Juso 15 and Juso 31 regardless from the year of the study are characterized with the lowest content of psychoactive substances (0,004-0,01 % Δ9THC). The mass of 1000 seeds is also very varied: from 43 g (He Bei) to 6,0 g (Wild Polish) for the reference variety of Białobrzeskie the value is 13,9 g (Photo 11).

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Photo 11. Sizes and mass of hemp seeds.

The genotypes in the collection of the Institute genotypes show a very wide variety of each of the studied feature. The collection includes varietie characterized by a long growing season (140-160 days), e.g. He Bei, Gigantheus, which in comparison with the variety Białobrzeskie give higher yields of straw up to 30%, but we must remember that in Polish climatic conditions, in principle, these cultivars do not give seed. The variety Finola and Jermakowska are characterized with short vegetation period in Polish climate-soil condition reaches about 1,5 m height giving a low yield of straw and high seed yield. Table 1. Comparison of development stages in selected hemp cultivars Cultivar

Beginning of flowering date 13.07 8.07 4.08

Full maturity date

Zenit Juso 11 Futura 77

Dioecious/ monoecios monoecious monoecious monoecious

Chamaeleon

dioecious

28.07

2.10

Diana

dioecious

1.07

5.09

Uniko B

dioecious

4.08.

8.10

Silistrenskie Fedora 17 Finola Juso 15 K - 195 Dnieprowska Fibrimon 24 Zolotonoska 26 He Bei Gigantheus Białobrzeskie

dioecious monoecious dioecious monoecious dioecious monoecious monoecious monoecious dioecious dioecious monoecious

10.07 7.07 30.05 8.07 10.07 2.07 12.07 20.07 1.09 16.08 15.07

20.09 15.09 20.08 15.09 20.09 10.09 20.09 25.09 29.09 29.09 20.09

20.09 15.09 2.10

At the Department of Biotechnology at INFandMP the following studies are carried out: the studies on polymorphism of the gene synthesizing THCA (1 – tetrahydrocannabinolicacid) what will allow for better characterization of the collected genotypes and differentiation between the industrial or narcotic group of hemp. It is assumed that the results

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will be used in breeding work and forensic science, as the result will indicate the cultivars that under no conditions can accumulate THC. Table 2. The fiber content in stem of selected varieties of hemp collected in the Gene Bank Cultivar Zenit Juso 11 Futura 77 Chamaeleon Diana Uniko B Silistrenskie Fedora 17 Finola

Average fiber kontent (%) 23,51 26,05 20,49 27,06 20,74 29,16 17,27 21,82 12,28

Cultivar Juso 15 K - 195 Dnieprowska Fibrimon 24 Zolotonoska 26 Gigantheus He Bei Białobrzeskie

Average fiber kontent (%) 28,36 16,48 19,11 22,28 22,54 17,62 12,00 26,60

CONCLUSION It must be emphasized that the collection of hemp genotypes at INFandMP is one of few such collections worldwide, very interesting not only because of the number of accessions but also because of the diversity of the accessions , what provides a rich source of material for breeding and research work. Breeding monoecious hemp is difficult, yet by complementing the traditional breeding methods with the techniques of genetic engineering will allow for obtaining earlier new varieties with specific targeted technological parameters. The genotype collection is continually expanded as a result of cooperation and exchange between research institutions and botanical gardens both in Poland and abroad.

REFERENCES Białousowa J., Bartosik A., Kurhański M., Nagórski A., Tumalewicz B. 1958. Konopie. Rośliny Włókniste, Pwril, Warszawa: 193-323. Bocsa I, Karus M. 1997. Der Hanfanbau. Heidelberg, C.F. Muller. Bytnerowicz H., Kurhański M., Nagórski A., Piertaszkiewicz K.,1968. Konopie, Pwril, Warszawa Grabowska L., Rebarz M., Chudy M. Hodowla I Uprawa Konopi Włóknistych W Polsce. Herba Polonica , Nr 3 2009: 328-334. Jagmin J. 1933. O Możliwo ciach Uprawy Konopi W Polsce. Towarzystwo Lniarskie, Wilno. Jaranowska B. 1962. Konopie Jednopienne, Pwril, Warszawa. Kilanowski W., 1974. Konopie, Rośliny Włókniste, Pwril, Warszawa: 143-201 Mańkowska G., Menesiak M., Grabowska L. 2007. Ocena Zasobów Genowych Konopi Zgromadzonych W Instytucie Włókien Naturalnych W Poznaniu Z Uwzględnieniem Nowych Kierunków Wykorzystania, Zeszyty Problemowe Postępów Nauk Rolniczych 2007 Z. 517: 853-860.

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Ustawa Z Dnia 29 Lipca 2005 O Przeciwdziałaniu Narkomanii, Dz.U. Z 2005 R. Nr 179, Poz. 1485. Wielgus K., Luwańska A., Lassociński W., Kaczmarek Z.Ś Estimation Of Cannabis Sativa L. Tissue Culture Conditions Essential For Callus Induction And Plant Regeneration, Journal Of Natural Fibres, Vol 5,Nr 3, S.199-207, Haworth Press, 2008. Van Der Werf H. 1994. Crop Physiology Of Fiber Hemp (Cannabis Sativa L.), Doctoral Thesis, Cabo Report 142, Wageningen. Ziemnicki Z., Wierzchowiecka K. 1984. Wpływ Przechowywania Nasion Konopi W Różnych Opakowaniach Na Zachowanie Zdolno ci Kiełkowania, Prace Ikwn, Rocznik Xxix, PoznańŚ 99-106,

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 25

PRODUCTION OF BIODEGRADABLE CARRIER MATERIALS FOR THE IMMOBILIZATION OF MICROORGANISMS FOR THE TREATMENT OF WATER Alvaro E. Gonzalias *, A. Werner and Hans-Joachim Radusch Centre of engineering sciences, Martin Luther University Halle-Wittenberg, 06099 Halle (Saale), Germany†

ABSTRACT The biocompounds were designed out of two polymers having different degradability. Batch experiment with a synthetic water clearly indicate that the biocompounds could remove ammonium and nitrate. Ammonium concentration was removed almost complete after 5 days while nitrate concentration was achieved after 2 days. In comparison to two matrix polymer (PCL) our biocompound showed the maximum denitrification rate.

Keywords: Biodegradable, Biocompound carrier, Biofilm, Simultaneous nitrification/ denitrifiction, Poly-3-hydroxibutirate-PHB, Policaprolactone-PCL

INTRODUCTION The biocompounds under consideration consist of one readily and one poorly biodegradable polymer (Figure 1). While the readily biodegradable component provides a *

Corresponding author. Tel.: +49 3461 46 2716; fax: +49 3461 46 3881. E-mail address: [email protected]. † www.kunststofftechnik.uni-halle.de.

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substrate for a fast set-up of microorganisms, the poorly degradable component serves as a matrix. The combination of readily and poorly degradable polymers leads to rapid growth of biofilms on the surface (Figure 2).

Figure 1. Biocompounds components.

Figure 2. Biofilm grows over the biocompound.

Through the diffusions processes in the carriers comes the formation of different environmental conditions. Thus, aerobic, anoxic and anaerobic zones may be formed in the biocompounds. The biocompound carbon makes simultaneous de-nitrification possible in the anoxic layers while nitrification takes place concurrently in the aerobic biofilm layers.

AIM The aim of this study was to investigate the ability of such biocompounds to remove ammonium and nitrate.

EXPERIMENTAL PART Biocompounds Carriers Straps were produced using an single-screw extruder Trusiograph (by Göttfert) with a compression ratio of 1:3 and a nozzle of 4 mm. Immediately after extrusion, the strands were cooled in the water bath and pelletised one day after production (Figure 3). The barrel

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temperature was selected so that the poorly biodegradable component (polycaprolactonePCL) would solely melt by treatment. The biocompounds consist 70% of poorly (PCL) and 30% of readily biodegradable (poly-3-hydroxybutyrate-PHB) component.

Figure 3. Biocompounds.

The biodegradable pellets were produced at the Centre of Engineering Sciences from the Martin-Luther-Univesität Halle-Wittenberg. Table 1 shows the main characteristics of the biocompounds carriers. Table 1. Characteristics of the biocompound carriers Parameter Material Diameter Height Density

PHBPCL 30_70 WB PHB 70%: PCL 30 % 4.00 ± 0.106 mm 3.78 ± 0.316 mm 1.0994 ± 0077 g/cm3

Experimental Conditions The experiment was conducted in a 1.5 litre glass reactor in which 10% of the volume was filled with biocompounds. 1 litre of mineral medium (ES ISO 113734) was added to the reactor. As a seed, a sludge of clarify was used from a wastewater treatment. The mixture was kept in the reactor by using bubbling air from the bottom.

Figure 4. Schematic diagram of airlift reactor.

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The synthetic substrate medium used for the experiments consisted of: 0.27 mg L-1 KH2PO4, 1.12 mg L-1 Na2HPO4 ּ 12H2O, 0.075 mg L-1 CaCl2 ּ2H2O, 0.10 mg L-1 MgCl2 ּ 6H2O, 0.02 mg L-1, 0.20 mg L-1 FeCl3 ּ4H2O, and 0.1 mg L-1 Na2S · 9H2O. Samples of ammonium (DIN 38406/5) and nitrate (DIN 38 405/9) were analysed during the experiment.The schematic diagram of the reactor is depicted in Figure 4.

RESULTS AND DISCUSSION Various biocompounds were produced utilizing different types from poorly degradable polymer (PCL 6500 and PCL 6506) and tested in different operational time for the microbiological activity (Table 2). Table 2. DNA of different biocompounds Biocompunds PCL 6500

Age 1 Jahr

DNA-amount 1,8 mg/g

Denitrification few

PHBPCL 30_70 WB

1 Jahr

4 mg/g

Very good

PCL 6506 WB

7 monate

1,2 mg/g

little

The biocompound, which showed best denitrification activity (PCL 30_70WB) was also studied in batch experiment in order to know the ability to remove ammonium and nitrate. In the first experiment, the ammonium concentration was reduced from approximately 71 mg NH4+-N/l at the beginning to almost zero after 5 days (Figure 5). The maximum specific load removal was 29 mg NH4+-N/m2-d.

Figure 5. Ammonium concentration during the time in batch experiment.

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In the second experiment, four different concentrations of nitrate were evaluated (Figure 6). The denitrification process was clearly observed in all reactors and the nitrate removal occurred faster at low concentrations. The maximum specific load removal rate was in a range of 0.7 to 1.3 mg NO3--N/m2-d.

Figure 6. Nitrate concentration during the time in batch experiment.

CONCLUSIONS The presented results demonstrate the ability of biocompounds on the basis of poorly biodegradable PCL as matrix filled with unmelted readily biodegradable PHB particles to remove ammonium and nitrate under the batch conditions. The Biocompound reached the maximum denitrification rate after 1 year of operation.

ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the Federal Ministry of Education and Research (BMBF-02WT0798).

REFERENCES Anton, W., Jank, M., Schnabel, R., Ochmann, C., 2002. Biocompounds as carrier material for complex wastewater treatment [Biocompounds als traegermaterial zur komplexen Abwasserreinigung]. KA – Wasserwirtschaft, Abwasser. Abfall 49 (9), 1222-1227. Boley, A., Müller, W.R., Haider, G., 2000. Biodegradable polymers as solid substrate and biofilm carrier for denitrication in recirculated aquaculture systems. Aquacultural Engineering 22 (1-2), 75-85.

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Hiraishi, A., Khan, S,T., 2003. Application of polyhydroxyalkanoates for denitrification in water and wastewater treatment. Appl. Microbiol. Biotechnol. (61), 103-109. Honda, Y., Osawa, Z., 2002. Microbial denitrification of wastewater using biodegradable Polycaprolactone. Polymer degradation and stability (76), 321-327. Walters,E., Hille, A., He, M., Ochmann, C., and Horn, H. (2009). Simultaneous nitrification /denitrification in a biofilm airlift suspension (BSA) reactor with biodegradable carrier material. Water Research Vol. 43, Issue 18, 4461-4468.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 26

THE CHANGES IN THE PROTEIN PROFILE IN RESPONSE TO CADMIUM STRESS IN FLAX (LINUM U SITATISSIMUM L.) Milena Szalata1*, Szalata Marlena †2 and Wielgus Karolina 1

Department of Biotechnology, Institute of Natural Fibres and Medicinal Plants, Poznań, Poland ²Department of Biochemistry and Biotechnology Poznan University of Life Sciences, Poznań, Poland

ABSTRACT Germination of flax (Linum usitatissimum L.) in the presence of cadmium induced the changes on the level of synthesized peptides or proteins. The elution process, using ion exchange chromatography, revealed presence of proteins/peptides rich in cysteine residues in the extract from seedlings growing in cadmium solution, which were not observed in the extract from seeds germinated in the control conditions.

Keywords: flax, cadmium, phytochelatins, phytoremediation

INTRODUCTION In many regions content of heavy metals in the soil is an environmental problem due to exceeding pollution limit, what excludes the usage of crop in food production. The possibility *

Department of Biotechnology, Institute of Natural Fibres and Medicinal Plants, ul. Wojska Polskiego 71B, 60-630 Poznań, Poland tel. (+48 61) 8455831, fax (+48 61) 8417830. † Department of Biochemistry and Biotechnology Poznan University of Life Sciences, ul. Wołyńska 35, 60-637 Poznań, Poland tel. (+ 48 61) 8487202, fax (+48 61) 8487211.

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of using plants for removing metals from the soil in the phytoremediation process, based on extraction, sequestration or decontamination, became an alternative to the soil restoration of polluted sites (Dary et al., 2010). Flax belongs to fibrous plants and its biomass depends on level of the heavy metal pollution. The earlier experiments revealed suitability of flax usage for phytoremediation purposes. Especially high accumulation and tolerance to cadmium contamination makes flax a valuable alternative as an energy crop (Jasiewicz and Antonkiewicz, 2003; Shi and Cai, 2009). The detoxification process comprise synthesis of peptides called phytochelatins or proteins called metallothioneins, which form complexes with metal ions (Li-Chan et al., 2002; Figueroa et al., 2008; Vázquez et al., 2009; Kavamura and Esposito, 2010; Yadav, 2010).

AIM AND BACKGROUND The aim of the study was determination of changes of the proteins/peptides profile induced by cadmium stress in flax (Linum usitatissimum L.). Plants developed mechanisms to protect against heavy metals such as: transport system of toxic elements to specific compartments or synthesizing peptides that bind them (Figueroa et al., 2008; Vázquez et al., 2009). The detoxification process includes chelation of metal ions and subcellular compartmentalization. Metal binding proteins are low molecular weight metal-thiolate peptides or proteins, which compounds belong to the class of phytochelatins (oligomers of glutathione) or class III metallothioneins (low molecular weight cysteine rich proteins) (LiChan et al., 2002). The main heavy metal chelators produced during the exposure to pollutants are phytochelatins (Kavamura and Esposito, 2010; Yadav, 2010). The phytochelatins belong to the family of small Cys-rich polypeptides and form complexes with heavy metal in the cytosol and transport ions into the vacuole. Plants exposed to the high level of cadmium (Cd) showed visible symptoms of chlorosis, growth inhibition, browning of root tips and finally death (Vadas and Ahner, 2009ś Barałkiewicz et al., 2009ś Yadav, 2010). The term ‗hyperaccumulators‘ defined plants with biological mechanism that permit to accumulate high amount of wide range of heavy metals. Generally, hyperaccumulators should have ability not only to tolerate high concentration of metals in their tissue but also to produce relatively high biomass. The standard amount of cadmium hyperaccumulation is 100 mg/kg of dry mass in shoots (Aibibu et al., 2010; Zhang et al. 2010). Cadmium is easily transferred from the polluted soil to the food chain through plants and it is toxic not only to plants but also to animals and humans (Shi and Cai, 2009). Plants used for phytoremediation cannot be used for food production purposes. The alternative is exploitation of contaminated plants as energy crops for biodiesel production. The study conducted on flax revealed high accumulation and tolerance to cadmium pollution. The application of genetic engineering for obtaining of transgenic plants such as Nicotiana tabaccum, Arabidopisis thaliana and Linum ussitatisimum with enhanced metal tolerance, is a very useful tool (Abhilash et al., 2009; Najmanova et al., 2007). Plant tissue cultures (callus, cell suspension or hairy roots) constitute an experimental model in studies on phytoremediation, which can be used to examination of selected plants in specified

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conditions, what reduces cost in comparison with conventional plant cultivation (Doran, 2009).

EXPERIMENTAL PART Flax seeds (Linum usitatissimum L.) of Cristal cultivar were germinated (50 seeds per Petri dish of 11 cm in diameter) on filter papers moistened with distilled water (control) or in the test solution (0.5 mM Cd(NO3)2) at 21°C in darkness. The experiment was carried out according to the modified method of Yoshida et al. (2006). The two-week-old seedlings were homogenized with 20 mM Tris/HCl buffer (pH 8.8) and the homogenate was centrifuged at 10,000 x g for 60 minutes. The proteins/peptides were purified by 1-step and 3-step ammonium sulphate saturation (0-30% and 0-30%, 30-50% and 50-80% respectively). After centrifugation, the supernatant was dialyzed against 20 mM Tris-HCl buffer (pH 8.8). Anionexchange chromatography on DEAE Cellulose equilibrated with 20 mM Tris/HCl buffer (pH 8.8) was performed. Proteins were eluted with a step gradient of NaCl (respectively 0.2, 0.3, 0.4, 0.5, 0.6 and 0.8 M NaCl) and 1.5 ml fractions were collected. The proteins/peptides content was examined in all fractions by measurements of absorbance, at 280 and 254 nm respectively.

RESULTS AND DISCUSSION The conducted experiment did not reveal differences in seeds germinated in the presence of the distilled water (control) or in the 0.5 mM Cd(NO3)2 test solution. Similar results were obtained by Belkhadi et al. (2010), who showed that flax plants can tolerate Cd concentrations higher than 100 μM without showing any visible toxicity symptoms. The proteins/peptides (Cys-rich) were extracted from two-week-old seedlings and purified by 1step (Figure 1) and 3-step (Figure 2) ammonium sulphate saturation (0-30% and 50-80%). The elution process, using ion exchange chromatography, revealed presence of proteins/peptides rich in cysteine residues in the extract from seedlings growing in cadmium solution, which were not observed in the extract from seeds germinated in the control conditions (absorbance 280 and 254 nm). These proteins/peptides were eluted in three extract fractions by 0.3 M, 0.4 M and 0.5 M NaCl gradient, respectively. The presence of the (Cysrich) proteins/peptides in the extract from seedlings growing in the 0.5 M Cd(NO3)2 solution could suggest formation of these proteins/peptides in response to the cadmium stress. The largest absorbance value of the proteins/peptides rich in the cysteine residues was observed at the extract fraction eluted by 0.4 M NaCl gradient and was probably related to seed germination in the test solution containing cadmium. The existence of different cadmium binding protein fractions eluted at 0.10, 0.25 and 0.50 M NaCl was observed by the team of Oomah (2007) in flax cultivated at three locations in Manitoba. Also Li-Chan et al. (2002) revealed presence of phytochelatin-like complexes eluted at 0.45 M and 0.5 M NaCl gradient.

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Figure 1. Elution profile (DEAE Cellulose) of proteins/peptides extracts from flax seedlings cultured in the absence or presence of 0.5 mM Cd(NO3)2 solution after 1-step ammonium sulphate saturation (030%) and dialysis.

Figure 2. Elution profile (DEAE Cellulose) of proteins/peptides extracts from flax seedlings cultured in the absence or presence of 0.5 mM Cd(NO3)2 solution after 3-step ammonium sulphate saturation (030%, 30-50% and 50-80%) and dialysis.

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CONCLUSIONS The experiment did not reveal difference in seeds germinated in control conditions and in the solution containing cadmium. Respective proteins/peptides rich in the cysteine residue were observed in three fractions, eluted by NaCl gradient (0.3-0.5 M), what suggests formation of protein complexes in the presence of cadmium. The highest absorbance value, indicating the high concentration of proteins/peptides synthesised in stress condition, was observed in fractions eluted with 0.4 M NaCl at both extracts purified by 1-step and 3-step ammonium sulphate saturation. Further research should allow to characterize changes in the proteins/peptides profile in response to cadmium stress.

REFERENCES Abhilash P.C., Jamil S., Singh N. Transgenic plants for enhanced biodegradation and phytoremediation of organic xenobiotics. Biotechnol. Adv. 27, 2009, 474-488. Aibibu N., Liu Y., Zeng G., Wang X., Chen B., Song H., Xu L. Cadmium accumulation in vetiveria zizanioides and its effects on growth, physiological and biochemical characters. Bioresour. Technol. 101, 2010, 6297-6303. Barałkiewicz D., Kózka M., Piechalak A., Tomaszewska B., Sobczak P. Determination of cadmium and lead species and phytochelatins in pea (Pisum sativum) by HPLC–ICP-MS and HPLC–ESI-MSn. Talanta 79, 2009, 493-498. Belkhadi A., Hediji H., Abbes Z., Nouairi I., Barhoumi Z., Zarrouk M., Chaïbi W., Djebali W. Effects of exogenous salicylic acid pre-treatment on cadmium toxicity and leaf lipid content in Linum usitatissimum L. Ecotoxicol. Environ. Saf. 73, 2010, 1004-1011. Dary M., Chamber-Pérez M.A., Palomares A.J., Pajuelo E. ―In situ‖ phytostabilisation of heavy metal polluted soils using Lupinus luteus inoculated with metal resistant plantgrowth promoting rhizobacteria. J. Hazard. Mater . 177, 2010, 323-330. Doran P.M. Application of Plant Tissue Cultures in Phytoremediation Research: Incentives and Limitations. Biotechnol. Bioeng. 103, 2009, 60-76. Figueroa J.A.L., Wrobel K., Afton S., Caruso J.A., Gutierrez Corona J.F., Wrobel K.: Effect of some heavy metals and soil humic substances on the phytochelatin production in wild plants from silver mine areas of Guanajuato, Mexico. Chemosphere 70, 2008, 2084-2091. Jasiewicz C., Antonkiewicz J. Assessment of common flax (Linum usitatissimum L.) usability for phytoremediation of soil contaminated with heavy metals. Ecol. Chem. Eng. 10(9), 2003, 901-907. Kavamura V.N., Esposito E. Biotechnological strategies applied to the decontamination of soils polluted with heavy metals. Biotechnol. Adv. 28, 2010, 61-69. Li-Chan E.C.Y., Sultanbawa F., Losso J.N., Oomah B.D., Mazza G., Characterization of phytochelatin-like complexes from flax (Linum usitatissimu) seed. J. Food Biochem. 26, 2002, 271-293. Najmanova J., Mackova M., Macek T., Kotrba P. Preparation of transgenic flax with enhanced metal tolerance. Abstr. / J. Biotechnol., 131S, 2007, S38-S39.

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Oomah B.D., Berekoff B., Li-Chan E.C.Y., Mazza G., Kenaschuk E.O., Duguid S.D. Cadmium-binding protein components of flaxseed: Influence of cultivar and location. Food Chem. 100, 2007, 318-325. Shi G., Cai Q. Cadmium tolerance and accumulation in eight potential energy crops. Biotechnol. Adv. 27, 2009, 555-561. Vadas T.M., Ahner B.A. Cysteine- and glutathione-mediated uptake of lead and cadmium into Zea mays and Brassica napus roots. Environ. Pollut. 157, 2009, 2558-2563. Vázquez S., Goldsbrough P., Carpena R.O. Comparative analysis of the contribution of phytochelatins to cadmium and arsenic tolerance in soybean and white lupin. Plant Physiol. Biochem. 47, 2009, 63-67. Yadav S.K. Heavy metals toxicity in plants: An overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants. S. Afr. J. Bot. 76, 2010, 167-179. Yoshida N., Ishii K., Okuno T., Tanaka K.: Purification and Characterization of CadmiumBinding Protein from Unicelluar Alga Chlorella sorokinian. Curr. Microbiol. 52, 2006, 460-463. Zhang X., Xia H., Li Z., Zhuang P., Gao B. Potential of four forage grasses in remediation of Cd and Zn contaminated soils. Bioresour. Technol. 101, 2010, 2063-2066.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 27

REGIONAL VALUE ADDED OF BIOENERGY ACTIVITIES A MATERIAL FLOW APPROACH Ruth Offermann , Thilo Seidenberger1 and Daniela Thrän 1,2 1

German Biomass Research Centre (DBFZ), 04347 Leipzig, Germany 2 Helmholtz Centre for Environmental Research (UFZ), 04347 Leipzig, Germany

ABSTRACT More than other renewable energies, biomass offers an opportunity for rural areas to establish sustainable regional development strategies. The increasing regional use of biomass does not only contribute to a reduction of energy related greenhouse gas emissions, but also can mean an attractive option for a region‘s economic development. In the context of the competition ‗bioenergy regions‘ launched by the Federal Ministry of Food, Agriculture and Consumer Protection, currently 25 model regions all over Germany aim to realise the development potentials of biomass use by establishing efficient regional networks. The progress of the individual regions is monitored and evaluated by accompanying research activities. A major focus of the technical-economic research is the estimation of regional economic impacts triggered by bioenergy activities.

INTRODUCTION AND BACKGROUND In economics gross value added expresses the ―additional value of goods and services that are newly created [during the accounting period] in the economy and are available for domestic final uses or for exports‖[1]. Generally, gross value added is defined as

Gross Value Added  Output - Intermediate Consumption 

Corresponding author, e-mail: [email protected], tel.: +49(0)341-2434-453.

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where output is the value of goods and services produced in the economy, and intermediate consumption is the cost of goods and services used in production. According to this definition regional value added can be understood as

Regional Value Added  Regional Output - External Intermediate Consumption [2]. The components of value added include all costs directly related to the production of a good or service. These are the compensation of employees, taxes less subsidies on production, consumption of fixed capital, rents and interest payments for land and subsoil assets used for the production. Costs for primary products or energy are not part of value added /1,3/. Outputs are valued at market prices or production costs in case there is no market price. While on national level the calculation of value added is a comprehensively specified procedure, no such standards exist for the estimation of regional value added. In our particular case, we also concentrate on the regional value added related to bioenergy activities rather than looking at the whole regional economy. The potential economic benefit a region could realise by developing its renewable energy generation capacities is currently widely discussed. In energy politics, the support of regional renewable energy initiatives is seen as an investment in a sustainable energy economy but also as a means to strengthen the economic situation of rural areas. Regional effects of renewable energies are especially accentuated related to industry clusters (i.e. Solar Valley Mitteldeutschland), or the development of decentralised energy generation structures including the operation of independent plants, the establishment of local resource markets and the extension of regional value added chains. Of all renewable resources, bioenergy provides the highest potential of developing regional energy supply structures in the latter sense. Unlike other renewable energies like wind and solar the supply of bioenergy also involves the procurement of fuels. Bioenergy resources in the majority are closely linked to agriculture and forestry; sectors that are of high economic meaning particularly in rural areas. Longdistance transport of most of these resources is hardly economic due to high water contents and relatively low energy densities of the fuels. A wide variety of different bioenergy technologies exist, ranging from more centralised facilities with a high amount of daily fuel input required down to decentralised applications with rather small-scale plants. Particularly small-scale up to middle-scale facilities offer the opportunity of integrating a high amount of local actors and organising multi-step regional value added chains. In the context of the competition Bioenergy Regions the German Federal Ministry for Food, Agriculture and Consumer Protection supports the development of 25 regional bioenergy networks in rural areas. These networks aim to promote bioenergy projects in their regions in order to realise regional development potentials related to an optimised use of bioenergy resources. In this regional context, bioenergy is first regarded as a means to increase the economic performance of rural areas but also as a contribution to a more sustainable energy system. Asked about the chances of their participance in the competition the majority of regions mentioned value added at first, followed by aspects like the creation of an independent and decentralised energy structure and the enhancement of regional identification with both considerably less mentions (Figure 1).

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Figure 1. Survey results of chances related to the bioenergy regions competition.

The competition is accompanied by interdisciplinary research activities including the analysis of social processes but also techno-economic aspects. The analysis of regional value added represents a central research activity within this context.

APPROACH In the past, only few estimations of regional value added directly related to bioenergy have been prepared. However, the available research used different methodologies for dealing with the problem. Top-down approaches like the construction of regional input-output tables represent mature economic procedures that enable various analysis possibilities but require extraordinary high efforts. Beyond that, data compilation is generally challenging as business figures are required that companies only unwillingly hand out. Besides top-down approaches, also bottom-up methodologies like material flow analysis have been used for estimating regional value added. However there is no standardised procedure of creating such analyses and these instruments are often not specifically made for economic analyses. In case there is sufficient access to data however meaningful results can be generated. In our case we aim to measure bioenergy activities in all 25 regions that take part in the competition Bioenergy Regions. These regions are spread all over Germany and are extremely heterogeneous regarding size, experiences with the use of bioenergy, ambitions and targets, size of agriculture and forestry sectors, number and function of persons involved in bioenergy activities, and many other aspects. Based on the relatively high number of observed regions, we initially disregarded top-down approaches for being too time-consuming. Given the opportunity of direct contact to the regions, we considered a largely standardised bottomup material flow analysis to be most appropriate to address the individual regional specifications while also achieving a high level of comparability. The approach (Figure 2)

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first involved a qualitative analysis of regional value added chains, including relevant segments, actors and possible indicators for each step of the chain. Based on this analysis standardised supply chains were formulated that were specified with the help of regional data. In a next step, principle actors of these chains, namely bioenergy plant operators and fuel producers were asked about major plant characteristics, the amount, type and origin of resources used and the energy output of their facilities. These data was used to construct major regional biomass flows and if monetized can also be used for a first assumption of regional financial flows. Further surveys concerning business figures however are required in order to state more precisely the effect of bioenergy on regional value added. During the competition, two repetitions of the survey are planned in order to monitor the effects of the bioenergy regions competition. Following from the material flow analysis, additional surveys concerning financial figures are planned to be carried out in selected regions. These are accompanied by secondary data collections so that finally the material flows can be valuated. Qualitative analysis of value added chains Development of standardised regional value added chains

Analysis of regional development concepts

Regional specification of value added chains

Validation of value added chains (regional survey)

Quantitative analysis of value added chains Status quo assessment (12/2009) Identification of plant operators and fuel producers (regional survey)

Survey of plant operators and fuel producers

Identification of regional biomass flows

Intermediate survey (12/2010) Analysis of changes Final survey (2012)

Valuation of regional biomass flows Survey of selected regions concerning financial figures

Secondary data (national statistics, etc.)

Figure 2. Approach to assess regional value added using material flow analysis.

REGIONAL VALUE ADDED CHAINS Relevant information was initially obtained from regional development concepts which each region had to formulate during the selection process of the competition. These concepts all had to include a chapter on the status quo bioenergy value added chains. Furthermore the regions named their measures and planned improvements of the mentioned value added chains. These data were used to construct bioenergy added value chains that are standardised but still can be adjusted to regional specifications. Validating these value added chains a survey was carried out in which each region was asked to verify the corresponding value added chains. Nearly every region described planned measures in the fields of biogas and energetic wood use. We thus separated the fuel characteristics. In this way, three different value chains

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were developed: wood (solid biofuels), biogas and a general one enabling the integration of also liquid biofuels or mixtures of agriculture and forestry resources. All in all we assumed the value added chains to consist of six independent steps including the major processes relevant to the creation of value added. According to the information given in the regional development concept the value chains were regionally specified. The validation in form of a survey of the regional managers again revealed that there are substantial differences between all regions. While some can largely base upon already existing experience and structures others only are at the beginning of a process. This is illustrated in Figure 3 where wood value added chains of two regions are compared. The second value added chain is characterised by a much more diversified resource base and also other segments of the chain are more developed than in the first one.

Figure 3. Wood value added chains of two different regions.

ANALYSIS OF MATERIAL FLOWS In all regions a survey was conducted asking for contacts to plant operators and fuel producers that are involved in activities related to the bioenergy region network. It is necessary to point that out as one of the objectives of the research is the investigation of network related effects. The plant operators and fuel producers again were contacted with a standardised written survey. The survey included questions about general technical parameters of the plant, output quantities, resource use, origin of resources and fuels and also questions concerning the attitude of towards the bioenergy region activities. The information collected was recorded in a database. Figure 4 shows the biomass flows of a selected region based on the collected data. In the region shown, biogas plants as well as heating plants and CHP plants are operated. Out of all regions the selected one had a relatively high number of local actors involved in their activities. Most of the substrates and wood fuels used are currently supplied from inside the region. In total, according to the survey data currently 11.7 GWh electricity are fed in per year while only 6.6 GWh of heat are used per year. Most

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likely though, the region does not fully realise their potentials, as a high proportion of heat produced in biogas and CHP plants can assumed to be currently wasted. Thus the obtained data allows a first very rough estimation of the region‘s bioenergy performance. However, large data uncertainties remain. The electricity and heat output of the CHP plant for example is not known, thus not included. Furthermore, only five out of seven heating plants provided figures of heat use and resource use. Data on biogas plants however is of much better quality as the respective respondents provided relatively comprehensive data. However, a continuous improvement of the data base is required.

Figure 4. Biomass flows of a selected region.

CONCLUSION Given a sufficient data base regional value added of bioenergy activities is possible to be estimated. Material flow analysis can provide good insight into the ongoing biomass activities of a region. The approach of analysing value chains in qualitative way first allow a first assessment of bioenergy activities and also functions as a plausibility instrument for the following material flow assessment. However, even if there is relatively good data access, information will hardly be complete making the result error prone. For being an instrument that is powerful and allows comparability between regions high efforts are required for achieving equivalent good data quality for all 25 regions. One option would be to concentrate on only a selection of regions however this would lower the meaning of results. In order to be able to calculate regional value added further improvements of the data base are certainly necessary. The calculation in itself according to its strict economic definition is only of marginal difficulty. What makes it most complex is the collection of data. As the regions are very heterogeneous an equivalent quality of regional data is hard to achieve. Particularly the illustration of very small regions is challenging. However, especially in these cases bottom-up approaches like material flow analysis have a clear advantage compared to top-down approaches. Regional specifications can be better measured and regional contacts may enhance the possibility of completing the data base. Furthermore, the

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material flow approach allows monitoring changes over time. All in all, the approach principally seems suitable, but improvements of the data base are required.

REFERENCES [1]

[2]

[3]

United Nations Department of Economic and Social Affairs: National accounts: A practical handbook. (http://unstats.un.org/unsd/nationalaccount/ handbooks.asp), United Nations: New York, 2003. Agentur für Erneuerbare Energien e.V.: Regionale Wertschöpfung durch die Nutzung Erneuerbarer Energien: Hintergrundinformationen. (http://www.unendlich-vielenergie.de/de/wirtschaft/detailansicht/article/16/ regionale-wertschoepfung.html), Berlin, 2009. Samuelson, P.A. und Nordhaus, W.D.: Volkswirtschaftslehre. (ISBN: 3636031120, 9783636031129), mi-Fachverl., 2007.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 28

SIMPLE KINETICS OF METHANE FORMATION USING MODEL SUBSTRATES: A SHORT DISCUSSION Robert Reinhard Pätz and Jan-Henryk Richter-Listewnik Innovation Laboratory Biotechnology Anhalt University of Applied Sciences Bernburger Straße 55, D-06366 Köthen/Anhalt

ABSTRACT In opposite to complex kinetics for anaerobic digestion a simple first-order kinetic is used to describe methane formation of different carbohydrates, proteins and oils. Rate values demonstrate that carbohydrates have high carbon dioxide contents at the beginning of the biogas production but methane formation rate is similar to proteins. Thus a twostep process should be best way to increase methane content.

Keywords: Carbohydrate, carbon dioxide, formation rate, two-step process

1. INTRODUCTION Biogas is one of the most important energy sources of the future of mankind. Its advantages are the possible use of natural resources like plant material as well as organic wastes and organic wastewaters from households, agriculture and food industry as substrates and the production of energy and fertilizer for sustainable plant production. But to use biogas effectively in technical scale there is the need of optimization of the methane formation procedure. One of most important problems is an optimized substrate mixture because different substrates give different quantities of biogas with different content of methane. There are a lot of papers with results of investigations of biogas yield and resulting methane content. The application of these values is only possible for long retention times in a one step process. But effective process arrangements need small reactors with short process times as hydraulic and sludge retention times. Under these circumstances the different gas formation

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of carbon dioxide and methane has to be considered. And even this requires better understanding of formation of the two gases and its formation rate development.

2. AIM AND BACKGROUND There are a lot of different models to describe methane formation. The simplest model is a two-step model with hydrolysis and acid formation to give methanogenic substrates in first step and methane formation in second step. All different substrates yield in one product, methane, independently from kind of substrate and intermediates. For the case that the methane formation is the rate-determining step one can assume a simple first-order kinetic as follows ([1], [2], [3]) S→C

(1)

with S as Substrate and s as substrate concentration and C as methane-carbon and c for methane concentration in mol L-1 or in mass concentration as g L-1 respectively. The firstorder rate in integrated form yields eq. 2 -kt

s(t) = s0 e

(2)

with s0 for initial substrate concentration and k as rate constant. Resulting for methane is equ. 3 and equ. 4 c(t) = s0 - s(t) c(t) = s0(1- e-kt)

= s0 - s0 e-kt

(3) (4)

Considering the fact that relative molar masses from methane and carbon dioxide are different there are differences in mass and volume development like demonstrated in figure 1.

Figure 1. Biogas formation as volume (red points) and as mass (blue points) over time. Crosses demonstrate methane mass development.

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Linearization results in equ. 5 ln[1 - c(t) /s0] = - k t

(5)

The term c(t) /s0 is the specific methane mass related to the initial substrate mass. It can be calculated by the specific biogas yield converted with methane content and calculated as mass. Figure 2 shows an example for calculation of k-value using this method.

Figure 2. Determination of rate constant k as shown in eq. 5.

3. EXPERIMENTAL PART / RESULTS Organic Mass For determined model substrates organic mass is calculated from molar composition as total organic mass. Dry matter of model plant material is determined gravimetri-cally and organic composition by incineration.

Biogas Formation Biogas formation is determined by VDI-guideline 4630 (German method) using Eudiometer. Methane and carbon dioxide content is measured with…

4. RESULTS AND DISCUSSION 4.1. Carbohydrates as Substrate Carbohydrates are hydrolysed to mono- and disaccharides and immediately acidified. Some of possible acidification reactions are

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Robert Reinhard Pätz and Jan-Henryk Richter-Listewnik Acidification (examples) Glucose 180 kg

... Acidic acid AA + (2) CO2 + H2 2* 22,4 m3

(6)

Glucose

… Butyric acid BS+

(7)

Glucose

… Lactic acid LA

 Propionic acid PA

(8)

BS, PS

... AA +

CO2 + H2

(9)

CO2 + H2

… AA

CO2 + H2

(10)

Methane formation (examples) AA

 CH4+ CO2

(11)

CO2 + H2

 CH4 + ..

(12)

As shown in these equation most of acidification reactions are connected with carbon dioxide formation, in case of acidic acid with two mols for acid formation and one mol for methane. Thus acidification step yields high amount of CO2 in biogas. Figure 3 demonstrates the fact.

Figure 3. Carbon dioxide mass and methane mass in g per 1000 g organic substrate during anaerobic digestion of carbohydrate.

Biogas formation rate seems high but real methane formation is not so fast. This is shown in figure 4, where carbon dioxide mass and methane mass is measured for amylose as total soluble carbohydrate.

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Maximum value of CO2 is about 80 % of gas after 2 to 4 days. Then methane content increases whereas CO2 content decreases. For that case that biogas and CO2 are separated out methane content may increase to 70 % and more. Other compositions written in papers are results of final determination without gas exchange. For technical application with carbohydrate as substrates it seems to be favourably to have a first reactor for CO2-rich gas production and a methane reactor.

Figure 4. Gas composition during amylose digestion over time (in h); black line is air.

4.2. Proteins as Substrates Amino acids are the product of hydrolyses of proteins. These amino acids must be deammonified to get organic acids as methanogenic substrates. These deammoni-fication reaction forms not only ammonia, but also carbon dioxide. Ammonia and some organic acids were used to produce microbial biomass. Other ones contribute to formation of methane. The resulting biogas composition for yeast extract as model substrate is shown in figure 5. Carbon dioxide is also formed in excess, but later on the methane formation is faster and higher than in case of carbohydrate substrates.

Figure 5. shows the gas composition (in %) for anaerobic treatment of yeast extract as model substrate over time (in hours).

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4.3. Fats and Oil According to theory the fatty acids as products of hydrolyses of fats and oil will under-go the beta-oxidation with formation of acetic acid. This is immediately a methano-genic substrate without any formation of carbon dioxide as by product. After that the methanogenes yield carbon dioxide and methane in same quantities. The use of sun-flower oil as model substrate agrees with this theory as it is shown in figure 6.

Figure 6. Gas composiotion for sunflower oil as model substrate.

4.4. Methane Formation Rate As described the rate constant is determined using eq.5. Table 1 summerizes the results for used model substates. Table 1. k-values of different model substrate in h-1 Substrate Glucose Amylose Microcrystalline cellulose Potato starch, raw Yeast extract Pepton Sunflower oil

k-value in h-1 0,011 0,029 0,035 0,023 0,030 0,047 0,021

There are some important results: a)Glucose seems not to be best substrate for methane formation b)Carbohydrates and proteins show similar rates for methane formation

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c)Plant material as substrates(e.g. raw potato starch) shows similar rates like model substrates

CONCLUSION The methane formation rate shows opposite results to biogas formation rates. The fastest formation is when applying proteins. Carbohydrates gives more biogas but with a high content of carbon dioxide at the beginning of biogas formation. Oils and fats when well distributed in reactor have similar methane formation rate but with a low content of carbon dioxide. For technical application there should be a necessary retention time for methane formation without new covered storage tanks. To determine this time it is important to make a mass balance using rate constants. For a continuous stirred tank reaction mass balance can be as follows V ds(t)/dt = Q0s0 – Q0 s(t) + V k s(t)

(13)

Q0 as influent substrate volume in m3 h-1, s0 substrate mass in influent in kg m-3 and V is reaction volume in m3. Hydraulic retention time th is defined as th = V/ Q0

(14)

For continuous cultivation conditions there is a direct correlation between th, substrate input and metabolic rate U (eq.15 and 16 resp.) th = (1/k)[(s0 – s(t))/s(t)]

(15)

th= U/[k(1-U)]

(16)

ACKNOWLEDGMENT All works were done by Tobias Schwalenberg and Anett Rennert

REFERENCES [1] [2]

LINKE, B. , P. MÄHNERT. Biogasgewinnung aus Rindergülle und nachwachsenden Rohstoffen. Agrartechnische Forschung 11(2005) Heft 5, S. 125 -132. CHEN, Y.R. Kinetic analysis of anaerobic digestion of pig manure and its design implication; Agricultural Wastes 8(1983) , 65 – 81.

278 [3]

Robert Reinhard Pätz and Jan-Henryk Richter-Listewnik HASHIMOTO,A.G.Methane from cattle waste; Effects of temperature, hydraulic rerention time, and influent substrate concentration on kinetic parameters. Biotechnol. Bioeng. 24 (1982),Pp. 2039 – 2052.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 29

BIOSAFETY OF TRANSGENIC POTATOES PRODUCING THE BIOPOLYMER CYANOPHYCIN Christoph Unger, Maja Hühns and Inge Broer Institute for Land Use – Agrobiotechnology and Applied Research in Bio- and Gene-Technology, Faculty for agricultural and environmental Science, University of Rostock, Justus-von-Liebig-Weg 8, 18059 Rostock, Germany

ABSTRACT Transgenic plants have been proposed to substitute several biopolymers generated so far from fossil resources. Broad acceptance of GMPs (genetically modified plants) in the public opinion can only be achieved by a careful analysis of their environmental interactions. In particular, our research examines the survival of transgenic potatoes functioning as bioreactor for cyanophycin synthesis over the winter period to exclude unintentional propagation in the ecotone.

Keywords: polyaspartate, polycarboxylate, cyanophycin, transgenic plants, biological safety, genetically modified plants (GMP)

INTRODUCTION The generation of transgenic plant opens a wide range of interesting possibilities for the generation of ‚bioplastics‗. Polycarboxylate is a non-biodegradable polymer with a worldwide consumption of 265.000 t/a (1997). A substitution with a biodegradable polymer can be achieved by replacing the polycarboxylates with chemical synthesized polyaspartate. Polyaspartate is a liquid polymer with several applications. It is already used to substitute polycarboxylates which have a broad range of industrial and agricultural applications, e.g. as dispersants, thickeners or additive to hydrogels (Oppermann-Sanio et al. 1999; Schwamborn, 1996). Up to now pure polyaspartate is produced only by chemical synthesis from 3-4

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manufactures worldwide with a scale up to 26.500 tons per year. In agriculture application of polyaspartate promotes the uptake of nutrients from soil and thereby the harvest and utilization of fertilizer. The Donlar Corporation received the ‗Green Chemistry Challenge Award‘ from President Clinton in 1996 in the USA for this product. Aspartate can also be used as starting substance for synthesis of different chemicals like 2-amino-1,4-butandiole, 3aminotetrahydrofurane, fumaric acid (used as polyester resin) and acrylamide (used as thickener, manufacture of dye stuffs or paper manufacture). Although the use of polyaspartate is very attractive, its chemical synthesis is very expensive compared to polyacrylate keeping the market volume for biodegradable polyaspartate low. An alternative source for polyaspartate is the microbial storage protein cyanophycin (multi-L-arginyl-poly-L-aspartic acid). Cyanophycin is composed of a poly-αaspartic acid backbone to which arginine residues are connected via isopeptide bonds. As CO2 - neutral strategy the gene of the cyanophycin synthetase from the cyanobacterium Thermosynechococcus elongatus BP-1 was introduced into the genome of potatoes (Neumann et al., 2005). Subsequently, the polymer producing enzyme was combined to a tuber specific promoter for a cyanophycin production basically in the potato tubers reaching contents up to 7.5% of the dry matter (Hühns et al., 2009). The second product present in cyanophycin – arginine - has an even higher market potential than polyaspartate. Primary, arginine is used in food- and pharmaceutical industry. Additionally arginine can be converted into different chemicals such as 1,4-butandiamine, which is used for nylon synthesis.

Polyacrylate

Polyaspartate

Arg

Arg

Arg

Cyanophycin

Figure 1. Chemical structure of polyacrylate, polyaspartate and cyanophycin.

PROJECT DESCRIPTION AND CONCLUSIONS Before commercial cultivation of transgenic potatoes, field studies are required to ensure the environmental safety of the GMPs. Due to the harvesting machines about 3 to 5 % of the potato tubers remain as ground keepers on the field, but in Northern Europe potato plants are not able to establish in the ecotone. Since integration of the cyanophycin production machinery might interfere with the carbohydrate metabolism it is possible that the amount of small sugar molecules serving as C-skeleton for an enhanced aspartate and arginine synthesis is changed. Consequently, these higher amounts of soluble sugar could lead to changes in

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frost tolerance. Secondly, cyanophycin, originally produced in cyanobacteria, is not a very common compound in the field. It has to be analyzed whether cyanophycin released by rotting potatoes does accumulate in soil. With our project we want to answer two major questions: Do the GMP have higher potential to resist cold temperatures and further, is the released cyanophycin completely degraded by microorganisms in the field. To work as close as possible under field conditions an exact number of potato tubers containing different constructs were wrapped in a net and buried (Figure 2). Later, these wraps were dug out and the potato tubers were scored and than analyzed. Additionally to the scoring, physiological parameters like sugar content, phenols and protein content were tested to find a relationship to frost tolerance. The concentrations of small soluble sugar molecules, among other cell solutes, in cells are discussed to be responsible for enhanced frost tolerance. Therefore, the total amount of soluble carbohydrates was analyzed in the intercellular fluid and the intracellular compartment separately. To approach the second question concerning a normal rotting of frozen ground keepers we analyzed peroxidase activity and phenolic compounds from potatoes that were dug out after enduring several month in the soil during the winter season. Future work will show whether the cyanophycin accumulates or is degraded in the soil.

Figure 2. Pit with potatoes. The mesh width was chosen very large to enable soil biota to pass the net without interference.

Summarizing, the data will reflect the eco-toxicology of the cyanophycin producing potatoes. An additionally ongoing study will test the human toxicity in mice, rat, rabbit and pig. Together the presented results form a decision platform for permission or rejection for large scale cultivation of the cyanophycin producing potato plants.

REFERENCES Bohmert, K., Balbo, I., Steinbüchel, A., Tischendorf, G. and Willmitzer, L. (2002). Constitutive expression of the beta-ketothiolase gene in transgenic plants. A major obstacle for obtaining polyhydroxybutyrate-producing plants. Plant Physiology 128, 1282-1290.

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Hühns, M., et al. 2009. Tuber-specific cphA expression to enhance cyanophycin production in potatoes. Plant Biotechnol J., 7(9): p. 883-98. 4. Neumann, K., Stephan, D.P., Ziegler, K., Hühns, M., Broer, I., Lockau, W., Pistorius, E.K. (2005). Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants. Plant Biotechnol. J. 3, 249-258. Oppermann-Sanio, F. B. et al. (1999). Biochemistry of polyamide metabolism. Biochemical Principles and Mechanisms of Biosynthesis and Biodegradation of Polymers: Proceedings of the International Symposium (Steinbüchel, A. ed. ) 185-193. Schwamborn, M. (1996). Polyasparaginsäuren. Nachr. Chem. Techn. Lab. 44, 1167-1179.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 30

PROVISION PATHWAYS FOR BIOMETHANE Diana Weigl, Katja Oehmichen, Michael Seiffert*, Franziska Müller-Langer and Frank Scholwin German Biomass Research Centre non profit research company (DBFZ), Leipzig

ABSTRACT Within the context of sustainability, one of the targets is to increase the share of biofuels for mobile and stationary applications. On that background, the question comes up, which of the many different options for instance for the provision of transportation biofuels seem to be particularly promising in the medium and long term. While liquid biofuels such as biodiesel and bioethanol are commonly available at the market, this is not the case for gaseous biofuels yet. However, the increasing use of natural gas (e.g. transportation fuel, to generate heat and electricity) and the available infrastructure for distribution are advantageous prerequisites for the market implementation of biomethane. There are two conversion routes given for the provision of biomethane. Bio-chemical produced biomethane (upgraded biogas) is using mainly substrates with a low content of lignocelluloses, while thermo-chemical produced biomethane (known as Bio-SNG) is utilising solid biofuels like wood. The provision pathways are applicable in promising combinations, since their production paths base on different types of feedstock and vary in terms of the plant capacity. Within a comprehensive study, different biomethane options have been analysed in depth. Based on a technical analysis promising concepts have been identified and then assessed by specific technical (e.g. efficiency, functionality and RandD potential), economic (e.g. costs of biomethane production and distribution) and environmental (e.g. greenhouse gas emissions and mitigation potential) criteria. It can be summarised that the provision pathways for biomethane are promising options to produce an efficient and sustainable energy carrier for mobile and stationary applications.

*

German Biomass Research Centre non profit research company (DBFZ) Torgauer Straße 116, D-04347 Leipzig. Phone: +49-341 2434-445, Fax: +49-341 2434-133, E-Mail: [email protected].

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Keywords: Biomethane pathways, biogas, Bio-SNG, biomass fermentation, biomass gasification, transportation biofuels, LCA, costs

ABBREVIATIONS EEC PEC CIS EU DE SRC STP w/o PSA PWW FICFB AER EF FM Ar

end energy consumption primary energy consumption Commonwealth of Independent States European Union (Belgia, France, Germany, Italy, Luxembourg, Netherlans) Germany short rotation coppice standard temperature pressure without pressure swing adsorption pressure water wash fast internally circulating fluidised bed absorption enhanced reforming entrained flow fresh mass as received

INTRODUCTION Biomass is of particular interest in the ongoing discussion on sustainable mobility due to its advantages concerning e.g. climate relevance and security of energy supply. Within the framework of sustainability, main targets relating biofuels are among others (i) efficiency regarding system technology and economics, (ii) environmental and climate protection (e.g. GHG emission reductions) and (iii) energy supply security regarding biofuel potentials and available resources. Within the context of sustainability, one of the targets is to increase the share of biofuels for mobile and stationary applications. The increasing use of natural gas (e.g. as transportation fuel, for heat and electricity generation) and the development of the available infrastructure for distribution may lead to a higher degree of market implementation of biomethane in the future. As shown for the consumption of natural gas among different sectors in Germany from 2000 to 2030 (cf. Figure 1), the share of the sector ―private households‖ is relatively high (around 39 %) and remains constant throughout the years. In the sector „trade and commerce‖, a decrease of 17 % in 2030 compared to 2005 is observed. In contrast, the natural gas consumption will increase to approximate 150 % in the electricity sector. The present targets for biomethane are confronted to the prognoses for natural gas consumption. In the context of the integrated energy and climatic protection program (IEKP) and the definitions of gas net entrance regulation (GasNZV) the feed of biogas/biomethane into the natural gas grid is planned with a volume of 6 billion m³/a to 2020 and 10 billion m³/a up to the year

Provision Pathways for Biomethane

285

Development of natural gas use per sector in DE in PJ/a

2030. In this context, the us of biomethane is strengthened in the combined heat and power sector as well as fuel. The upgraded biogas and Bio-SNG can be applied in promising combinations. 3.500 3.000

EEC-private households

2.500

EEC-trade and commerce EEC-transport

2.000

EEC-industry 1.500

District heating

1.000

PEC-electricity Target biomethane

500 0 2000

2010

2015

2020

2025

2030

Figure 1. Development of (bio)methane demand in Germany [1].

BIOMETHANE PRODUCTION PATHWAYS There are two conversion routes for the provision of biomethane. The bio-chemical route using organic substrates with a low content of lignocelluloses as feedstock. It is based on the anaerobic fermentation of the substrate to produce biogas that is subsequently upgraded to natural gas quality. Second, the thermo-chemical route, using lignocellulosic feedstock, it is based on the gasification of the feedstock and the subsequent cleaning and conditioning of the gas to natural gas quality (Bio-SNG). The two biomethane pathways use different types of feedstock and vary in terms of plant capacity. Concerning the resource use, biomethane generation permits the utilisation of almost all significant biomass fractions (woody biomass, herbaceous biomass, agricultural residues, livestock waste, cereals, etc.). An overview about the single steps of the biomethane production, subsequent distribution and use is given in Figure 2. The generation technologies for biogas and Bio-SNG are distinct from one another in various aspects, including:      

suitable raw material principle and components of the procedure technical maturity and need for research and development output range residual materials and recycling options expenditure on gas purification for ensuring gas quality

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Biogas substrates

Solid biofuels

(e.g.energy crops, residues)

(e.g. woody, herbaceous)

Bio-chemical anaerobic digestion

Thermo-chemical gasification (e.g. fluidised bed, pyrolysis/ torrefaction + entrained flow)

(e.g. wet fermentation)

Gas clean-/conditioning (e.g. oil / alkaline wash, activated coal, ZnO)

CHP (heat / electricity)

(e.g. fluidised, fixed bed) Raw Bio-SNG

Raw biogas

Bio-SNG upgrading

Biogas upgrading (e.g. PSA, water wash, amine)

(e.g. acid / MEA wash, TEG drying, H2 membrane)

Biomethane

Biomethane

Biomethane feed in and distribution Feed in TransferDistribution G260 /G262 / station G685

© DBFZ, 2009

Synthesis: Methanation

(Energetic) biomethane use Fuel station DIN 51624

Mobility

Electricity / CHP

Heat

Bilder: DBFZ, Repotec, Google

Figure 2. Overview of biomethane options.

Table 1 shows basic characteristics of biomethane production options (from biogas and Bio-SNG). Biogas plants and gas treatment technology are already commercially available. In the comparison, the Bio-SNG production technology is currently not available in a commercial scale. Demonstration tests were realised in 2008 and in June 2009 a pilot and demonstration unit of 1 MW has started operation in Güssing / Austria. Commercial availability is expected in the medium term. Table 1. Comparison of characteristics for the biomethane production routes Biomethane Suitable raw materials

Biogas Biogas substrates (liquid, pasty) energy crops, residues

Raw materials demand (fresh) Transport distance raw materials Area specific biomethane yield State of technology

approx. 7,000 - 17,000 t per year and MW CH4, th 5 to 30 km for residues, energy crops up to approx. 100 km 3,000 to 4,500 m³ N / (ha a) (e. g. maize silage) Biogas plants and gas treatment technology commercial available

RandD demand

Further development of process integration; need for increased efficiency Germany: > 240 MW CH4, th EU: approx. 430 MW CH4,th (Main regions: Sweden, Netherlands, Switzerland) up to 20 MW CH4,th (up to 16 mn m³ N/a)

Installed overall plant capacity Germany / EU

(Expected) plant capacities

Bio-SNG Solid biofuels, predominantly wood (e. g. residues, energy crops), in the future probably also herbaceous biomass possible approx. 3,500 - 4,500 t per year and MW CH4, th Unimodal up to 150 km, adapted logistic concepts required; harbour locations unlimited 3,500 to 5,000 m³ N / (ha a) (e. g. SRC willow); lower for forest wood Gasification systems available for fossil fuels, 1st demonstration plant in 2008, commercial availability in the years to come Demonstration: overall chain, plant availability and reliability, use of approved system components Germany: EU: 1 MW CH4,th (Austria), plans in Switzerland and Sweden up to 300 MW CH4,th (up to 240 mn m³ N/a)

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287

Before it is possible to feed biogenous gases into the natural gas grid there are many procedural stages to go through. The cleaning and/or preparation of gases to natural gas quality as well as the pressurizing of the gas on the required grid pressure are necessary. Through feed-in of biomethane into the gas grid, there is decoupling between the areas of biomethane production (e.g. rural areas) and demand (e.g. urban areas). Currently, there are approximately 120 biomethane plants based on biogas, landfill gas and sewage gas in Europe. Biomethane already accounts for about 55 % of the overall use of methane in road transport in Sweden. The respective share in Switzerland is around 18 %. The technical biomass potential describes the share of available biomass for energy purposes under consideration of technical restrictions, i.e. including available production technologies, their efficiency and availability of suitable land, use competition. Out of the technical biomass potential (taking into consideration the regarded biomass fractions), the technical biomethane potential based on bio- and thermo-chemical conversion pathways can be determined considering the single conversion efficiencies of the regarded biomethane provision routes. In the year 2005 an annual technical biomethane potential of about 30 bn m³STP was foreseen in Germany. The estimated potentials for Europe (i.e. EU-28 and CIS) are about 300 bn m³STP/a. Until the year 2020 a rising energy crop potential is expected due to increasing area specific yields and available land for biomass production. For 2020 a technical biomethane potential of 39 bn m³STP/a can be applied in 2020 in Germany (i.e. approx. 34 % of the total German natural gas demand). For Europe a technical biomethane potential of approx. 480 bn m³STP per year can be expected in the year 2020. Figure 3 gives an overview on the development of the technical biomethane potential, classified by the regarded feedstock and geographical area. 320 Bio-SNG base Biogas base

240

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Industrial wood residues

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Figure 3. Technical biomethane potentials in 2005 and 2020 [2].

ASSESSMENT OF BIOMETHANE CONCEPTS Promising concepts for the provision of biomethane were identified by a technical analysis. These concepts were subsequently by specific technical, economic and

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environmental criteria assessed in order to identify significant advantages and disadvantages for a potential market implementation of biomethane.

1.1. Biomethane Concepts The objective of the development of different provision concepts is to illustrate the improvement and optimisation potential of existing biomethane pathways concerning feedstock variety, biomass conversion as well as subsequent gas-treatment. The regarded plant concepts differentiate in terms of feedstock (e.g. biogas - increase of bio waste share over the time; Bio-SNG - integration of short rotation wood and straw over the time), process components (e.g. biogas – differ in the gas cleaning systems; Bio-SNG – vary in the gasifier types) as well as plant capacity (e.g. rise of the plant capacity). The time horizons investigated are the status 2005 (Status), short term 2010 (ST), medium term 2020 (MT) and long term 2030 (LT). Table 2 summarises the main characteristics of the concepts. Table 2. Overview of biomethane concepts Biomethane Status 2005 (H gas quality) Based on Biogas Raw materials Manure and mix of energy crops 46 / 27 kt FM/a Processes 2,5 MW (main PSA / PWW differences) Based on Bio-SNG Raw materials -

Processes (main differences)

-

Short term (ST) Mix of energy crops and manure 47 / 48 kt FM/a 5 MW PWW / Amine

forestry wood residues, 98 kt ar/a 22 MW FICFB gasifier, Activated coal / ZnO alkaline gas cleaning

Medium term (MT)

Long term (LT)

Mix of energy crops, biowaste and manure 77 / 74 kt FM/a 10 MW PWW / Amine + combustion of ferm. residues

Mix of energy crops, biowaste and manure 73 / 71 kt FM/a 10 MW PWW / Amine + combustion of ferm. residues

forestry wood residues, willow, (straw) 330 / 328 kt ar/a 75 MW FICFB gasifier 77 MW AER gasifier

Forestry wood res., willow, straw 1.673 kt ar/a 380 MW FICFB gasifier 293 MW EF gasifier with pyrolysis slurry

1.2. Economic Aspects In addition to technical aspects, the decision on a preferable fuel is mainly driven by economic reasons. Thus, the production costs of biomethane have been analysed and compared. The calculation is based on the annuity method, which includes an annuity factor that enables non-recurring payments (e.g. investment costs) and regular payments (e.g. material costs) in a project to be consolidated into an annual average payment over the assessment period. Costs are separated into capital related, feedstock related, operation related

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(auxiliaries, labour, maintenance) and other costs (administration, insurance). Revenues from marketing of by-products are also considered. For all concepts, a calculatoric time frame of 15 years is considered and an inflation rate of 2 %. Capital costs include capital investments for biomass pre-treatment, conversion, conditioning, building and other auxiliary installations. Plant-specific costs for up-scaled concepts are calculated based on existing facilities with the help of scaling factors (e.g. 0.8 for fermenter). Figure 4 shows that with increasing capacities, the specific total capital investment costs are decreasing as a result of economies of scale as well as improved and more efficient technologies. While biomass treatment and biomethane upgrading influence the total capital investments for biomethane via biogas, for Bio-SNG biomass conversion is one of the most important due to the complexity.

Biomethane / SNG

SNG-LT-293MW SNG -MT-77MW SNG -MT-75MW SNG-ST-22MW

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Capital Operation Raw material Revenues by-products Propane Total 11,6

11,3 13,6

BG - Status- 2,5MW -2

price spread biomethane household

Biomethane / SNG

Figure 4. Specific total capital investments of different biomethane concepts.

-

2 4 6 8 10 12 14 16 18 Biomethane production costs free plant in EURct/kWhbiomethane

Figure 5. Biomethane production costs.

20

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Figure 5 shows the production costs of the investigated biomethane concepts in comparison to the current selling price of biomethane to households. Costs also include the addition of propane for the increase of the heating value of biomethane prior to the feed-in to the gas network. Because of increasing capacities and increased concept specific overall efficiencies, the total biomethane production costs decrease.

1.3. Environmental Aspects The environmental impacts of a product are not limited to their use or the production process. Substantial environmental impacts may also occur within the pre-chains. The most important method to assess selected environmental impacts is the life cycle assessment (LCA), which can be applied to consider environmental impact categories such as the ―anthropogenic green house effect‖ indicated by the greenhouse gas emissions in form of CO2 equivalents. The goal and scope definition describes the considered system boundaries and defines the functional unit. The functional unit is 1 kWh biomethane and all results refer to this unit. The system boundary of the assessment follows the well-to-tank (WTT) approach and includes all primary and secondary process chains from biomass production to biomethane provision. By-products have been taken into account by the method of allocation. Figure 6 shows GHG emissions for the reference concepts compared to the fossil reference of the EU directive 2009/28/EC (EU RED). Values are comparable for both biomethane based on biogas and Bio-SNG. Due to the technology development and diversified inputs of raw material, the emissions decrease with increasing capacities. In addition to raw material production, the stage of biomass conversion influences the total GHG emissions significantly. 302 Raw material Raw material supply Conversion Distribution methane Credit manure handling Total

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Fossil reference EU RED 102

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-50

0

50

100

150

200

250

Specific GHG emissions (WTT) in g CO2-Eq./kWhbiomethane

Figure 6. Specific GHG emissions of developed concepts.

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Figure 7 shows, the GHG mitigation potential of different biomethane applications (transport, heat and electricity). Biomethane for transport, for heat as well as for electricity has great GHG mitigation potential compare to their corresponding fossil reference. 100 90

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Rape Diesel

Ethanol from wheat

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Electricity mix Germany 2008

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Use for electricity production in CHP

GHG-balance of different biomethane applications compared to fossil reference systems in %

Figure 7. GHG balance of different biomethane applications.

CONCLUSION In the effort to provide sustainable alternative energy carrier, biomethane emerges as an option that could substitute significant amounts of natural gas in the future. Biomethane can be produced through two different routes: the bio-chemical route (from biogas), which is already commercially available and the thermo-chemical route (from Bio-SNG), which is in demonstration phase. Overall, economic and environmental aspects strongly depend on regional conditions and applied concepts. In this paper exemplarily eight different concepts of biomethane production have been described and assessed in economic and environmental terms. The results of the analysis show promising effects on costs and GHG emission reductions in comparison to the fossil reference. For a successful market implementation of biomethane, further RandD activities as well as interest of the consumer market on biomethane as fuel for transport are strongly required. Through further specific technological development, it is possible to (i) reduce biomethane production costs and to (ii) achieve higher GHG-mitigation potential. An important factor for a sustainable production of biomethane is beside the feedstock availability the optimal interaction of the entire process.

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ACKNOWLEDGMENT The authors would like to thank the European Commission as well as the Fachagentur Nachwachsende Rohstoffe e. V. for their financial support to elaborate these results. Any interpretations or opinions contained in this paper are those of the authors and do not necessarily represent the view of the supporters.

REFERENCES [1]

[2] [3]

[4]

[5]

EWI/PROGNOS: Energiereport IV – Die Entwicklung der Energiemärkte bis zum Jahr 2030, Energiewirtschaftliche Referenzprognose. Energiewirtschaftliches Institut an der Universität zu Köln (EWI), Prognos AG, Untersuchung im Auftrag des Bundesministeriums für Wirtschaft und Arbeit, Köln, Basel, 2005. Thrän, D., Seifert, M., Müller-Langer, F., Plättner, A., Vogel, A.: Möglichkeiten einer europäischen Biogaseinspeisungsstrategie. Teilbericht. January 2007. Müller-Langer, F., Oehmichen, K., Majer, S., Rönsch, S., Scholwin, F., Weithäuser, M., Seifert, M.: Erdgassubstitute aus Biomasse im Überblick – Ökonomische und Ökologische Parameter im Vergleich. Fachagentur Nachwachsende Rohstoffe e.V. (FNR) „Erdgassubstitute aus Biomasse – eine Bestandsaufnahme“, Güstrow, August 2008. Müller-Langer, F., Rönsch, S., Weithäuser, M., Oehmichen, K., Seiffert, M., Majer, S., Scholwin, F., Thrän, D.: Erdgassubstitute aus Biomasse für die mobile Anwendung im zukünftigen Energiesystem. Endbericht zum Forschungsvorhaben FZK 22031005 Fachagentur Nachwachsende Rohstoffe e.V. (FNR), April 2009. Müller-Langer, F., Rönsch, S., Weithäuser, M., Oehmichen, K., Scholwin, F., Höra, S., Scheftelowitz, M., Seifert, M.: Ökonomische und ökologische Bewertung von Erdgassubstituten aus nachwachsenden Rohstoffen. Endbericht im Auftrag der Fachagentur Nachwachsende Rohstoffe e.V. (FNR), June 2009.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 31

FLAMMABILITY OF POLYMERS REINFORCED WITH LIGNOCELULLOSIC RAW MATERIALS Maria Władyka-Przybylak* and Krzysztof Bujnowicz Institute of Natural Fibres and Medicinal Plants, Poznań, Poland

ABSTRACT Reinforcing or filling of thermoplastics with natural particles, e.g. flax, hemp has been gaining importance recently. The addition of lignocellulosic materials to polymers can change properties of the resulting composites, which depends on properties of the natural materials and of the polymers. The flammability of composites play an important role, especially in a building industry and transportation. In this work, the flammability of polypropylene composites reinforced with hemp shives was analyzed. The share of hemp and flax shives was 5%, 10% and 30%. The samples were tested using Cone Calorimeter at heat flux of 35 kW/m2.

INTRODUCTION Recently, in the composite materials more attention has been put on technologies of biodegradable materials [1-3]. The use of lignocellulosic materials in the form of fibres or shives results not only in an increase of a composite biodegradability, but also changes its flammability. Such composites find application in automotive and building industries. Therefore, the flammability characteristics of the composites based on a synthetic polymer and natural fillers are of essential importance [4-6]. One of the methods for testing fire performance of different materials is a cone calorimetry method, for which measurement conditions are given in ISO 5660 standard [7]. The cone calorimeter is the most advanced apparatus among all bench-scale reaction-to-fire test instruments. The main property determined during the tests is the heat release rate (HRR). The rate of heat release is determined by measuring oxygen consumption derived from oxygen concentration and flow *

Institute of Natural Fibres and Medicinal Plants, ul. Wojska Polskiego 71 B,60-630 Poznań, Poland.

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rate in the combustion product stream. This test method is based on the observation that, generally, the net heat of combustion is proportional to the amount of oxygen required for combustion, namely heat energy of approximately 13.1 MJ is released per kilogram of oxygen consumed, irrespectively to the type of organic material tested [8]. The sample can be tested in two positions - horizontal or vertical (depending on application) in a wide range of heat flux intensities from 5 to100 kW/m2.

EXPERIMENTAL Hemp and flax shives (unmodified hemp and flax shives, length 2-4 mm) were located in the polypropylene matrix using twin screw extruder Leistritz MICRO 27 GL/GG-44D with Brabender gravimetric feeding system. Samples for flammability tests were prepared with pressing moulding. The samples were tested in accordance with ISO 5660 standard at heat flux of 35 kW/m2. The composite samples had dimensions of 100 x 100 x 4 [mm], and were held in a retaining frame. The following parameters were measured using the cone calorimeter: heat release rate, time-to-ignition and mass loss rate.

RESULTS AND DISCUSSION Parameters determined in the tests are shown in Figures. 1- 4. Results for composites containing 5, 10 and 30 wt. % of hemp and flax shives were compared to the results for polypropylene (PP). Heat release rate (HRR) curves show that thermal decomposition and combustion of the composites varies depending on shives content. When shives content was more than 10%, the curves became similar to those of typical lignocellulosic materials with two characteristic peaks. The addition of hemp and flax shives resulted in advantageous changes in fire performance of composites in comparison with PP. The most visible effect can be seen for the HRR peak (Figure 3). For the composites containing 30% of hemp and flax shives, the HRR peak was by 50% lower than these of PP.

Figure 1. Heat release rate (HRR) of PP/hemp shives composites compared to PP.

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Figure 2. Heat release rate (HRR) of PP/flax shives composites compared to PP.

Figure 3. Total heat release rate [THR], average heat release rate [HRR aver] and heat release rate peak [HRR peak] of PP/hemp and flax shives composites compared to PP.

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Figure 4. Average mass lose rate [MLRaver], average heat of combustion [HOCaver], time to ignition [TTI] of PP/hemp and flax shives composites compared to PP.

Other measured parameters such as average mass loss rate [MLR] and average heat release rate (HRR aver) (Figures. 3-4) also showed a reduction as a result of hemp and flax shives addition. This advantageous effect became even more significant when hemp and flax shives content was increased to 30%. Only two disadvantageous effects of shives addition were observed - the composites showed a shorter time to ignition (IT) and higher total heat released (THR).

CONCLUSIONS The addition of hemp and flax shives, especially in the amount of 30 wt. %, resulted in a decrease of heat release rate peak [HRR peak]. Also other parameters characterizing heat released during combustion – mass lose rate [MLR], average heat release rate [HRR] were

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reduced. However, time to ignition of composites and heat of combustion [HOC] became slightly worse in comparison with PP. To increase the fire retardancy of composites reinforced with natural fillers treatment of hemp shives with selected fire retardants could be very promising solution.

REFERENCES [1] [2] [3]

[4] [5] [6] [7] [8]

A.K. Błędzki, J. Gassan, Prog. Polym. Sci., 24 (1999) 221. K. Błędzki, A. A. Mamun, O. Faruk, eXPRESS Polymer Letters, 1 (2007) 755. R. Kozłowski, M. Władyka-Przybylak, K. Bujnowicz, Latest Achievements in the Area of Composites Reinforced With Natural Fiber, Fiber Reinforced Composites 2007, Port Elizabeth 9-12.12.2007. A.P. Mouritz, Z. Mathys, A.G. Gibson, Composites: Part A, 37 (2006) 1040. Sz. Matko, A. Toldy, S. Keszei, P. Anna, Gy. Bertalan, Gy. Marosi, Polym. Degrad. Stab., 88 (2005) 138. L.B. Manfredi, E.S. Rodríguez, M. Władyka-Przybylak, A. Vázquez, Polym. Degrad. Stab., 91 (2006) 255. ISO 5660-1:2002 Fire Tests - Reaction to Fire. Part 1: Rate of Heat Release from Building Products (Cone Calorimeter Method), 2002. M. Helwig, R. Kozłowski, Review of flammability test methods useful for evaluation of composites based on wood and natural fibres, 5th Global Wood and Natural Fibre Composites Symposium, Kassel, 27-28.04.2004.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 32

FEEDSTOCKS AND (BIO) TECHNOLOGIES FOR BIOREFINERIES Joachim Venus 1* Leibniz-Institute for Agricultural Engineering Potsdam-Bornim Dept. Bioengineering, Potsdam

ABSTRACT Renewable resources can be utilized directly, e.g. as energy carriers, as packaging materials, as fibres, for the production of colouring agents or as lubricants. However, they can also be converted biotechnologically by enzymes and microorganisms, giving us access to a multitude of biocompatible products and possible uses. The carbon sources from agricultural feedstocks and residuals can be utilized by a lot of microorganisms, e.g. lactic acid bacteria to produce lactic acid. Lactic acid, its salts and esters have a wide range of potential uses and are extensively used in diverse fields. The goal is to develop a fermentation process based on the substitution of expensive nutrient supplements by cheaper materials from renewable resources due to their main proportion of the whole process costs.

Keywords: renewable resources, biorefineries, biotechnology, bio-based products, lactic acid; fermentation; bioengineering

INTRODUCTION The world is facing many serious challenges. A fast-growing human population and the consequent growing demand for food, energy and water are the most serious. In addition, anthropogenic climate change requires that we significantly reduce our greenhouse gas *1

Leibniz-Institute for Agricultural Engineering Potsdam-Bornim. Dept. Bioengineering, Max-Eyth-Allee 100, D14469 Potsdam. Fon/Fax: +49(0331)5699-112/-849; e-mail: [email protected].

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emissions to avoid further damaging effects for the globe. Only the use of new technologies in combination with changing the feedstock base will allow us to bridge the gap between economic growth and sustainability in the long run (WEF, 2010). The increasing demand for a sustainable supply of food, raw materials and fuels, together with recent scientific progress, is the major economic driving force behind growth of the Knowledge Based Bio-Economy (KBBE) in Europe over the last few decades. The bioeconomy – the sustainable production and conversion of biomass, for a range of food, health, fibre and industrial products and energy, where renewable biomass encompasses any biological material to be used as raw material - can play an important role in both creating economic growth, and in formulating effective responses to pressing global challenges. In this way it contributes to a smarter, more sustainable and inclusive economy (KBBE, 2010). Sustainable economical growth requires safe resources of raw materials for the industrial production. Today‘s most frequently used industrial raw material petroleum, is neither sustainable, because limited, nor environmentally friendly. While the economy of energy can be based on various alternative raw materials, such as wind, sun, water, biomass, as well as nuclear fission and fusion, the economy of substances is fundamentally depending on biomass, in particular biomass of plant. Special requirements are placed to both, the substantial converting industry as well as research and development regarding the efficiency of raw material and product line as well as sustainability (Kamm et al., 2006).

AIM AND BACKGROUND Biorefineries in theory would use multiple forms of biomass to produce a flexible mix of products, including fuels, power, heat, chemicals and materials. In a biorefinery, biomass would be converted into high-value chemical products and fuels (both gas and liquid). Byproducts and residues, as well as some portion of the fuels produced, would be used to fuel on-site power generation or cogeneration facilities producing heat and power.

Figure 1. Biorefinery Concept [PNNL, 2007].

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Integrating the production of higher-value chemical/material co-products into the biorefinery‘s fuel and power output will improve the overall profitability and productivity of all energy-related products (PNNL, 2007). At the highest level, biorefineries input biomass as carbon sources and can generate fuel, power and products as output (Figure 1). In this construct, biomass is separated into its component parts: sugars (as cellulose, hemicellulose or starch), lignin, protein and oils. In various current biorefinery concepts, the sugar or oil fractions are used to produce liquid transport fuel or bio-based products. At present, biorefineries are classified based on, technological (implementation) status, type of raw materials used or main type of conversion processes applied. A search of the literature revealed a variety of terms describing biorefineries (IEA, 2009).The biorefinery systems are classified by quoting the involved platforms, products, feedstocks and, if necessary, the processes:         

Conventional Biorefineries 1st, 2nd, and 3rd Generation Biorefineries Whole Crop Biorefineries Thermochemical Biorefineries Advanced Biorefineries Lignocellulosic Feedstock Biorefineries Marine Biorefineries Two Platform Concept Biorefineries Green Biorefineries

Figure 2. Overview of current platforms, products, feedstocks and conversion processes (IEA, 2009).

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It can be seen from the network on which the biorefinery classification system is based (Figure 2) that – coming from the different biogenic feedstocks like starch and sugars, lignocellulosics, proteins, oil/fat containing material, residues and wastes - (Bio)Technologies (marked as the orange ones, e.g. fermentation, enzymatic conversion, anaerobic digestion) are only a part of the whole mixture of several conversion technologies. There are also thermochemical (e.g. gasification, pyrolysis), chemical (e.g. acid hydrolysis, synthesis, esterification) and mechanical processes (e.g. fractionation, pressing, size reduction) used in terms of biomass (pre-) treatment and subsequent processing, respectively. If we have a closer look into the scientific literature there is a noticable increase of research papers related to biorefineries in general during the last 10 years (Figure 3).

Figure 3. Biorefinery related paper within the ISI database.

On the other hand it has been stated in a lot of documents (at national, EU, global/US level) that not only academic research will be necessary to meet the already mentioned global challenges of the future to bring all the positive effects of renewable resources as feedstocks in view of     

Global warming and climate change (reduction of GHG emissions), Sustainable, secure and affordable supply of energy and other resources, (Global/EU/national) competitiveness, job creation and innovation, Reduction of the dependence on fossil feedstocks, Alternatives of employment and income for the rural areas

to reality. The development of biotechnology processes and their uptake by the industry are not yet optimal. Aside from underfunding, which is regularly highlighted by the industry, technology transfer appears to be insufficient. In combination with EU policies on innovation, this should be as a priority for the Strategy, with support actions for research and the uptake

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of new technologies. Therefore the following measures were already discussed a couple of years ago [EU Commission, 2007]: 



In cooperation with industry, Member States and other funding bodies, engage schemes to finance/promote the establishment of multifunctional pilot plants to demonstrate the potential of bio-based applications and facilitate their market penetration, subject to a proportionate impact assessment and in accordance with EC rules in the field of competition and internal market Support the setting up of demonstration/pilot projects and integrated bio-refineries, which are flexible installations at pilot or industrial scale for the production of biofuels and other biomaterials, based on a variety of feedstock. Giving support to demonstration projects is important since SMEs active in this area do not have the resources to set up a real proof-of-concept. It would also help to test logistical solutions and form value chain coalitions between actors.

Bio-based products are prepared for an economical use by a meaningful combination of different methods and processes (physical, chemical, biological and thermal). It is therefore necessary that biorefinery-based technologies should be developed in technical related facilities. For that reason the Leibniz-Institute for Agricultural Engineering Potsdam-Bornim (ATB) has been applied for a project at the EU (ERDF - European Regional Development Fund) to build-up a pilot plant facility for the lactic acid fermentation based on renewable feedstocks. After the successful application and funding procedure the pilot plant (Figure 4) was taken into operation at the site of ATB in 2006 (Venus/Richter, 2007).

Figure 4. Pilot plant facility for the development of biotechnological/fermentation processes, e.g. lactic acid as a basic chemical.

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Lactic acid assigns to bio-based organic intermediates of high interest. Up to date it is produced on industrial scale and is available on the market at a competitive price level. In chemical industry it serves as a building block for a variety of key chemicals or bulk polymers, e.g. acrylic acid, poly(lactic acid) (PLA). Today, lactic acid is widely used in the food, cosmetic, pharmaceutical, and chemical industry [Vijayakumar et al., 2008] but has received increasing attention as a monomer for the production of PLA, a bio-based and potentially biodegradable polymer (Jim Jem et al., 2010). On the assumption of a worldwide lactic acid production in the range of 350.000 tons, global lactic acid consumption is estimated to increase significantly at a rate of about 12-15% per year (Corma et al., 2007). Growth on demand for lactic acid and its salts and esters in industrial applications will be driven mainly by lactic acid–based polymers and, to a lower degree, lactate solvents (Malveda et al., 2006). Meanwhile a lot of effort has been put on the field of biorefinery concepts, systems, and projects connected with already existing pilot and demonstration facilities, respectively. For the EU level a database exists where everybody could search for a country, specification or product (please refer to Table 1 with the related information of ATB‘s pilot plant). Table 1. European database for already existing biorefineries, e.g. ATB pilot plant

http://www.bio-economy.net/bioeconomy/member_states/index_bioeconomy_member_states.html Country :

Germany

Place :

Postdam

Type :

Pilot

Accessibility :

Open to all

General information :

Raw materials: starchy materials (e.g. cereals) Products: Lactates, lactic acid, starter cultures

Services :

Stirred vessels (up to 1000 L), 450-L-fermentor, power, steam, water, compressed air

Funding :

€ 3,2 million (75% funding by EU/ERDF; 12,5% Federal and 12,5% Regional Government respectively Investitions-Bank des Landes Brandenburg - ILB)

Contact :

Dr. Joachim Venus, [email protected]

Web :

www.atb-potsdam.de

RESULTS AND DISCUSSION For several years, ―bioconversion of raw materials produced in agriculture into chemicals, microbial biomass and active substances― has been the subject of intensive study in the Bioengineering department of the ATB. These research projects are highly complex.

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Alongside technologically oriented studies of process design, they include fundamental research into strain optimization and the kinetics of microbial processes. Interdisciplinary cooperation both within the department and with partners outside the institute ensure that viable application solutions are found not only for the fermentation process phase, but also for all other up- und down-stream processes involved. For the now existing pilot plant with the following aims   

swift transfer of new biotechnological processes into practice often fails due to the lack of a reference facility that can be used for multiple applications pilot facility for production of lactic acid at the ATB consequently fills a gap in the various phases of bioprocess engineering provision of product samples is intended to open up the possibility of interesting partners in industry with specific product requirements in the various applications

some basic results will be presented in the following section. Although the lactic acid fermentation is a well known process also in industrial scale the following aspects have been identified from a recent evaluation (Bozell/Petersen, 2010) of so called ―top candidates‖ in the field of biobased productsŚ   

Optimization of bioconversion of carbohydrates; bioprocesses with high rate, yield, titer, product, pH and inhibitor tolerance; engineering of organisms to produce single materials

The worldwide research is advancing focused on the use of renewable raw materials as carbon sources (Li/Cui, 2010). In this context, there is a strong interest to reduce costs for raw materials and to use renewable resources. The cost effectiveness of bioprocesses is still a major issue because, in the case of bulk products (building blocks for the already existing product trees in the chemical industry), the price is mainly affected by raw material costs. The overall process for the renewable-based lactic acid manufacture is illustrated in Figure 5.

Figure 5. Bioconversion of renewable materials into lactic acid.

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Biotechnological production of lactic acid as an example of these ―building blocks‖ is carried out with several feedstocks serving both as carbon as well as nutrient source. From laboratory to pilot scale, our research aims at the development of a fermentation process based on the substitution of expensive nutrient supplements by alternative and cheaper materials from renewable resources (Figure 6).

Figure 6. Alternative carbon and nutrient sources for lactic acid fermentation (from left to right: rapeseed meal, starch, straw, sugar, barley, lupins, shredded grain, rye, bagasse).

With respect to the development of a renewable based biotechnology for non-food applications, one of the first steps is the preparation of a suitable carbon source containing the broth for fermentation processes. In most cases, starch cannot be used by lactic acid bacteria directly. For this purpose, the big starchy macromolecules have to be converted into glucose molecules by enzymatic hydrolysis. Whereas the fermentation of glucose can be carried out efficiently, the bioconversion of the pentose fraction presents a challenge. A lot of attention has therefore been focused on genetically engineering strains that can efficiently utilize both glucose and pentoses, and convert them to useful compounds. The metabolic engineering objectives so far have focused on higher yields, productivities and expanding the substrate and product spectra (Ho et al., 1999; Picataggio et al., 1998; Aristidou, 2000). At the moment there is a second part of nutrients (yeast extract, salts) necessary to cover the nutritional requirements of the lactic acid bacteria. The sterilization of the glucose (e.g. biomass hydrolyzates) and the other components is carried out in 400-L and 250-L-vessels. After sterilization, these media can be used in this form as a basic nutrient broth in fermentation. Further investigations deal with the availability of the several substrates for fermentation and the effects on the subsequent down-stream processing steps. In existing commercial facilities, lactic acid is produced in batch operations. In the present case, a continuous operating mode is used. Due to the continuous loss of viable microorganisms in chemostat mode there is a limit of the lactate productivity Pr, which is defined as the product of the dilution rate D [h-1] and the lactate concentration P [g/L]. To increase the overall yield of the whole fermentation process it is necessary to enhance whether one or both of the above mentioned parameters. Depending on the input concentration of the main substrate (glucose content of the hydrolyzate) the lactic acid bacteria need a certain time (residence) to degrade the carbon source nearly completely. In respect to the phenomenon of product inhibition on the one hand and substrate limitation on the other hand there is a need to optimize the productivity. Therefore an important parameter to improve the lactic acid production seems to be the amount of active biomass (cfu) in the bioreactor. One of the usual ways to keep the biomass inside of the system is the cell retention with hollow fibre membranes. The main portion of the fermented medium leaves the bioreactor as a cell-free filtrate through a membrane module, while a second process (containing biomass) is necessary to maintain a stable steady

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state. The main parameters of a continuous mode lactic acid fermentation is shown in the following Figure 7. 1,E+ 11 biomass lactic acid glucose cell number

100

1,E+ 10

50

1,E+ 09

0

cell number [ cfu/ ml]

biomass, substrate, product [ g/ L]

150

1,E+ 08 time [ hours] 20 30 40 50 60 70 80 90 100 110 120 130 140

Figure 7. Time course of a typical fermentation with cell retention.

When lactic acid is produced from the complex raw materials mentioned above, the culture filtrate solution obtained after fermentation still contains other substances or impurities that have undesirable effects in specific applications (further processing) of lactic acid.

Figure 8. Effect of several downstreaming steps on the purity of lactic acid.

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The fermentation broth from the lactic acid fermentation was firstly filtered to remove the cells in order to prevent a deposition of bacteria on the membrane surface and the creation of bacteria clusters in the space between the membranes of the electrodialysis unit. All cells of the fermentation broth were removed and cellfree permeate was obtained by ultrafiltration (hollow fiber membrane). After the conversion of sodium lactate into lactic acid by electrodialysis (EDR), the product is finally upgraded by ion exchange resins, whereby polluted effluents of rinsing water and consumption of regenerants are minimized. Conventional processes for down-streaming are based on precipitation steps that generate large amounts of chemical effluents. Consequently the environmental impact and the operating costs of traditional processes can be reduced by using alternative technologies, such as electrodialysis with monopolar and bipolar membranes. Selected components of sodium lactate and lactic acid solution after different downstream steps are illustrated in Figure 8. The results confirm that the two-stage electrodialysis is a suitable and efficient technique for recovering lactate ions from the pretreated fermentation broth and for the subsequent conversion into lactic acid considering environmental aspects. Ultrafiltration and softening of the sodium lactate solution are required in order to operate the electrodialysis properly. Chemical impurities such as inorganic cations and compounds of nitrogen were considerably reduced. Additional de-ionization and decolorization process steps using ion exchange resins were integrated to polish the free lactic acid for further applications in industry.

CONCLUSION The future of industrial biorefineries is a positive one. It has been described that biorefineries may play a major role to play in supplementing our growing demand for sustainability, whether it is to tackle climate change or to create novel energy sources, biobased products and fossil replacements. The construction of a pilot facility for production of lactic acid at the ATB as one specific example of a biorefinery consequently fills a gap in the various phases of bioprocess engineering from applied fundamental research through application research to the launch of biotechnological processes in practice. First results for the manufacture of lactic acid illustrate the conformity with existing investigations. Exploitation of high quality lactic acid for the production of biodegradable polymers is one of the recent applications.

REFERENCES Aristidou, A.; Penttila, M.: Metabolic engineering applications to renewable resource utilization. Current Opinion in Biotechnology 11, 2000 (2) 187-198. Bozell, J.J.; Petersen, G.R.: Technology development for the production of biobased products from biorefinery carbohydrates - the US Department of Energy‘s ―Top 10‖ revisited. Green Chem., 2010, 12, 539–554. Corma, A.; Iborra, S.; Velty, A., Chemical Routes for the Transformation of Biomass into Chemicals. Chemical Reviews 2007, 107, 2411-2502.

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EU Commission: Communication from the commission to the council, the European Parliament, the European Economic and Social Committee and the Committee of the Regions on the mid term review of the Strategy on Life Sciences and Biotechnology, {SEC(2007) 441}. Ho, N.W.Y.; Chen, Z.; Brainard, A.P.; Sedlak, M.: Successful Design and Development of Genetically Engineered Saccharomyces Yeasts for Effective Cofermentation of Glucose and Xylose from Cellulosic Biomass to Fuel Ethanol, Advances in Biochemical Engineeing/Biotechnology, Vol. 65, 1999, 163-192. IEA Bioenergy: Biorefineries: adding value to the sustainable utilisation of biomass. Task 42 Booklet, 2009. Jim Jem, K.; van der Pol, J.; de Vos, S., Microbial Lactic Acid, Its Polymer Poly(lactic acid), and Their Industrial Applications. In Microbiology Monographs 2010; Vol. Plastics from Bacteria Natural Functions and Applications, pp 323-346. Kamm, B., Kamm, M., Venus, J.: Principles of biorefineries: the role of biotechnology, the example lactic acid fermentation. – In: Edwin C. Hearns (Ed.), Trends in Biotechnology Research (pp. 199-223), Hauppauge, N.Y.: Nova Science Publishers, 2006 (ISBN 160021-224-7). Li, Y.; Cui, F.: Microbial Lactic Acid Production from Renewable Resources. In Sustainable Biotechnology Sources of Renewable Energy, 2010; pp 211-228. Malveda, M. P.ś Blagoev, M.ś Kishi, A., CEH Marketing Reasearch Report ―Lactic Acis, Its Salts And Esters‖. SRI ConsultingŚ Melo Park, 2006. Picataggio, S.; Zhang, M.; Franden M.A.; Mc Millan, J.D.; Finkelstein, M.: Recombinant lactobacillus for fermentation of xylose to lactic acid and lactate. US Patent 005798237A, 1998. PNNL (Pacific Northwest National Laboratory): Top Value-Added Chemicals from Biomass. Volume II - Results of Screening for Potential Candidates from Biorefinery Lignin (Prepared for the U.S. Department of Energy under Contract DE-AC05-76RL01830, October 2007). The Knowledge Based Bio-Economy (KBBE) in Europe: Achievements and Challenges. Summary, 14 September 2010. Vijayakumar, J.; Aravindan, R.; Viruthagiri, T.: Recent trends in the production, purification and application of lactic acid. Chemical and Biochemical Engineering Quarterly 2008, 22: 245-264. Venus, J.; Richter, K.: Development of a Pilot Plant Facility for the Conversion of Renewables in Biotechnological Processes. Eng. Life Sci. 2007, 7, No. 4, 395-402. WEF (World Economic Forum): The Future of Industrial Biorefineries, 2010.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 33

NON-FOOD CROPS AS A FEED STOCK FOR MODERN BIO-BASED INDUSTRY Maria Mackiewicz-Talarczyk*, Krzysztof Heller, Przemyslaw Baraniecki and Irena Pniewska Institute of Natural Fibres and Medicinal Plants (INFandMP), Poznan, Poland

ABSTRACT The paper presents the point of view and future estimation of the non-food crops as a feed stock for modern bio-based industry, based on the European Commission project Crops2Industry - Non-food Crops-to-Industry schemes in EU27. Agreement Nr: 227299, coordinated by Dr. Myrsini Christou of CRES - Center for Renewable Energy Sources and Saving, Pikermi, Greece. The project aims to estimate among others the possibility of application on renewable resources to replace the fossil and non-renewable feed stock for industry.

I. INTRODUCTION The project is conducted from September 2009 to March 2012, with the involvement of fourteen partners from universities, institutes and SME, including the Institute of Natural Fibres and Medicinal Plants, Poland. The countries involved: Austria, France, Germany, Greece, Italy, Netherlands, Poland, Romania and UK. The Crops2Industry project is focused to explore the potential of non-food crops, which can be domestically grown in EU27 context, for selected industrial applications, namely oils, fibres, resins, pharmaceuticals and other specialty products and outline and prioritise cropsto-products schemes, suitable for the different Member States, which will support sustainable, economic viable and competitive European bio-based industry and agriculture. These will *

Institute of Natural Fibres and Medicinal Plants (INFandMP), ul. Wojska Polskiego 71b, 60-630 Poznan, Poland. Contact: [email protected].

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offer tremendous market opportunities for EU farmers and hold the potential of transforming a significant portion of our fossil fuel based economy to a bio-based economy of the 21 st century. [1] The following deliverables of the project are planned:  

   

Explore the potential of non-food crops, which can be domestically grown in EU27 countries, for selected industrial applications, namely oils, fibres, resins, pharmaceuticals and other specialty products (to be addressed in WP1) Identify current molecular genetics technologies (genomic and biotechnological tools) and suggest their potential applications in a crop-specific manner to address a wide range of breeding constraints regarding yields and tolerance to abiotic and biotic stress (to be addressed in WP2). Explore the potential and feasibility of the European industry to make high-value biobased products from renewable agriculture and forestry feedstock and biotechnological routes (to be addressed in WP3). Perform supply chain cost analysis, identify best business opportunities and assess the socio-economic impacts of selected crop-to-product schemes at EU-27, regional and country levels (to be addressed in WP4). Assess selected production and environmental impacts and identify a ‗core‘ list of standards and criteria for the environmental and socio-economic sustainability of selected non-food crops-to-industrial-products systems (to be addressed in WP5). Perform an overall assessment aiming to select and prioritise crops-to-products schemes in technical, socio-economic and environmental terms (to be addressed in WP6).

Develop a sound dissemination plan for distributing the information collected to targeted audience; provide a mechanism for bringing stakeholders together to force a coherent strategy for the promotion of bio-based products in Europe and link with other relevant projects (to be addressed in WP7). [1] The ultimate objective is to explore the potential of non-food crops, which can be domestically grown in EU27 context, for selected industrial applications, namely oils, fibres, resins, pharmaceuticals and other specialty products and outline and prioritise crops-toproducts schemes, suitable for the different Member States, which will support sustainable, economic viable and competitive European bio-based industry and agriculture. [1] The website of the project: www.crops2industry.eu The proposed project is carried out in 8 WPs.  

WP1 will report on non-food crops for selected industrial applications. Information will refer to main physical traits, cultivation areas, inputs, supply and logistics, yields, quality issues. WP2 will identify current molecular genetics technologies and suggest their potential applications in a crop-specific manner to address a wide range of breeding constraints. Improvement of non-food crops will entail breeding for agronomically important traits i.e. yield and tolerance to abiotic and biotic conditions.

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WP3 will explore the potential and feasibility of the European industry to make highvalue biobased products from non-food crops and biotechnological routes. WP4 will perform supply chain cost analysis, identify best business opportunities, and assess the socio-economic impacts of selected crop-to-product schemes at EU27, regional and country levels. WP5 will assess selected production and environmental impacts and identify a ‗core‘ list of standards and criteria for the environmental and socio-economic sustainability of selected crops-to-product schemes in a global and country-specific perspective. WP6 will perform an integrated technical, environmental, and economic assessment to help selecting and prioritising non-food crops. WP7 will address dissemination issues.

The expected output is to identify whether and under which terms Europe has the potential and the technical competence to develop a competitive bio-industry fed by a sustainable agriculture. The tasks which is INFandMP responsible for are: WP1 and WP3. [1]

II. CHOSEN WORK PACKAGES AND TASKS OF CROPS2INDUSTRY PROJECT

II.1. WP1. Non-Food Crops The Institutions involved in conducting this task - the leader: CRES and UNIBO- Italy, INFandMP-Poland, NCPRI- Romania, ITERG- France. This WP will serve to explore the potential of non-food crops, which can be domestically grown in EU27 countries, for selected industrial applications, namely oils, fibres, resins, pharmaceuticals and other specialty products. The work will be divided in six tasks: Task 1.1 Oil crops, Task 1.2 Fibre crops, and Task 1.3 Carbohydrate crops and Task 1.4 Other specialty crops. Information is collected referring to their main physical traits, cultivation areas, inputs, supply and logistics, yields, quality issues. Research gaps, prospects and recommendations to widen the range of potential feedstock for the understudy industrial uses are tackled. Within Task 1.1 Oil crops: CRES will undertake the leadership of the task and provide input on the rapeseed, sunflower, Ethiopian mustard. ITERG will report on oil crops for liquid biofuels, whereas UNIBO will report on the oil crops for lubricants, solvents and polymers. In the scope of task Task 1.3 Carbohydrate crops: UNIBO will undertake the leadership of the task and provide input on the maize, potatoes, cassava and sugar beets, whereas CRES will report on sweet sorghum and Jerusalem artichoke. The Task 1.4 Other specialty crops will deal with medicinal and aromatic herbs (i.e. hollyhock, marigold, caraway, celandine, St. John's Worth, plantain, common sage, French marigold, common yarrow, coriander, sweet fennel, lavender, lemon balm, peppermint, etc).

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In the horizontal Task 1.5 Multiple end use potentials and allocation factors the multiple use possibilities for the under study non food crops will be recorded and the best range of end use allocation factors will be defined. In Task 1.6 Available land area for the cultivation of non-food crops in EU27, the available lands for the cultivation of - food and - non-food crops will be estimated for now, 2020 and 2030 based on spatial agro-climate data and performed in GIS software. [1] Up to May 2010 the INFandMP, within Task 1.2. analyzed fibre crops such as flax, industrial hemp, nettle and kenaf. WP addresses the following topics:         

Plants morphology and anatomy Areas of origin and current cultivation Growing conditions – input requirements Logistics (harvesting – handling) until the industrial plant gate Yields Quality Applications: current – potential Factors restricting growth and yielding potential Research gaps

II.2. WP3. Bio-Based Products The next Work package, the INFandMP is involved directly is WP3 Bio-based products. Within WP3 the main target is to explore the potential and feasibility of the European industry to manufacture high-value biobased products from renewable agriculture and forestry feedstock and biotechnological routes. The work will be divided in four tasks: Task 3.1 Oils, Task 3.2 Fibres, Task 3.3 Resins, Task 3.4 pharmaceutical and other specialty products. In this WP, the bio-industry demands in oils, carbohydrates, resins, pharmaceutical and other specialty products will be reported and restricting factors that inhibit broader industrial use of the feedstock will be identified. Research gaps, prospects and recommendations to procure bio-based products will be tackled. Within WP3 - INFandMP has started from the elaborations regarding the detailed chain of raw materials and derived semi-products and products from fibre plants. The graphical presentation of such chain is presented below. The similar chains would be prepared for other fibrous plants. [2] In the scope of ―WP3 Bio-based products‖ the following institutions will contribute: Institute of Natural Fibres and Medicinal Plants (INFandMP) - Poland, ITERG - France, KEFI-Italy, HEMPFLAX - the Netherlands, CHIMAR, Greece, and NCPRI- Romania) In the traditional commodity markets in plant-derived products, a growing number of companies encounter difficulties in rejuvenating their product offerings, the main reason being that new means of extracting, processing or (chemically) modifying raw materials are running out. On the other hand, according to the ‗Plants for the Future Platform‘, all stakeholders – consumers, industry, academia, society, etc - are aware that in the 21st century the ‗bio-economy‘ will emerge, with increasing demands in healthier and safe products,

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whereas the bio-sciences are rapidly producing the know-how to enable the development of the technologies. [1]

The main target of this WP is to explore the potential and feasibility of the European industry to make high-value biobased products from renewable agriculture and forestry feedstock and biotechnological routes. [1] In this WP, the bio-industry demands in oils, carbohydrates, resins, pharmaceutical and other special products will be reported and restricting factors that inhibit broader industrial use of the feedstock will be identified. Prospects and recommendations to widen the range of potential feedstock for the understudy industrial uses will be tackled. The work on this WP will start from the product concept with an identified market and work back to the feedstock rather than start with the feedstock and seek products for markets. The following tasks will be conducted, coordinated by the relevant companies. [1] Task 3.1 Oils (ITERG) ITERG‘s contribution will be drawn from their activities in the fields of biofuels (oleaginous crops and oils for biodiesel production, bio lubricants, biopolymers and paintings, involving their analytical expertise and quality control for fat and vegetable oils, for industries including fat and oil industry (vegetable oil producers, manufacturers of animal fats, margarine and spread industry), and derived products users (food industry, non-food industry and component manufacturers. [1]

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Task 3.2 Fibres (KEFI, HEMPFLAX) The contribution of Kenaf Eco Fibers Italy S.p.A., will be based on their long experience with natural fibre products and their industrial applications: soundproofing systems, thermal insulation of walls, floors and roofs, automotive components, and ecological building, but above all for a sustainable future, in order to safeguard the environment. Hempflax Company will report on a wide range of activities, including harvesting and processing methods for industrial hemp, designing machinery to separate the bast fibres from the stalks' wood core, a process called decortication. HempFlax' processing line does not just separate the bast from the wood, but also the leaves, the seeds and the remaining substance. These five raw materials constitute the basis for HempFlax' currently marketed products. Their contribution will cover to the whole processing, manufacturing, application and marketing chain of hemp. [1] Task 3.3 Resins (CHIMAR) The use of wood panel products contributes to more efficient forest utilization and provides thus a cost effective solution to related environmental problems. To utilize large quantities of forest residues for conversion into low cost panel products, it is necessary to develop less expensive adhesives with secured availability, in order to derive meaningful advantage. Adhesives from renewable (non-petroleum) raw materials have a noteworthy role to play in this direction. To date, CHIMAR has tested a great variety of natural products as phenol substitutes at various levels. In particular, the natural-derived products studied so far are:

 

From wood and forestry residues: Crude Bio-oil and various chemicals derived from it, Lignin, Tannin, Pulping spent liquor, Lignosulphonate From agricultural resources: Cellulose, Amylum (from wheat, corn, etc), Proteins (soy), Bio-Liquids derived from various agri-wastes (olive stone, cashew nut shell, and sorghum).

Natural products with self adhesive properties like mastic gum. [1] CHIMAR, has achieved successful production of panels like plywood (PW), Oriented Stand Boards (OSB) and particleboards (PB), at various scales (lab, pilot, industrial), utilizing some of the above mentioned materials as phenol substitutes in PF resins, without deterioration to the properties of the boards. More resins with the rest of the above mentioned products are still under development, while CHIMAR also carries out research for the development of a totally natural adhesive. [1] In this task CHIMAR Hellas will be able to provide advice on the possible effective utilisation of various natural materials in products related to the wood-based panels industry, based on its long-lasting research on binding systems from natural-derived renewable materials and the skilled personnel with experience on this subject. [1] Task 3.4 Pharmaceutical and Other Specialty Products (NCPRI) NCPRI‘s contribution will be drawn from their main competences, which areŚ fundamental and applicative research regarding technological development in biotechnology, medicine, pharmaceutical areas, production at small scale that sustains the scientific research

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activity and technological development. The Pharmaceutical Biotechnologies department has a notable experience in the following areas: 



Highly complex technologies (preparation of pharmacologically active vegetable extracts for drugs and cosmetics, preparation of human use drugs, active substances as well as pharmaceutical formulations, improvement of various technologies or their replacement with up-to-date procedures, according to the international requirements and production of veterinary drugs and food-additives, animal growth stimulants). Analytical and pharmacological studies and determinations for all products issued by the technological departments.

Technical assistance, provided by the senior researchers, to other specialized companies for the experimentation and implementation of the technologies performed in the institute. [1] The output of all tasks will be to:     

Review on the product yielding capacity from various industrial crops streams Identify desirable quality characteristics that feedstock has to meet for mature industrial processes Report on current alternative resources (including petroleum-based or chemical counterparts) for each industrial use. Set prospects to widen the range of potential feedstock for the understudy industrial uses, based on the technology improvements Identify restricting factors that inhibit broader industrial use of the biomass feedstock (supply, costs, physical traits, consistency in quality, technical performance, research gaps, etc)

Set forth research gaps, prospects and recommendations to procure bio-based products will be tackled. [1]

III. THE WORKSHOPS WITHIN CROPS2INDUSTRY AND THEIR INPUTS REGARDING THE RESTRICTING FACTORS LIMITING BROADER INDUSTRIAL USE OF FIBRE CROPS, THE RESEARCH GAPS AND RECOMMENDATIONS The Workshops which took already part within the project Crops2Indstry: In Athens (September 2009), in Poznan at INFandMP (in November 2009) and in the Netherlands in March 2010. The Workshop at INFandMP, held on 18 November 2009 was entitledŚ ―Can fibre crops offer a viable alternative land use option and could theysupport a competitive industry?‖ During this Workshop - the special round table took part addressed among others to:  

the restricting factors limiting broader industrial use of fibre, prospects to widen the range of potential feedstock,

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research gaps and recommendations

1. Restricting Factors Limiting Broader Industrial Use of Fibre Crops Restricting factors limiting broader industrial use of fibre crops were identified in technology, ecology, society and in finance. A. Restricting Factors in Technology:

      

Concentration of processing industry around cultivation zones Organization of cultivation (high fractionation, especially in new member states) Supplies strongly depend on weather conditions Quality of raw material depends on weather conditions, harvest date, method of fibre extraction and storage conditions High level of manual operations in extraction and handling Low efficiency of flax and hemp processing compared to chemical fibres – result of currently used technologies Without proper management, fibre extraction and processing can be a source of environment pollution (consumption of water, energy, waste products).

For the fibre to be utilized by industry, the quality and quantity must be consistent over time. This requires a reliable supply chain to be in place with both the quality and quantity of raw material to be consistent over time. There must also be sufficient profit available throughout the production pipeline. [3] B. Restricting Factors in Ecology





Physico-chemical factors: the quality of basic raw materials is subordinated from environmental conditions. Adverse conditions cause lower fibre yields, dry mass and fibre quality. In addition, there is a problem with "fidelity of flax yielding" which implies that it is hard to obtain high yields of good quality raw material. Biological factors: The recommended regionalization of flax growing leads to excessive concentration of breeding having thus a narrow genetic pool as a result. In addition, there is low heritability of functional traits and difficulties with correction of functional traits. [3]

C. Restricting Factors in Society

  

The low level of consciousness on the subject: advantages of natural fibres, The use of synthetic fibres and The import of cheap fibre from third countries,

resulted in the decrease of profitability of fibre crops cultivation and in shrinkage (decay in some countries) of the textile industry.

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In addition,   

the product price (and not the product quality) is the most important factor that affects buying decision for many market segments (Price is not so important for clothing, but more important for home) the conflict between crop selling price (farmers income) and buying price of raw material (fibre), as well as the poor knowledge about industrial hemp and marihuana

resulted in not satisfying the demand on products from natural fibres. [3] D. Restricting Factors in Economics

       

Competition from other cultures (wheat, maize - market fluctuations) Products diversity (fibres + shives; fibres + waste fibres) Low specialization of industry (often factories cover the whole production chain from fibre to final product) – low production volume (no textile industry in Europe), = big investments Quality maintenance (field-field; region-region; year-year) Complex and low efficiency processing technologies Competition from tropical natural fibres Competition from man-made fibres Political barriers (subsidies inequality). [3]

2. Research Gaps and Recommendations A. Research Gaps and Recommendations in Technology As 70% of the biomass processed is waste, we have to think about using it. Wastes can be utilised far better:



  

The environmental impact of natural fibres also relies on how by-product management is organized. In principle renewable resources will be fully bioconvertible and may be reutilised as source for carbon in the form of carbohydrates (sugars), lignin or nitrogen (protein) and minerals. Often agricultural production utilises only a small part of the total fixed carbon in the biomass produced or harvested. Eco-effective design of products requires reuse of wastes to make new products. The suggestion is made that limitless economic growth can be obtained when the resources are properly reused (without quality loss). Since primary production is paid for quantity rather than quality, the breeding is often focussed on yield improvement and disease resistance. Concerns about the safety of genetically modified organisms or GMO-crops has resulted in fierce political discussions

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  

Concentration of cultivation and processing that will improve homogeneity, concentration of raw material production, control of sowing material and mechanization on highly plant producing areas. This will also contribute to developing new harvest techniques e.g. using combines. New harvesting and processing technologies (more efficient and controllable e.g. using ultrasound, enzymes, osmosis, plasma and other unconventional methods) Improvement for further processing steps (spinning) Searching for new applications (e.g. production of viscose fibres from shives). [3]

Comments from the round table were as follows: Is the technology mature and sufficient to produce fibres? 







 

The shortage of science and technology is not an issue but putting the supply chain together. There are growers equipped with modern machineries which have agreements with industries for shive production. The real problem is how to attract the market, which is fed by imports of low-cost raw material. They don‘t know how to sell their products, which they try and promote all over the country. General awareness for the general public awareness is needed. Fibre crops combine hundreds of applications to produce a number of products. Therefore, we have to use the entire feedstock, with all their components. Some of them may be more valuable than others; nevertheless, all components are valuable for several end uses. The biorefinery concept is a good option, because it improves the overall efficiency of the industrial plant by exploring all by-products for several uses. In this case, we need better linking with other sciences and the chemic industry, in contrast to biofractionisation, that is carried out physically. Hemp cultivation is new for Poland but the crop is easy to cultivate, has high yields and low requirements in chemicals and pesticides. It is advisable to use the fibres to cover the market demands for fibre products and the co-products for energy. The market shows more demand for hemp for industry and co-products for energy. How to prioritise bio-products needs? There is a wide range of different uses of hemp, flax, kenaf, etc. Mature markets rank the first and the biggest market for hemp for instance is the paper and pulp. The niche market is the building industry. Hemp could also be used for energy but in Poland hemp is a prescribed prohibited plant and we cannot exploit all its potential uses. A structure for prioritarisating the uses of products is however needed.

A whole range of products can be produced from fibrous crops through the biofractionisation or biorefining concept, which implies innovation in industry, priorities in demand. These require investment and policy changes. [3]

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B. Research Gaps and Recommendations in Ecology

   

Knowledge of genetic mechanism plants immunity on drought. Breeding of new cultivars more resistant to drought and high temperature. Widen of genetic pool (interspecific crosses). Research for improvement of dew retting process for different weather conditions. Limitation of environmental conditions influence on raw material quality (biotechnology).

Conduct of research concerning bio-stimulators. [3] C. Research Gaps and Recommendations in Society

  

Education and PR for increasing knowledge about advantages of natural fibres. Promotion of the own textile production in EU countries. Market research for estimate hiding market segments for bio-product made from fibre crops.

Conduct of research concerning possibilities of cost reduction in each period of processing and biological production (drop of price). [3] Comments from the round table 





Public awareness has to be increased by organising the dissemination of information. The workshops are important for the industry as well as for the investments; there have to be more targeted workshops like this, open to farmers and end-users. More information has to pass through the investor level, so industries and big investors have to be contacted separately in order to initiate business and then the farmers will follow. More representatives from the industrial part should be participated in this workshop but though many invitations were sent, their participation was limited due to language problems. In the targeted workshops like this one, policy stakeholders like representatives from the Ministry of Industry, Ministry of Agriculture, Ministry of Economics, etc. should also be invited. It would be helpful to have a strategy document on the opportunities for fibre crops to provide to parliamentarians, to help them draft strategies and policy measures. It is a good time for promoting the production of energy crops in Poland. Now there are only 10,000 ha energy crops plantations but another 100,000 ha could be released in the future for growing energy crops. However, farmers don‘t know too much on energy crops. More meetings like this are needed in order the relevant partiers to be informed about the possibilities of cultivating energy crops.

Farmers learn from other farmers, and that is a slow process. The way of getting farmers involved is to have demo farms and free access to them, field dates, and invite farmers to come along. The farmers can not be in meetings like this because they are busy and they don‘t

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speak English. Maybe INF andMP‘s demonstration plantations could be used for such a purpose for the farmers in Poland. [3] To attract investors the projects have to be bankable, else the investor is not going to have any support from the banks. There is not enough confidence on the investment at this stage of development; there are lots of uncertainties as to where to find the raw material, how long will be the investment, what is the market for the products. [3] D. Economic Factors

   

Yield improvement Breakthrough extraction and processing technologies (cost and volume efficient) Development of new/niche products Involving bast fibres and marketing specialists together in marketing activities

Comments from the round table   

 

 

Security of investment is a long-term EU - governmental issue. We have to pass the message to policy makers that we need stability of science and stability of investment. From the industrial part, more innovation in product development is needed, in order to promote investments Long term supply is needed. How long a contract at a certain price do the farmers wish to have and how long a contract with a delivered quantity are the industries willing to sign? In this economic crisis long term contracts are not secure; industries may stop to pay before the end of the contract. From the farmers‘ side a 5 year contract is enough. From the industrial side, usually it is a 10 years contract, which currently refers to length of the contract and not to the price. Fibre crops can have several end uses, so for an investment to be viable the whole feedstock should be used and all possible bio-products should be marketed. That needs innovation, which is one way to add value to the crops we cultivate. Speaking of subsidies, hemp is not considered as an energy crop and as such it cannot be subsidised accordingly; therefore a delegation of INFandMP will participate in the negotiations of hemp‘s inclusion as energy crop that would give a motive for farmers to grow it. The constraint is not in the Ministry of Agriculture but in the Ministry of Health, because hemp is considered as a narcotic. Assessment of cost variables is required in order to estimate profits of the investments. From the investors‘ point of view, an investor wants to maximise profits and minimise risks. Due to the crisis, investors have no assurance they will compensate for the loss. Technologies rapid development is an issue for the investor as he risks investing on a technology that after five years would be out of date. A full understanding of the rate of development of technology is therefore required. The risk taker might be the innovator.

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323

If EU wants fibre industries, it has to provide economic supports. However, markets will globalise after 2013 so the EU bio industry will have to be more competitive and not depended on subsidies and other financial support schemes.

Starting from small, local, farmers‘ owned, cooperative factories that develop markets for a number of products is an example that could be replicated to fibre industries. That was the case of milk dairies in NZ, which started small and local with few farmers proving the milk and producing only a couple of products, but overtime the farmers became more and the products were more and then farmers became the owners of the factories which developed a number of products. Starting small and local avoids complicated logistics and can be proved more efficient than going to large biorefinery plants starting from scratch. [3]      

   

Market appraisal and sensitivity of the costumers to the product quality and price, as well as diversification of the products is needed. Prospects to widen the range of potential feedstock Should we apply for research of more crops? Should we apply for research of more plants, unknown plants? We need focus on research items. Industries like KEFI should extend their experience and business plan to other places, like Jordan or Poland and to new cops. Market legislation and policies will monitor the users of the new crops, like kenaf. In certain areas kenaf maybe attractive to be grown. In Jordan, an energy crops map is going to be developed which would specify/propose crops for specific locations, climate and soil conditions, without taking away the good agricultural lands from food crops. This overarching plan for Jordan will assure the integration of the food, feed, fibre and fuel crops in the national level. The challenge behind the long strategy for energy plants is to research on possible crops to be grown in Jordan. Over time we will experience climate change so adaptation of existing crops /acclimatisation of new crops into different environments will assure crop supplies in the future. Fibre crops have different logistics depending on the length of fibres they produce. For producing long fibre tissue, innovation is needed in the field of transport and logistics, whereas for short fibre production the logistics are solved. In Poland local scale is efficient. The rule in Poland is to have the farms and the factory located within 50km distance, so transportation is negligible.

Farmers desperately look for an extra income and new crops so once they have the chance and the contract with industries, they will grow everything. The problem is who is going to invest on the factory, the farmers or the factory? On the one hand the business plans are not positive and on the other hand international markets supply vast quantities at cheap prices. Somebody has to take the risk (time scale for buildings, machinery, tax incentives that allow industry to grow without costing much to the EU/governments). [3] The afore mentioned restricting factors, research gaps and recommendations how to overcome the barriers in the fluent application of non food crops, especially fibre crops a feed

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stock for modern bio-based industry will be further elaborated, the similar recommendations would be worked out for carbohydrate and oil crops as well.

REFERENCES [1] [2]

[3]

Myrsini Christou, CRES, Greece: Description of work, FP7-KBBE-2007-2B „Crops2Industry‟ Coordination Action. November 2008. Jozef Wasko, M. Praczyk. The chain of raw materials and derived semi-products and products from fibre plants. Institute of Natural Fibres and Medicinal Plants, Poznan, Poland. 2000, up dated in 2009. Myrsini Christou, CRES, Greece: The report 1st Thematic workshop of Crops2Industry EU Project : “Can fibre crops offer a viable alternative land use option and could they support a competitive industry?”, held on 18 November 2009, at the Institute of Natural Fibres and Medicinal Plants, Poznan, Poland. January 2010.

InŚ Renewable Resources and Biotechnology… Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski

ISBN: 978-1-61209-521-9 © 2011 Nova Science Publishers, Inc.

Chapter 34

MULTISCALE METABOLIC MODELING OF CEREALS: AN INTEGRATED SYSTEMS BIOLOGY APPROACH FOR RESEARCH BIOMASS Mohammad R. Hajirezaei1, Mohammad R. Ghaffari1, Björn H. Junker1, Johannes Müller2, Björn Usadel3, Michael Leps 4, Rainer Lemke5 and Falk Schreiber1,6 1

Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany 2 Martin-Luther-University Halle-Wittenberg, Crop and Ecological Physiology / Plant Systems Modeling, Betty-Heimann-Str. 5, D-06120 Halle (Saale), Germany 3 Max Planck Institute of Molecular Plant Physiology, Integrative Carbon Biology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany 4 SunGene GmbH, a BASF Plant Science Company, Corrensstr. 3, 06466 Gatersleben, Germany 5 Julius-Kühn-Institute, Federal Research Centre for Cultivated Plants, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany 6 Martin-Luther-University Halle-Wittenberg, Institute of Computer Science, Von-Seckendorff-Platz 1, D-06120 Halle (Saale), Germany

INTRODUCTION An important goal of climate and energy policy of the Federal Republic of Germany is a significant increase in the cultivation of crops for the production of biomass for energy purposes. Modern plant breeding is a key technology to increase efficiency of metabolic processes which are responsible for the storage of photosynthetic products into the plant biomass. Particularly, those metabolic pathways are of great importance, which lead to the formation of cellulose, starch and sucrose. The identification of possible starting points and bio-technological strategies can be supported by simulation studies based on mathematical models of metabolic networks underlying biomass formation. An essential part of model

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development is the proper validation of the models based on experimental data on stoichiometry, mass dynamics and mass balances of the related metabolic processes. The key points outlined above are the main objective of the 2021-bioenergy network project ―multiscale metabolic modelingŚ an integrative system biological approach for biomass research (short: MMM)‖, which is developed by an interdisciplinary consortium from academic and industrial research. Data collected within the project will be used to parameterize different metabolic models which are hierarchically interconnected with each other (multiple scale modeling, see Fig 2). Dynamic total models of carbon and nitrogen budget are fundamental to whole plant level. For selected tissues, stoichiometric models of primary metabolism will be designed which in turn will be refined by detailed quantitative flux models of the central metabolism. Finally, detailed enzyme kinetic models are developed for metabolic routes crucial for biomass accumulation such as photosynthesis, starch and nucleotide biosynthesis. This hierarchical approach, where one model refines the previous one, will lead to a combined model which provides both an overview of the biological processes and detailed practice-relevant predictions.

ITERATIVE EXPERIMENTAL AND SIMULATED METABOLIC DATA LINK The validation and optimization of metabolic models created in silico requires an iterative match with experimental data. In the present project, barley (Hordeum vulgare L.) is used as model plant since (1) its genetics and metabolism are comparatively well studied, (2) it represents not only an important traditional food plant but usually is included also into agricultural rotations of energy plant production, and (3) results obtained for barley in future may be transferred to other important cereal energy crops like, e. g, rice, wheat, rye or triticale. To this end, different genotypes of barley differing in biomass potential and growth habit will be investigated with regard to metabolite concentration and dynamics, enzyme activities, metabolic fluxes and cell wall composition. Measured data will be used to create, parameterize and validate stoichiometric, kinetic, and mass balance models. The findings and models will be transferred to the crop plant rice (Oryza sativa ) that will be validated using available transgenic lines with high yield. In particular following questions should be answered: (1) modifications of which metabolic pathways in which tissues may be expected to lead to an increase of the yield of usable biomass? (2) Which strategies can be used to increase the yield of primary photosynthetic energy storage by optimizing the metabolic fluxes in the primary metabolism? (3) Which relationships exist between phenotypic characteristics (growth habit, growth height, and biomass) and metabolic processes (carbon and nitrogen budget of primary metabolism, metabolic fluxes into storage pools and cell wall metabolism)? (4) Are there any starting points in the nucleotide sugar metabolism for a change in cell wall composition with the goal of improved energy storage by biomass formation?

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INTEGRATED MODELING AT MULTIPLE LEVELS The development and validation of models of the different metabolic processes related to plant biomass formation and energy storage occupies a central role within the MMM. Generally, models of plant mass budget and metabolism (mass balance models, stoichiometric networks, metabolic flux models and kinetic models) show considerable great differences in the abstraction level (process resolution in time and biological structures) depending on the available a priori knowledge (e.g. enzymatic figures) and on the scope of the model (see Figure 1).

Figure 1. Hierarchical levels of metabolic and mass balance models.

Figure 2. Resolution degree of biological modeling levels. While mass balance models aim to a more complete description of biomass formation, stoichiometric models focus on the primary metabolism and kinetic models simulate selected metabolic pathways.

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The mass balance models describe the dynamics and balances of major functional components of biomass formation in a more aggregated manner, focusing mainly on carbon and nitrogen household in an architectural context. Stoichiometric and flux models refine this approach for selected tissues and metabolic pathways. This way, for example, the complete primary metabolism will be represented. Kinetic models that allow simulating metabolic situation of selected metabolic pathways (see Figure 2) show the highest level of detail. The horizontal (within a hierarchy level) and vertical (via multiple levels) coupling of biophysical and biochemical process models represents a new challenge in plantphysiological systems.

DATA INTEGRATION AND VISUALIZATION Since the development of the modeling framework requires linking various experimental and model generated data types, the integration of data and their user-friendly visualization occupies a central role in MMM project. To support this a data pipeline is created, which already provides access to several of the models described and which can be used to integrate additional models developed within this project. Stoichiometric models of primary metabolism developed from various plant organs (source and sink leaves, stem and grain) will be combined with enzymatic parameters into MetaCrop, an integrated information system which is used for a detailed presentation of metabolic networks (http://metacrop.ipkgatersleben.de).

Figure 3. Interactive visualization of stoichiometric networks in VANTED (FBA-SimVis Add-on ) showing specific fluxes.

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All metabolic pathways available in MetaCrop, such as photosynthesis, cell wall biosynthesis, and other specific models are linked together within the system. Based on MetaCrop, they can be transferred easily into externally developed analysis and simulation tools or directly into the visualization and analysis software VANTED (http://vanted.ipkgatersleben.de, see Figure3) using standard exchange formats such as SBML. In addition, the VANTED Add-on FBA-SimVis (http://fbasimvis.ipk-gatersleben.de) allows analyzing the metabolic networks visually and via Flux Balance Analysis. Software tools such as Copasi (http://www.copasi.org) can be employed for kinetic modeling

PRACTICE-RELEVANT FORECASTS AND FUTURE USE Due to the novel vertical and horizontal integration of biological metabolic and mass balance models, a hierarchical modeling framework will be established that enables both at the same time a detailed practice-relevant prediction and an overview of the related plant physiological processes. The models will be linked in a way that key points can be identified for predictive breeding and targeted genetic changes to optimize and modulate metabolic pathways relevant for biomass formation. These project strategies are based on universally valid rules so that in principle they can be transferred to other energy crops. In a first step, this will be done by validating the models in rice.

CORRELATION BETWEEN METABOLITES AND MORPHOLOGICAL TRAITS: A POWERFUL PREDICTIVE TOOL It has been repeatedly shown that there is either a positive or a negative correlation between metabolic traits and biomass in Arabidopsis (Meyer et al. 2007; Sulpice et al. 2009). This opens the possibility to use identified metabolites and/or genes as biomarkers to predict plant biomass. However, a remaining challenge will be the consideration of different species as well as fluctuating environmental conditions which have great impact on the composition of metabolites and thus on those pathways regulating the outcome of biomass. We, therefore, take advantage of natural varieties differing in their growth and biomass production and will perform a multivariate metabolic and enzyme profiling at various developmental stages of barley plants to address the questions whether firstly a metabolic signature can be found in correlation to plant growth in the vegetative phase as well as in the generative phase and secondly whether metabolic traits and/or related genes might also correlate positively or negatively with the final yield. Preliminary results revealed that indeed specific metabolites identified in leaves (see Figure 4A) or seeds (see Figure 4B) of barley plants correlate positively or negatively with morphological traits demonstrating the power of metabolic composition for biomass accumulation and yield production (see Figure 4). Whether these metabolic traits and/or related genes and proteins directly determine biomass and yield production or are involved in specific metabolic pathways regulating the final yield remains to be elucidated.

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Figure 4. Pearson coefficients of metabolic traits against yield components in Leaves (A) and seeds (B). Significant correlations are shown by one asterisk (p9

6

Quality of farmland

Crop species diversity

Index

> 2.2

1.25

Field size

Median field size

ha

= 10

40

Quality of farmland

Energy balance of farm

GJ/ha

= 80 – 75*GV/ha

50 – 75*GV/ha

GV/ha, AZ

Energy balance of crop

GJ/ha

80 + AZ-20

50

AZ

special GHG emission

kg CO2-Äq/GJ MP

136

BN 1 * 1.5

GV/ha, AZ

4

P-content classification

BK-region

according to ABAG with a C-factor of 0.03 (seed grassland). contamination by technique (PT) divided by pollution load capacity of soil (PB). 3) % of the regional standardized treatment index. 4) from regional guideline value. 5) ÖLF = ecologically and culturally significant area. 6) depends on livestock density (i.e. at 0.8 RGV/ha = 80 kg CO2/GJ market product (MP)). 2)

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The 14 criteria from 6 problem areas (categories) listed in the table, record and assess all major risks standing in the way of these aims. The criteria have been scientifically acknowledged (VDLUFA standpoint 1998), sufficiently put to the test (more than 400 analysed farms), and are professionally supervised by an expert committee consisting of 8 scientists from 6 German States. The result of the analysis is printed out as a graph (Figure 3).

Figure 3. Graph of a KUL analysis result (sector environmental compatibility).

This graph illustrates the 14 test criteria in the sector environmental compatibility (ecology) and their evaluation by rating. The centre of the circle marks the targeted optimum (rating 1, the adjacent light area shows the tolerance range (ratings 2 to 6), and the red area an increasingly critical situation (ratings > 6). In Figure 4 the latter applies to the surface N balance, the field size (cp. Figure5) and the specific, product-related, greenhouse gas emission, indicating exceeding contamination and consequently a need for action or consultation. In the course of analysis the farms receive a detailed consultation and interpretation report, which describes the calculation of criteria, evaluates the ecological status, points out causes for identified shortcomings, and proposes suitable countermeasures. A farm is environmentally compatible if it seeks its economical optimum within the ranges of tolerance. That this is compatible with high productivity has been demonstrated in many cases. Thus, indicators for environmental compatibility are the easily recordable effects, emanating from the farm to the subjects of protection soil, air, water, and biodiversity.

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2.1.2. Sector Social Compatibility - Criteria For Socially Compatible Agriculture Aims:  protection of basic needs (health, work, income)  safeguarding the social capacity for development and ability to function (qualification, equal opportunities, etc.)  participating in social shaping processes

The farm‘s compliance with these aims is tested by 9 criteria from 3 categories. (Table 2) Table 2. Test criteria, targeted optima and tolerance thresholds Category

Test criteria

Dimension

Optimum Rating 1

Tolerance threshold rating 6

Availability of jobs

%7

= 100

70 8

Age distribution

% of AK

variable

variable8

Percentage of women

%

> 43 or < 57

23 or 74

Qualification

%

variable9

variable9

Holidays

Working days

= 30

20

Working conditions

Points

= 12

6

Gross wage level

%10

= 95

70

Social activities

Points

= 11

6

Percentage of owner

%

= 66

51

Occupation

Conditions of employment

Participation 7)

% of the required staff. depending on age group (up to 30, 30 to 50, > 50). 9) depending on level of education (training, with agricultural training, HND or lower secondary leaving certificate). 10) % of gross wage level of the German economy. 8)

Figure 4. Graph of a KSL analysis result (sector social compatibility).

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The KSL criteria are at present still under scientific discussion and in a test phase. Up to now 11 farms were analysed by using this system, which proves its general practicability for the time being. The results are represented graphically, analogously to KUL (Figure3). The goal conflict between the criterion ―income level‖ (gross wages), rated 7, thus exceeding the tolerance range, and the excessive ―availability of jobs‖, rated 1, is obvious here. This means that the farm employs more staff than required by its structure, which in consequence is compensated by deductions from wages.

2.1.3. Sector Economic Compatibility – Criteria of Economically Compatible Agriculture Aims:



ensuring material basics and resources along with maintaining funds for a long-term agricultural production  efficient use of the factors soil, work and funds  preservation of rural areas by value added and creation of agricultural jobs  earning an appropriate income and participating in the general welfare development in favour of agricultural staff  reproduction of innovation capacity and competitiveness in agriculture The 11 criteria of KWL (Tab. 3) cover the above-mentioned aims in a measurable quantity and evaluate them using tolerance ranges. Figure 7 demonstrates that in this example the test criteria for economic compatibility with the exception of the equity ration can be found within the tolerance range; 5 criteria are, however, close to the tolerance threshold (rating 6). The test criteria of KWL are generally accepted and ascertained according to standardized and coordinated algorithms for calculation within the context of the annual financial statement and/or the voluntary accountancy evaluation/five-year accountancy analysis. When assessing data it should be noted that the economic efficiency of a farm does not only depend on the farm‘s management. Decisive determinants are moreover the underlying conditions of agricultural policy, the developments on the global agricultural market, technological progress, the innovation of agricultural production, the progressive structural change, and the increasing division of labour. This requires the continuous reviewing and updating of KWL criteria and the evaluation procedure. A total of 630 farms have been analysed by the system KWL in the fiscal year 2005/06 in Thuringia. The mean of the total random test marks an economic situation that lies on the verge of the tolerance range, and even exceeds it in parts (cp. also para. 3.3).

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Table 3. Test criteria, targeted optima and tolerance thresholds Category

Test criteria

Dimension

Optimum rating 1

Tolerance threshold rating 6

Profitability ratio (ordinary)

%

> 10

0

Return on total assets

%

>5

0

Return on net assets

%

> 10

0

Rel. factor remuneration

%

> 130

90

Debt service competence

%

31

150

Cash flow III

EUR/ha LF

500

50

Equity ratio

%

> 95

60

Changes in equity

EUR/ha LF

> 160

0

Net investments

EUR/ha LF

> 150

0

Income per AK

TEUR/AK

> 50

25

Revenue

EUR/ha LF

> 1200

700

Profitability

Liquidity

Stability

Added value

2.2. Tolerance Ranges as Evaluation Method The 34 test criteria of KSNL (14 KUL, 9 KSL and 11 KWL) are subject to a uniform evaluation procedure based on tolerance ranges.

Figure 5. Graph of a KWL analysis result - sector economical compatibility.

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Figure 5. Principle for the determination of tolerance ranges.

Table 4. KSNL criteria profile illustrated by surface N balance Surface N balance [kg N/ha Bilanzfläche] Definition Method of evaluation Dimension Evaluation Protection goal System level Intent to steer Required data

Difference between supplied and sold N Farm gate balance (kg N/ha) minus NH3-losses (kg NH3-N/ha) kg N/ha balance area (LF minus untilled fallow) Rating 1 = 0 … 20 kg N/haś Rating 6 = 30 … 50 kg N/ha Water, air (N2O), biodiversity, soil fertility Total enterprise Avoidance of N-surplus, preservation soil fertility Receipts for bought-in and sold products (fertilizer, market, feed products etc.)

Table 4. KSNL criteria profile illustrated by surface N balance Data sources Survey costs Location factor Plausibility check Reproducibility Controllability Justiciability Communicability Practicability Scientific consensus

Accountancy, multiple application Low to medium Leachate quantity (mm) Integrated in analysis programme High Given (accounting records) Given for external, objective analysis Balance and evaluation are easy to impart Tested extensively (> 400 farms, 675 analyses) Vote expert committee; VDLUFA standpoint confirmed

Ranges of tolerance (Figure 5), partly determined depending on the location of each test criterion, indicate the margin between a targeted optimum (rating 1) and an only just acceptable impact or an only just tolerable condition (rating 6). Basis of this classification is the overall concept of a sustainable agriculture aiming at

Criteria System of Sustainable Agriculture (CSSA - KSNL)      

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ensuring economic performance with simultaneous high efficiency, preserving soil productivity and reproduction beyond that, limiting adverse effects on the ecosystem to an acceptable level, remuneration for the factors soil, work and funds according to demand, maintaining man-made landscape and a necessary level of biological diversity, and protecting social functions and their reproduction.

The tolerance threshold (rating 6) separates inevitable or tolerable effects or conditions from critical risks - risk increases with higher ratings.

2.3. Profile There is a profile for each KSNL test criterion (Table 4), givening an overview of the characteristics of the respective criterion (cp. KTBL 2008).

3. APPLICATION AND RESULTS 3.1. Possible Fields of Operation Even if individual sectors or KSNL criteria groups may be used for specific problems (cp. para. 4), generally, farmers are advised to apply the entire KSNL criteria set. A sound counselling is only possible when all operational aims are taken into account. With affordable costs for data acquisition, analysis, evaluation, and counselling, KSNL is in a position to     

point out and assess all significant risks jeopardizing sustainable development, recognize conflicts of goals regarding economic, ecological and social interests in order to weigh them up in a responsible manner, reliably provide for an outcome quality -objectively and, if necessary, justiciablewhich permits external use of the results and the issuing of certificates, carry through goal-oriented sustainability consultations on farms, ensure that administrative controls can be completed efficiently.

3.1.1. Data Collection The system boundary for data collection is basically determined by the reliability of data and/or their verifiability, so that for most criteria the farm itself represents the valuation level. If several criteria apply and in case of internal counselling, however, as a matter of routine, the plant level is divided into the sublevels plant cultivation, animal husbandry and bio energy. Besides the farm itself, depending on the test criteria, the balance area (LF minus untilled fallow), livestock (GV), staff (AK) or market product, etc. can be used as a reference parameter. Generally, the type of data collection shall ensure that besides internal counselling tasks the results can be also used for external presentation in agricultural reporting, evaluation of grant policy measures, political advisory work, discussions with environmentalists, water

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managers, etc. Therefore, reliable, reproducible and, if necessary, also justiciable results are required. To secure this claim for security, data collection is based mainly on allocatable data, and the analysis is generally carried through externally -with the exception of KWL- by the central, independent project office at the VAFB in Jena, in order to guarantee a neutral, nationwide standardized and objective analysis and evaluation. KWL uses standardized and verified algorithms for calculation, which are analysed by accounting services and also within the context of voluntary accountancy evaluation/five-year accountancy analysis.

Figure 6. Weighted mean of Thuringian reference farms 2005/06 .

3.1.2. Selected Results The entire KSNL has been so far tested and optimised extensively on TLL reference farms on an area of approx. 10,000 ha in a three-year cycle. The selected reference farms characterize the situation of agriculture in Thuringia, even without the right to be statistically representative. In comparison to the top-level farms in Thuringia, the following basic statement can be derived from the analysis (Figure 6): From an economic point of view the sustainability assessment signifies quite a critical situation for Thuringian farms in regard to crop years 2005 and 2006, reflecting the high economic pressure the farms feel exposed to. There are various causes for the repetitive occurrence of critical, i.e. not sustainable, conditions, requiring analysis on a case-by-case basis. The question remains if and to what extent this dissatisfying situation can be improved by agricultural adjustment reactions. As Thuringian top-level farms can be called economically stable and future proof (Figure 7), according to their mean evaluation results, an economically sustainable development seems to be basically possible under the present general conditions.

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Figure 7. Weighted average of Thuringian top-level farms 2005/06.

As to environmental compatibility, results show (Figure 6) that with the exceptions of nitrogen balance, erosion disposition and specific emissions from greenhouses, Thuringian agriculture does not present a significant risk either for the sustainability of production function or for adjacent ecosystems, even if in individual cases tolerance levels of numerous criteria have been exceeded. Particularly, the bearers of the VDLUFA certificate ―Environmentally compatible farm ‖ prove that toe holds for an environmentally compatible performance can mainly be found in farm managements, and that high productivity, i.e. a high energy balance in plant cultivation, and environmental compatibility are not mutually exclusive (cp. Figure 8; para. 3.3.). On the agro-social sector, only the results of Thuringian reference farms covering an area of approx. 10,000 ha are available (Figure 6) up to now. On average, this choice of farms observe the tolerance range of all criteria, with the exception of gross wage levels. This is a sign of a content agro-social situation, without ignoring that some of the nine test criteria are on verge of of tolerance thresholds, even exceeding them in individual cases. On the social sector Thuringian top level farms demonstrate also that under present general conditions future compatible agro-social structures can be implemented (Figure 7).

3.3. Cause Analysis Confirmed exceedance of a tolerance range indicates that in the respective criteria sector sustainable development is not provided any longer. This fact makes a specific operational cause analysis necessary. The causes for critical states are mainly farm management problems or goal conflicts between criteria. Management problems are among the dominating causes for non-sustainable processes. Examples to prove this fact can be found primarily in the results of the environmental compatibility analysis KUL, due to its comparatively wide scope of inspection. Figure 8

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demonstrates on the basis of 228 analysed farms how the influence of local and operational factors, which are commonly believed to have an effect on environmental compatibility, is superimposed by management factors. On the left, the diagram shows the relevant influencing factors. The farm‘s percentage in a certain criterion is illustrated by the bars, e.g., 30 % of the inspected farms‘ quality of the soil is up to 35, approx. 50 % of the farms quality is between 35 to 60, and 20 % of the farms have a soil quality of over 60; topmost bar. The diagram shows an appropriate distribution of the listed influencing factors covered by the 228 farms.

Figure 8. Influence of local and operational factors on environmental compatibility (issuing of certificates) of farms (n = 228).

The points in the bars symbolize the distribution of the 32 certificates awarded by the VDLUFA to farms, which demonstrably operate within the specified tolerance ranges and thus represent an integral measure of environmental compatibility. The certificate has only been awarded since the year 2000; the KUL analysis however, has been carried through since 1994. Insofar, the ratio of 32 certificate bearers in proportion to 228 analysed farms is not realistic. The percentage of certificate awards in the total analyses exceeds 20 % very rarely. It becomes apparent that the certificate is awarded independently from the influencing variables to be considered. The certificate is received by farms in suboptimal and optimal locations, by big and small farms, farms with low and high intensity, etc. The certificate is received by comparatively many organic farms (bottom right); however, also farms with an extraordinarily high productivity of over 120 GJ/ha obtained the certificate. Although the mentioned factors (Figure 8 left) have undoubtedly a certain influence on environmental compatibility, the effect is overlaid by respective farm managements which determine - as the dominating factor - environmental compatibility and also, according to initial results, sustainability on an operational level. Thus, the results show also that it is unacceptable to draw conclusions regarding environmental compatibility of farms solely from

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the mentioned influencing factors or from the level of intensity or even consider such factors an indicator for decision-making in agricultural policy. In fact, the results demonstrate that starting points in view to environmental compatibility can be found particularly in farm managements. An improvement of the ecological and also economical and social situation can consequently only be expected if farmers will be enabled to recognize operational weaknesses and their causes by means of suitable valuation systems and derive together with their advisor appropriate adjustment reactions from that. While management problems can be solved comparatively easy – as a rule, already by identifying them – goal conflicts require professional need for action and sometimes also reference to the policy regime. Identifying these conflicts and putting them up for political and scientific discussion is therefore the actual challenge of the sustainability concept. Goal conflicts due to differing interests either require to build a consensus after legally protected interests have been evaluated or a solution brought about by resorting to natural science or political instruments. Goal Conflicts within The Sectors Goal conflicts can occur within or between sectors. An example of a frequent goal conflict within the sector environmental compatibility is the partly vehement claim for soil cultivation without plough on the one hand, and the involved increased plant protection product consumption on the other hand. Such conflicts have to be registered, discussed and solved deliberatively within the scope of individual sectors. In case of the above-mentioned example, the circumstances are reasonably transparent. Soil cultivation without plough is interesting for the majority of farmers because it increases the vigour and is possibly cheaper. On the other hand the consumption of plant protection products increases in most cases, sometimes even beyond the tolerable dosage. On a site, which is disposed to erosion, this increased ecological impact can possibly be justified by the decreased erosion disposition. In these cases, the exceedance will be indicated but not assessed unfavourably (white instead of red bar). On the field level, however, this happens less frequently, so that balancing processes must be performed site-specifically, taking all variables into account, in order to justifiably solve the conflict. With this approach other conflicts within the sectors can also be analysed, like

    

job supply vs. wage level, technologically optimal field sizes vs. biodiversity, extensification vs. glasshouse emission per produced unit of market product, high revenue/ha vs. return on equity and assets, high net investments vs. ability to pay debts etc.

Goal Conflicts between the Sectors There are often goal conflicts between the sectors economic interests and ecological or agro-social goals. Examples are

  

return on capital vs. job supply, conditions of work, wage level, holidays changes in equity and/or net investments vs. wage level return on capital vs. fruit species diversity, ÖLF provision, soil protection.

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Farmers are striving primarily for economic success, like all other entrepreneurs. This is a normal and sensible attitude if certain limits, mainly found on the environmental and agrosocial sector, are observed. If these limits are pointing towards irreversible processes they have generally priority over economic interests. Like this, quasi guide rails are given along which the farmer can optimise his entrepreneurial goal (Figure 9).

critical impacts and/or states – optimisation of agricultural production process = leeway for farmers = tolerance ranges. Figure 9. Guide rail principle: optimisation of entrepreneurial goals, considering non-exceedable limits.

Goal Conflicts with Agro-Political Causes Besides the conflicts discussed above, goal conflicts triggered by general agro-political conditions and implemented via subsidies have to be kept in mind. These include mainly measures for market relief, like land set-asides and extensification, decreasing the valuecreation potential and the volume of work in rural areas. The economic losing generated in such a way has to be compensated by public funds. This applies generally also to specifications with a guiding character, which often appear to be regimental, restricting the farmer‘s necessary freedom of decision. This is why, in many cases, practicable instruments that set controllable goals without prescribing the way to them are more convincing and promise progress along with creative handling of goal conflicts.

3.4. Conclusion and Summary of Results The statements made here on all three sustainability sectors apply, mind you, only to the mean of the inspected farms. Considerable differences can be found between farms to some extent. This fact points out that the focus should be on the individual farm and not on an abstract average value if sustainable development shall be put into practice.

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Hence, with the described test procedures the scientific and technical pre-conditions are available to measure and analyse sustainability on farms which differ completely in regard to their organisation and location and to prompt the necessary actions, irrespective of whatever optimisations might be necessary in individual cases. This procedure meets the claim for a reliable assessment procedure that     

recognizes avoidable shortcomings and their causes, enables a goal-oriented optimisation of operational procedures and measures, contributes to the reduction of costs by pointing out efficiency reserves, puts the farmer in a better argumentative position to defend himself against general reproaches, and provides advantages in the market through demonstrable sustainable management certificate.

However, allowances are also made for agro-political demands on a system     

that makes objective reporting on sustainable development of the agricultural sector possible, by means of which the effects of agricultural and/or grant policy measures can be analysed (political control of success), which is administratively easily controllable by external, i.e. objective, analysis and assessment, that brings transparency and conceptional clarity into the present sustainability discussion, and by which the argumentative position and the public image of agriculture can be improved.

And ultimately such a procedure succeeds in accompanying and supervising the future sustainability challenges in the face of agriculture. Such challenges refer mainly to    

the extremely growing need for biomass, triggering an increasing competition for surfaces between food, energy and resource plants, the consequences of structural change, which is accelerated considerably by growing stress of competition in the liberalised agricultural market, the effects that result from increased demands on the quality of products and on the ecological and social environment, the generally unknown consequences of an emerging climatic change.

In order to sustainability create these extremely complex and controversial processes, an extensive analytical system like KSNL is required to reliably recognize non-sustainable movements and occurring goal conflicts. This is regarded as a precondition to optimise operational and also agro-political decision-making processes in due time.

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4. KSNL AREAS OF APPLICATION On the grounds of a predominantly provable database and an external, objective analysis, KSNL guarantees an outcome quality, which besides the operational advisory tasks also allows applications with external effects. At the same time, the modular set-up permits also the use of individual sectors and/or test criteria for special problems. The following areas of application are mainly coming into question:     

tested farm network sustainability issuing of certificates evaluation of grant programmes limitation of regimenting instruments application during studies and training

4.1. Tested Farms Network Sustainability A decisive instrument to create the complex processes of future developments in a sustainable manner is the set-up of a tested farms network sustainability, which on the basis of representative farms makes the required data available at a local, regional, but also at federal level. Such a tested farms network can be definitely considered an advancement of the existing economic tested farms accountancy: it can be a far-reaching decision-making aid in agricultural politics to sustainably shape farming, serving simultaneously as a means of supervision for orientation and optimisation of farm-related and also agro-political decisions. Due to its consistent outcome quality, KSNL is especially suited for this task. Based on KUL, several years of experience have been gained with the environmental tested farms network in Thuringia. In a nutshell, a tested farms network ―sustainability‖ can    

facilitate objective coverage on sustainable development in the agricultural sector, analyse the effects of agricultural and/or grant policy measures (political control of success), improve the argumentative position and public image of farming, and bring transparency and conceptual clarity into the present sustainability discussion.

4.2. Issuing of Certificates The initial version of the KSNL certificate is the KUL certificate ―Environmentally compatible farm‖ introduced in the year 2000. This certificate is awarded to farms by VDLUFA if they have controllably met the respective test conditions. With this certificate a scientific association operating nationwide attests environmental compatibility to farms. This certificate can be used to obtain a trading advantage but also to document environmental compatibility to administrations, nature conservationists, water supply and distribution boards, etc.

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Figure 10. Test criteria for the VDLUFA certificate ―Environmentally compatible farm‖.

Based on these experiences, a KSNL certificate ―Environmentally compatible farm― is currently being developed together with the TÜV (Technical control board) Thuringia and will be completed very shortly. The KSNL certificate will include inclusion 15 to 20 test criteria (Figure 11). Their tolerance ranges shall not be exceeded. The obtainment of this KSNL certificate documents verifiably that a farm meets the basic conditions for a sustainable, future proof development. Such farms are reference farms, which demonstrate controllably sustainable farming in their region.

Figure 11. Test criteria of the KSNL certificate "Sustainable farming‖.

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4.3. Evaluation of Development Programmes The appropriate evaluation of development programmes gains increasing importance in the face of limited financial means. It is to be anticipated that in future only programmes, which have proved to be purposeful in view to such evaluations, will be continued. Therefore, in many cases, target-oriented development programmes instead of the usual guidelineoriented represent a more practical solution. If so, objectively controllable KSNL test criteria can be drawn on as a precondition for assistance and also for evaluation. The Directive of the Thuringian Ministry of Agriculture, Nature Conservation and Environment on the current agricultural investment development programme (AFP) contains a rating procedure to determine if farms are eligible for assistance, using selected KSNL criteria from the sector economical compatibility (KWL) (Figure 12).

4.4. Limitation of Regimenting Instruments Increasing importance should be attached to the development of practicable instruments, which outline controllable aims, taking different locational and structural cause and effect mechanisms into account. This includes criteria systems setting revisable aims but depending on the situation and locational conditions - giving farmers plenty of rope to achieve them. This maintains entrepreneurial freedom, supports creative search for optimal solutions and involves agricultural sciences and agricultural advisory services in decisionmaking processes. The successful appliance of KSNL and/or individual criteria groups could therefore attract interest as an incentive, e.g. in risk assessment for CC-controls but also in cross compliance advisory services (Figure 13).

Figure 12. Test criteria to examine farms before getting gouvernmental-support.

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Figure 13. Test criteria for cross compliance advisory service.

4.5. Application during Studies and Training Last but not least, criteria systems like KSNL, which dissolve sustainability into concrete, controllable parts, are suitable as course contents for trainings. In consequence, it is will be possible to  illustrate complex contexts visibly and comprehensibly,  pursue and supervise sustainability on all farm levels,  recognize goal conflicts, and  acquire skills for conflict resolution. Moreover, systems by which different scenarios can be evaluated in a transparent way are didactically very suited to sensitise trainees and students and to introduce them to the complex issue sustainability.

5. SUMMARY AND CONCLUSION With KSNL a serviceable instrument is made available, allowing farmers at affordable costs to take the test and find out if they meet the requirements of sustainable development or not. Thus, the foundations are laid to lead farming altogether to the road of sustainability and to accompany future challenges by supervision. As ultimately the necessary sustainable development of the agricultural sector can only be implemented on a farm level, in future, primarily agricultural politicians are requested to develop scientifically acknowledged sustainability assessments for farms and/or to support pilot projects regarding tested farms network ―sustainability‖. That would optimise

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practicability of the systems in use, accelerate the scientific discussion on sustainability criteria, and also assist pressure groups in rural areas to cooperate in solving goal conflicts.

REFERENCES Agenda 21, 14.25 (1992): Documents on conference of United Nations on Environment and Development. Rio de Janeiro, June 1992. Committee of enquiry ―Human and environmental protection – aims and basic conditions of a sustainable future compatible development‖ (1997)Ś Sustainability concept– foundations for tomorrow‘s society. Federal Diet printed paper 13/7400 Criteria system sustainable agriculture (KSNL). KTBL script in print. VDLUFA viewpoint (1998): Criteria for environmentally compatible agricultural management (KUL); Eckert, Breitschuh, Hege, Sauerbeck. Publisher: Association of agricultural institutes for studies and research. Breitschuh, G.: Eckert, H. (2000): Problems of and approaches to a sustainable development of agriculture. In: Congress volume 2000 Stuttgart-Hohenheim- Sustainable agriculture part 1. VDLUFA-series 55/2000, p. 17-22

INDEX 2 21st century, 312, 314

A absorption, 11, 16, 17, 19, 53, 116, 196, 198, 284, 337 abstraction, 327 access, 243, 244, 265, 268, 299, 321, 328 accessions, 238, 241, 243, 244, 245, 249 accounting, 263, 374, 382, 384 acetic acid, 193, 203, 276 acetone, 148, 153 acetylation, 20 acid, 41, 42, 43, 44, 47, 51, 52, 54, 55, 61, 93, 94, 99, 113, 131, 132, 135, 136, 138, 139, 149, 192, 193, 194, 201, 202, 203, 205, 207, 210, 211, 212, 214, 236, 238, 239, 241, 242, 261, 272, 274, 276, 280, 299, 302, 303, 304, 305, 306, 307, 308, 309, 334, 347, 348, 351, 352, 353, 354, 360 acidic, 205, 236, 274 acidity, 194 acne, 334, 335 acrylic acid, 304 active compound, 11, 59, 60 additives, xii, 76, 220, 226, 317, 365, 366 adhesion, 12 adhesive properties, 316 adhesives, 211, 316 adjustment, 212, 241, 384, 387 adsorption, 165, 205, 284 advancement, 390 adverse effects, 383 agar, 224, 333 age, 149, 153, 330, 379 aggregation, 61

agricultural market, 380, 389 Agricultural residue, 68, 69, 70 agricultural sector, 389, 390, 393 agriculture, xiv, 21, 50, 86, 89, 98, 100, 104, 123, 128, 264, 265, 267, 271, 280, 304, 311, 312, 313, 314, 315, 357, 372, 373, 374, 375, 376, 380, 382, 384, 385, 389, 394 Agrobacterium, 226 air pollutants, 158 albumin, 212, 221 alcohol production, 112 algae, 347, 348, 349, 350, 354 alimentary canal, 100 alkaloids, 204 allergic reaction, 57 alternative energy, 229, 291 alternative fuels, 167, 168, 169 alters, 193, 202 aluminium, 51 ambient air, 350 amino, 210, 211, 275, 280 amino acid, 210, 211, 275 amino acids, 275 ammonia, 81, 82, 83, 84, 85, 86, 275 ammonium, 81, 82, 85, 86, 251, 252, 254, 255, 259, 260, 261 amputation, 358 amylase, 132, 140 anaerobic digesters, 63, 64 anaerobic digestion, 271, 274, 277, 302 analgesic, 334 anatomy, 314 animal husbandry, 375, 383 annealing, 224 antibiotic, 342, 344 antioxidant, 61, 64, 65, 148, 150, 151, 155, 334, 337 antipyretic, 334 APC, 132, 136, 137, 138 apoptosis, 65

396

Index

aptitude, 359 aquaculture, 210, 255 aqueous solutions, 213 Arabidopsis thaliana, 331 arginine, 280 arsenic, 262 Art of the Renewable Resources, viii Asia, 12, 59, 78, 95, 334, 363 Asian countries, 363, 364 aspartate, 280 aspartic acid, 280 assessment, 42, 43, 79, 187, 268, 288, 290, 303, 312, 313, 355, 375, 384, 389, 392 assets, 264, 381, 387 astringent, 60 atherosclerosis, 61 atmosphere, 86, 87, 123, 158, 160, 221, 230, 364 atoms, 232, 354 Austria, 79, 142, 149, 238, 286, 311 authorities, 365, 370 authority, 375 Automobile, 358 awareness, 1, 55, 320, 321

B Bacillus subtilis, 132, 337, 344 bacteria, xiii, 61, 96, 106, 224, 225, 226, 299, 306, 308, 337 bacteriostatic, 106 balance sheet, 109, 112 barriers, 101, 319, 323 base, 70, 94, 116, 198, 202, 226, 267, 268, 283, 300, 316, 317, 336, 337, 347, 348, 350, 357, 363, 364 basic needs, 379 basic raw materials, 318 bedding, 113 beet molasses, 113 Belgium, 103 bending, 16, 17 beneficial effect, 41, 46, 124 benefits, 12, 229, 230 bioavailability, 212 bio-based products, 299, 301, 308, 312, 314, 317 biochemistry, 157 Biocompound carrier, 251 bioconversion, 304, 305, 306 biodegradability, 193, 202, 293 Biodegradable, ix, 251, 255 biodegradable materials, 293 biodegradation, xiii, 261 biodiesel, xv, 180, 210, 232, 236, 239, 258, 283, 315, 347, 348, 349, 353, 354, 355

biodiversity, 2, 243, 376, 378, 382, 387 bioenergy, xiv, 68, 79, 89, 238, 263, 264, 265, 266, 267, 268, 326, 348 Biofilm, 251, 252 biofuel, 59, 99, 101, 118, 284, 348 biogas, 1, 2, 3, 4, 6, 8, 22, 81, 84, 86, 87, 90, 94, 95, 98, 128, 131, 140, 142, 266, 267, 271, 273, 274, 275, 277, 283, 284, 285, 286, 287, 288, 289, 290, 291 Biogas crop, 1 biological consequences, 148 biological control, 148, 149 biological processes, 326 biologically active compounds, 11 biomarkers, 329 biomaterials, xiii, 21, 23, 303, 357, 358, 359, 360, 361 biopolymer, 110 biopolymers, 19, 279, 315, 358 biosphere, 377 biostimulator, 41, 42, 43, 45 biosynthesis, 148, 326, 329 biotechnology, xiii, xv, 105, 299, 302, 306, 309, 316, 321, 350 birds, 63, 64, 245 black liquor, 147, 149, 151, 153 bleaching, 13, 158, 194, 197, 206 blends, 107 blood, 60, 61, 65, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156 blood plasma, 147, 148, 149, 150, 151, 152, 153, 154, 156 bonding, 16, 26 bonds, 26, 163, 236, 280, 337, 354 branching, 2, 43, 45, 46 breast cancer, 334 breathing, 42 breeding, xii, xiii, 8, 39, 60, 61, 100, 105, 181, 241, 242, 243, 244, 249, 312, 318, 319, 325, 329 Britain, 98 brittleness, 18 building blocks, 305, 306 butyl ether, 101 buyers, 102 by-products, 11, 12, 105, 210, 220, 289, 320, 363

C cadmium, 257, 258, 259, 261, 262 Cairo, 104 calcium, 61, 86, 193, 202 calcium carbonate, 86 calibration, 149

Index caloric intake, 12 calorie, 231 calorimetry, 293 cancer, 61, 64, 65, 334 cancer cells, 61 candidates, 305, 348, 354 capillary, 197, 198 capsule, 230, 233 carbohydrate, 97, 204, 231, 274, 275, 280, 324 carbohydrate metabolism, 280 carbohydrates, 230, 231, 237, 271, 281, 305, 308, 314, 315, 319 carbon, 24, 25, 26, 68, 91, 95, 168, 174, 230, 231, 232, 236, 237, 252, 271, 272, 273, 274, 275, 276, 277, 299, 301, 305, 306, 319, 326, 328, 347, 349, 354 carbon atoms, 354 carbon dioxide, 68, 91, 95, 230, 231, 237, 271, 272, 273, 274, 275, 276, 277, 347 carbon monoxide, 168, 236, 237 carbon neutral, 349 carboxylic acid, 93, 242 carotenoids, 114, 204 catalyst, 194, 203, 204, 236 cattle, 209, 210, 278 C-C, 26 cell line, 64, 337 cell phones, 358 cellulose, xiii, 12, 13, 61, 131, 132, 149, 241, 242, 247, 276, 301, 325 Census, xi, 60 Central Europe, 106 certificate, 375, 379, 385, 386, 389, 390, 391 challenges, 19, 209, 299, 300, 302, 389, 393 chemical, 12, 13, 32, 33, 42, 55, 69, 71, 75, 109, 113, 132, 133, 147, 148, 151, 154, 155, 158, 193, 194, 197, 204, 211, 221, 229, 230, 231, 234, 235, 237, 238, 242, 244, 247, 279, 280, 283, 285, 287, 291, 300, 301, 302, 303, 304, 305, 308, 317, 318, 337, 348, 360 chemical characteristics, 69 chemical industry, 113, 304, 305 chemical properties, 42, 71, 229, 230, 234, 235, 237, 238 chemical reactions, 42 chemicals, 12, 33, 124, 203, 280, 300, 304, 316, 320 China, 62, 90, 104, 168, 364 chlorine, 24 chloroform, 193, 203, 351, 352 chlorophyll, 204 cholesterol, 61 chromatograms, 93 chromatography, 91, 215, 257, 259, 353

397

circulation, 42, 230 CIS, 284, 287 cities, 158, 159 citizens, 363 clarity, 341, 389, 390 classes, 74, 226, 355 classification, 53, 57, 166, 182, 184, 212, 302, 335, 359, 361, 369, 370, 371, 377, 382 cleaning, xii, 90, 192, 194, 202, 285, 287, 288 clients, 145 climate, 1, 59, 89, 90, 119, 168, 232, 233, 241, 248, 284, 299, 302, 308, 314, 323, 325, 357, 371 climate change, 299, 302, 308, 323 clone, 93, 95, 181, 182, 184, 187, 188, 223, 224, 225, 226, 336 cloning, 188, 224 clothing, 53, 55, 57, 319 clusters, 73, 264, 308 C-N, 16 CO2, 1, 28, 68, 77, 82, 84, 123, 141, 142, 143, 144, 145, 167, 168, 169, 180, 214, 221, 232, 274, 275, 280, 290, 347, 348, 350, 372, 377 coal, 24, 25, 70, 90, 126, 127, 288, 347 coatings, 142, 211, 212 color, 49, 51, 52, 53, 54, 194, 204, 206, 212 combined effect, 156 combustion, 21, 22, 23, 24, 26, 27, 28, 67, 70, 72, 73, 74, 75, 76, 77, 79, 86, 127, 142, 167, 168, 169, 176, 179, 231, 236, 237, 288, 294, 296, 334, 347 combustion processes, 75 commerce, 284 commercial, 38, 115, 168, 184, 210, 219, 220, 234, 280, 286, 306, 337 commodity, 39, 314, 358 commodity markets, 314 Commonwealth of Independent States, 284 communication, 190, 374 compaction, 67, 124, 377 compatibility, 193, 202, 375, 376, 378, 379, 380, 381, 385, 386, 387, 390, 392 compensation, 142, 264 competition, 109, 263, 264, 265, 266, 287, 303, 389 competitive advantage, 142, 145 competitiveness, 302, 380 compilation, 265 composites, xii, 11, 12, 14, 16, 17, 18, 19, 20, 293, 294, 295, 296, 297 composition, 1, 2, 6, 7, 8, 12, 23, 69, 75, 91, 93, 150, 192, 201, 202, 210, 211, 220, 229, 230, 231, 235, 236, 237, 238, 239, 241, 242, 244, 247, 273, 275, 326, 329, 347, 349, 350, 351, 353, 354, 365 composting, 22, 24, 25, 26, 27, 28

398

Index

compounds, 42, 43, 44, 45, 59, 87, 93, 106, 166, 185, 210, 230, 231, 237, 258, 281, 306, 308, 360, 361 compression, 176, 252 conditioning, 16, 143, 213, 285, 289 conductance, 330 conference, xi, xii, xiii, xiv, xv, 20, 78, 394 conflict, 319, 380, 387, 393 conflict resolution, 393 consciousness, 118, 318 consensus, 382, 387 consent, 375 conservation, 90, 95, 234, 238 constituents, 335, 337 construction, 31, 37, 53, 105, 265, 308 consumers, 142, 314 consumption, 21, 101, 102, 107, 109, 110, 112, 117, 124, 143, 144, 145, 168, 169, 175, 179, 230, 231, 237, 264, 279, 284, 293, 304, 308, 318, 387 contaminated soil, 262 contaminated soils, 262 contamination, 189, 223, 224, 258, 376, 377, 378 content analysis, 33 control condition, 257, 259, 261 control group, 60, 150, 152 controversial, 389 cooking, 217, 229, 230, 348, 351 cooling, 14, 60, 134, 143, 245 cooperation, x, 41, 42, 132, 249, 303, 305, 358, 360, 361, 371, 373 corns, 41, 44, 47 correlation, 37, 75, 77, 148, 150, 152, 195, 233, 277, 329 correlation coefficient, 152, 233 correlations, xiii, 148, 153, 154, 330 corrosion, 23, 24, 75, 77 cortex, 119, 120 cosmetic, 31, 106, 304, 335, 337, 341 cosmetics, 53, 113, 114, 115, 212, 317, 333, 334, 335, 344 cost, 12, 13, 182, 193, 198, 202, 212, 259, 264, 305, 312, 313, 316, 320, 321, 322, 374 cost effectiveness, 305 cotton, 13, 51, 186, 189 cotyledon, 210 Council of Europe, 95 course content, 393 covalent bond, 12 covering, 31, 143, 385 creep, 359 critical state, 385 crop, xiv, 1, 2, 6, 8, 25, 42, 43, 68, 89, 97, 99, 103, 107, 122, 123, 124, 125, 126, 127, 145, 181, 210,

232, 243, 257, 258, 287, 312, 313, 319, 320, 322, 323, 326, 330, 377, 384 crop production, 124 crop residue, 25, 68 crop rotations, 1 crops, xii, xiv, xv, 1, 2, 5, 21, 22, 23, 24, 25, 26, 29, 41, 42, 43, 62, 78, 79, 81, 89, 97, 98, 103, 106, 113, 116, 118, 119, 123, 124, 125, 126, 127, 128, 129, 145, 181, 186, 229, 230, 238, 258, 262, 286, 288, 311, 312, 313, 314, 315, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 329, 348 crude oil, 102, 193, 194, 195, 196, 229, 230, 232, 234, 235, 236, 347 crystalline, 12, 13, 134 crystallinity, 360 cultivar, 3, 4, 5, 32, 34, 37, 59, 60, 247, 259, 262 cultivars, 1, 2, 3, 4, 5, 6, 7, 8, 32, 33, 37, 38, 47, 99, 106, 110, 181, 248, 249, 321 cultivation, 1, 5, 8, 38, 39, 43, 47, 86, 89, 90, 92, 97, 99, 100, 101, 103, 104, 105, 107, 109, 111, 113, 114, 115, 116, 119, 121, 123, 124, 125, 126, 127, 128, 223, 232, 237, 243, 259, 277, 280, 281, 312, 313, 314, 318, 320, 325, 334, 336, 350, 351, 354, 383, 385, 387 cultivation conditions, 89, 277 culture, xii, 106, 223, 307, 353 culture conditions, 353 cycles, xii, 183, 184, 224, 230, 335 cycling, 182 cysteine, 257, 258, 259, 261 Czech Republic, xii, 47, 79, 131

D data analysis, 331 data collection, 266, 383 database, 267, 302, 304, 390 debts, 387 decay, 161, 162, 318 decision makers, 229, 230 decision-making process, 389, 392 decomposition, 294 decontamination, 258, 261 decortication, 316 decoupling, 287 deficiency, 2 deficit, 2, 9, 42, 43 deflate, 143 deformation, 18, 234 degradation, 61, 79, 157, 165, 256 degraded area, 106 degumming, 204 denitrification, 87, 251, 254, 255, 256

Index Denmark, 14 density values, 74 Department of Agriculture, 333 Department of Energy, 102, 308, 309 deposition, 308 deposits, 61, 236, 364 depth, 35, 283 derivatives, 93, 102 desolventizing, 209, 213, 214, 216, 217, 219, 220, 221 destruction, 71, 95 detectable, 174, 175, 178, 224 detection, xiii, 224, 226 detergents, 52, 113, 212 detoxification, 212, 214, 221, 258 developed countries, 238 developing countries, 95 deviation, 4, 14, 18, 377 diesel engine, 167, 169, 173, 179, 180, 236, 353 diesel engines, 167, 169, 236, 353 diesel fuel, 101, 169, 171, 175, 178, 179, 180, 236, 237, 348 diffusion, 86, 333, 336, 341, 342, 343, 344 digested residue, xiv, 81, 82, 83, 84, 85, 86 digestibility, 4 digestion, 114, 271, 274, 275, 277, 302, 351 dioecious hemp, 241, 242, 243 discomfort, 334 diseases, 3, 61, 114, 148, 334 dispersion, 27, 108, 193, 194, 196, 202, 211 displacement, 196, 199 disposition, 377, 385, 387 distillation, 37, 38, 199 distilled water, 203, 213, 259, 350 distribution, 67, 68, 69, 70, 71, 74, 75, 283, 284, 285, 379, 386, 390 divergence, 189, 238 diversification, 323 diversity, 181, 186, 187, 241, 249, 319, 377, 383, 387 DNA, viii, xiii, 95, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 181, 182, 183, 184, 185, 186, 188, 189, 190, 224, 225, 226, 254 DNA damage, 148, 155 DNA polymerase, 182, 183, 184 DNA sequencing, 184 domestic markets, 108 dosage, 22, 27, 387 dosing, 11, 14, 15, 19 double bonds, 236, 354 drinking water, 148, 149, 151, 153 drought, 1, 2, 41, 42, 43, 44, 45, 47, 90, 321 drug addict, 243

399

drug addiction, 243 drugs, 115, 317 dry matter, 1, 2, 3, 4, 5, 6, 8, 85, 90, 94, 106, 124, 127, 280 dry matter content, 1, 2, 3, 5, 8 dry matter yield, 1, 4, 8, 85 drying, 26, 33, 63, 71, 100, 203, 219, 234, 367, 370 durability, 44, 47, 67, 69, 73, 77, 78 dusts, 27 dyeing, 49, 51, 53, 55, 62, 64, 114, 116 dyes, 49, 50, 53, 55

E East Asia, 334 ecology, 12, 318, 378 economic crisis, 322 economic development, 263 economic efficiency, 380 economic evaluation, 79 economic growth, 300, 319 economic performance, 264, 383 economics, 124, 263, 284 economies of scale, 289 education, v, 379 Efficiency of Four Crop Species, viii, 123 effluent, 5 effluents, 308 Egypt, 104 elaboration, 111 election, 340 electrical properties, 360 electrical resistance, 20 electricity, 62, 86, 87, 142, 144, 229, 230, 267, 283, 284, 291, 347, 348 electrolysis, 142 electron, 157, 165, 337 electrophoresis, 184, 224 elongation, 45 e-mail, 21, 59, 81, 103, 104, 263, 299 embryogenesis, 223, 224, 227 emerging markets, 168 emission, 12, 24, 77, 83, 84, 123, 168, 169, 174, 236, 284, 291, 359, 377, 378, 387 employees, 264 employment, 167, 302, 379 emulsions, 210, 211 encapsulation, 212 endothelial dysfunction, 61, 65 end-users, 321 energy consumption, 124, 143, 144, 145, 284 energy density, 67, 69, 74

400

Index

energy efficiency, 67, 69, 73, 123, 124, 127, 128, 129, 141, 142, 144, 145 energy input, 123, 124, 126, 128 energy supply, 145, 264, 284 engineering, xiii, 89, 132, 249, 251, 258, 305, 306, 308, 358, 365 England, 155 Enhanced Oil Recovery, 192, 194, 199, 207 entrepreneurs, 388 environment, xiv, 12, 21, 29, 42, 52, 108, 229, 232, 233, 236, 237, 316, 318, 357, 373, 389 environment factors, 232, 233 environmental aspects, 291, 308 environmental conditions, 242, 252, 318, 321, 329 environmental factors, 233 environmental impact, 290, 308, 312, 313, 319, 348 environmental protection, 50, 102, 394 enzyme, 11, 12, 13, 14, 15, 16, 19, 60, 132, 133, 134, 136, 137, 138, 140, 193, 202, 212, 280, 326, 329 enzymes, 11, 13, 20, 131, 132, 134, 136, 137, 138, 140, 299, 320 epidermis, 31 equilibrium, 161, 195 equipment, 62, 63, 116, 142, 144, 193, 203, 204, 218, 229, 234, 238 equity, 380, 381, 387 erosion, 90, 91, 95, 371, 385, 387 erythrocytes, 149 ESI, 261 ESR, 157, 158 ester, 16, 113, 169, 171, 173, 191, 193, 194, 195, 197, 199, 202, 203, 204, 205, 206, 207, 236, 353, 354 ethanol, xiv, 3, 102, 110, 111, 117, 118, 149, 193, 203, 336, 340, 342, 343, 352 ethers, 101 ethyl alcohol, 110 EU, 98, 99, 104, 106, 108, 109, 110, 112, 115, 183, 185, 209, 212, 242, 284, 286, 287, 290, 302, 303, 304, 309, 312, 313, 321, 322, 323, 324, 357 Europe, v, 9, 12, 29, 62, 95, 98, 102, 103, 104, 106, 119, 210, 242, 280, 287, 300, 309, 312, 313, 319 European Commission, 98, 103, 104, 292, 311 European Community, 22 European market, 118 European Parliament, 309 European Regional Development Fund, 303 European Union, 284 evaporation, 336 evidence, 188, 360 exchange rate, 230 expectorant, 334 expertise, 315, 355

exploitation, 95, 258, 337 exports, 98, 263 exposure, 51, 149, 152, 163, 258 extraction, xv, 91, 101, 116, 185, 186, 189, 204, 209, 212, 213, 214, 215, 216, 219, 220, 229, 230, 234, 238, 258, 318, 322, 333, 336, 337, 341, 342, 343, 347, 349, 350, 351, 352, 353, 354, 355 extracts, 260, 261, 317, 333, 335, 337, 341, 342, 343, 344, 351 extrusion, 71, 75, 252

F factories, 101, 104, 108, 319, 323, 348, 369 families, 334 farm land, 91, 92, 93, 94 farmers, 118, 128, 312, 319, 321, 322, 323, 372, 383, 387, 388, 392, 393 farmland, 357, 372, 377 farms, 100, 321, 323, 372, 373, 374, 375, 378, 380, 382, 383, 384, 385, 386, 388, 389, 390, 391, 392, 393 fat, 6, 7, 12, 13, 16, 17, 204, 238, 242, 302, 315, 348 fatty acids, xv, 201, 202, 236, 238, 247, 276, 348, 353, 354 feedstock, xv, 68, 102, 201, 202, 214, 215, 283, 285, 287, 288, 291, 300, 303, 312, 313, 314, 315, 317, 320, 322, 323, 348 fermentation, 89, 95, 98, 214, 220, 284, 285, 299, 302, 303, 305, 306, 307, 308, 309, 355 fertility, 5, 124, 382 fertilization, 32, 36, 86, 94, 232 fertilizers, 33, 124, 126, 128 fiber, 6, 13, 20, 61, 97, 103, 104, 105, 106, 107, 108, 109, 186, 214, 242, 247, 249, 308 fiber content, 6, 247, 249 fibers, 104, 106, 109, 121, 210, 211 fibra flax, 41 fibre plant, 223, 314, 324 filament, 365 fillers, 211, 293, 297, 360 films, 211, 212, 220 filters, 77 filtration, 215, 218, 336 financial, 8, 104, 111, 255, 266, 292, 323, 380, 392 financial condition, 111 financial support, 8, 255, 292, 323 fingerprints, 95, 181 fire retardancy, 297 fire retardants, 297 first generation, 169 fish, 209 fission, 300

Index flame, 168, 360, 361 flame retardants, 360, 361 flammability, xiii, 293, 294, 297 flavonoids, 60, 61, 64, 114 flax fiber, 106 flexibility, 62, 143, 233 flooding, 90, 199 flour, 74, 363 flowers, 52, 60, 232, 242 fluid, 195, 198, 218, 219, 281 fluidized bed, 209, 218, 219, 220, 221 fluorescence, 149 foams, 210, 211 folic acid, 61 food, xii, xv, 12, 21, 31, 59, 61, 63, 89, 90, 97, 98, 99, 100, 101, 109, 111, 112, 113, 128, 148, 209, 211, 212, 243, 257, 258, 271, 280, 299, 300, 304, 306, 311, 312, 313, 314, 315, 317, 323, 326, 348, 363, 389 food chain, 258 food industry, 21, 31, 59, 63, 271, 315 food production, 257, 258, 348 food products, 97, 98, 111 force, 70, 77, 99, 194, 195, 197, 198, 234, 300, 312, 371 formation, xiv, 23, 28, 32, 33, 91, 92, 93, 94, 157, 162, 165, 167, 168, 169, 191, 192, 193, 194, 195, 197, 202, 206, 207, 210, 215, 236, 237, 252, 259, 261, 271, 272, 273, 274, 275, 276, 277, 325, 326, 327, 328, 329 formula, 4, 42, 43, 44, 193, 197, 202, 232 foundations, 393, 394 fragments, 182, 184, 224 France, xii, 69, 98, 104, 284, 311, 313, 314 free energy, 194 freedom, 388, 392 friction, 52, 176 frost, 3, 90, 91, 281 fruits, 59, 60, 61, 63, 116, 232, 238 fuel cell, xiv, 142 fuel consumption, 21, 124, 168, 169, 175, 179 fuel prices, 68 functionalization, 214, 221 funding, 303, 304 funds, 109, 374, 380, 383, 388 fungus, 96 fusion, 300

G gas, nutrient loop, 81 gasification, 70, 86, 168, 284, 285, 302, 334 GC-content, 185

401

gel, 210, 215, 224 gel formation, 215 gene bank, xiii, 241 genes, 90, 190, 329 genetic diversity, 181, 186, 187 genetic factors, 37 genetic marker, 190 genetic resource, xiii, 233, 241, 243, 244, 245 genetically modified plants (GMP), 279 genetics, 186, 312, 326 genome, 280 genotype, 33, 229, 241, 242, 249 genotyping, 183, 185, 187, 188 genus, 60, 118, 354 geometry, 12 Georgia, v, 47 Germany, v, xi, xii, xiii, xiv, xv, 1, 2, 4, 8, 9, 11, 13, 14, 20, 24, 67, 70, 77, 78, 79, 81, 95, 98, 104, 108, 118, 141, 167, 182, 191, 201, 221, 223, 251, 263, 265, 279, 284, 285, 286, 287, 304, 311, 325, 333, 347, 350, 369, 374 germination, 259 glass transition, 360 glass transition temperature, 360 global warming, 2, 68, 347 glucose, 60, 65, 132, 133, 134, 136, 137, 138, 139, 306 glucosidases, 60 glucoside, 64 glucosinolates, 212 glutamate, 113 glutathione, 258, 262 glycerol, 193, 194, 205, 236 goods and services, 263, 264 governments, 323 Grain by-product, 11, 12 granules, 13, 14 graph, 233, 378 grass, 12, 21, 89, 90, 94, 95, 140 grasses, 89, 94, 118, 262 grazing, 3 Great Britain, 98 Greece, 311, 314, 324 green alga, 355 green land, 98 greenhouse, xiv, 21, 68, 81, 82, 84, 86, 245, 246, 263, 283, 290, 299, 347, 348, 372, 378 greenhouse gases, xiv, 68, 81, 82, 84, 372 greenhouses, 385 grounding, 76 growth, 2, 3, 9, 41, 42, 43, 44, 45, 46, 47, 59, 60, 68, 82, 83, 85, 91, 92, 96, 98, 134, 231, 232, 241, 242, 252, 258, 261, 300, 314, 317, 319, 326, 329,

402

Index

330, 331, 334, 335, 336, 337, 338, 339, 344, 348, 350 growth rate, 60, 331, 348 growth temperature, 330 guidelines, 124 Guinea, xii, xv, 229, 233, 235, 238

hydrophobic properties, 211, 212 hydroxide, 14, 135 hydroxyl, 148, 162, 163 hydroxyl groups, 162, 163 hyperglycemia, 61 hypothesis, 37

H

I

habitat, 42, 43 halogen, 360 hardness, 90, 91, 191, 192, 234 harvesting, xiii, 38, 39, 93, 99, 105, 116, 242, 245, 247, 280, 314, 316, 320, 333, 336, 337, 355 health, 59, 61, 223, 300, 363, 364, 379 health care, 59 heat release, 293, 294, 295, 296 heat transfer, 74 heating rate, 14 heavy metals, 69, 108, 257, 258, 261 height, 3, 43, 60, 242, 248, 326, 337, 338, 339, 344 hematopoietic system, 147 hemicellulose, 12, 16, 61, 301 hemisphere, 334 hemp, xiii, 13, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 49, 53, 54, 55, 97, 98, 103, 104, 105, 106, 107, 108, 127, 181, 186, 189, 241, 242, 243, 244, 245, 247, 248, 249, 293, 294, 295, 296, 314, 316, 318, 319, 320, 322 hemp fiber, 106, 242 herbal medicine, 114, 119 heritability, 318 heterogeneity, 151 hexane, 193, 203, 213, 216, 218, 351, 352, 353, 354 high school, v history, 95 hives, 294 homeostasis, 147 homes, 144 homogeneity, 44, 47, 320 horizontal integration, 329 human, 12, 57, 61, 64, 65, 106, 115, 116, 148, 155, 209, 210, 221, 281, 299, 317 human body, 106 human health, 61 humidity, 14, 35, 244, 245 humus, 63, 64, 82, 86, 336, 377 husbandry, 375, 383 hybridization, 60 hydrocarbons, 168, 230, 236 hydrogels, 279 hydrogen, 16, 141, 142, 193, 203, 337 hydrolysis, 131, 132, 133, 204, 272, 302, 306, 352

ideal, 67, 95, 348 identification, 65, 97, 181, 189, 232, 264, 325, 374 illumination, 350 image, 55, 71, 77, 172, 360, 389, 390 image analysis, 71, 77 images, 145, 172, 179 immunity, 321 impact assessment, 303 impact strength, 14, 17, 361 imports, 320, 357 improvements, 197, 214, 221, 266, 268, 317 impurities, 194, 204, 206, 232, 307, 308, 348 in vitro, xiii, 106, 155, 182, 185, 186, 223, 224 in vivo, 155 income, 302, 319, 323, 357, 379, 380 independence, 357 India, 47, 61, 90, 102, 168, 239, 367 individuals, 182, 184, 185, 186, 189 Indonesia, xii, xv, 191, 192, 195, 201, 203 industrial processing, 110, 112 industries, 12, 21, 31, 59, 68, 110, 113, 191, 192, 198, 202, 293, 315, 320, 321, 322, 323, 334, 337 industry, xv, 12, 13, 20, 21, 31, 41, 59, 62, 63, 64, 98, 100, 101, 103, 104, 105, 106, 108, 109, 110, 112, 113, 115, 141, 144, 168, 202, 264, 271, 280, 293, 300, 302, 303, 304, 305, 308, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 324, 357, 358, 359 inequality, 319 inflammatory cells, 337, 344 inflation, 289 inflorescence harvest, 35, 37, 38 infrastructure, 141, 283, 284 ingredients, 337, 365, 369 inhibition, 42, 61, 65, 182, 258, 306, 342 inhibitor, 83, 84, 85, 305, 342 inorganic fillers, 360 institutions, 249, 314 insulation, 107, 316 insulin, 60 integration, 13, 170, 267, 280, 286, 288, 323, 328, 329, 330 interface, 12, 194, 211 interference, 281

Index intervention, 155 investment, 264, 288, 289, 320, 322, 355, 392 investments, 289, 319, 321, 322, 381, 387 investors, 321, 322 iodine, 193, 194, 203, 204, 205, 206, 207, 236 ionization, 308 ionizing radiation, 147, 148, 154 ions, 197, 258, 308 IR spectra, 14, 163 Iran, 364 irradiation, 148, 149, 151, 152, 153, 156 irrigation, 336 isolation, 144, 183, 185, 186, 189, 190, 245, 355, 366 isomers, 360 isotope, 85 issues, 312, 313 Italy, 13, 284, 311, 313, 314, 316

J Japan, 91, 92, 93, 94, 149, 364 jatropha curcas, 201, 202 job creation, 302 Jordan, 323

K KBr, 14 kinetic model, 326, 327, 329 kinetic parameters, 278 kinetics, xiv, 147, 157, 166, 271, 305 KINS program, 149 KOH, 193, 194, 203, 204, 205, 206, 207

L laboratory tests, 32, 44, 83 labour force, 371 lactic acid, 299, 303, 304, 305, 306, 307, 308, 309, 360 lactobacillus, 309 landscape, 235, 377, 383 leaching, 82, 86 lead, 1, 84, 86, 136, 143, 151, 168, 169, 175, 176, 178, 214, 217, 221, 223, 236, 261, 262, 280, 284, 325, 326, 359, 393 lead-acid battery, 143 leadership, 313 learning, 35 legality, 23

403

legislation, 110, 167, 323 lentigo, 61 leukemia, 61 level of education, 379 liberation, 70, 76 life cycle, 290 lifetime, 142 light, 2, 49, 51, 53, 55, 143, 169, 170, 172, 183, 184, 224, 231, 363, 364, 365, 367, 378 lignin, xv, 12, 13, 16, 17, 53, 61, 69, 71, 79, 106, 157, 158, 160, 161, 162, 163, 164, 165, 301, 319 linen, 49, 51, 52, 53, 54, 55, 57, 103, 104, 105, 107, 108 lipid peroxidation, 65, 147, 148, 155, 156 lipids, 16, 17, 148, 150, 155, 348, 349, 350, 355 liquid fuels, 101, 110, 168 liquid phase, 196, 205 liver, 61, 149, 150 liver cancer, 61 livestock, 101, 285, 371, 377, 383 logistics, xii, xiv, 79, 86, 141, 142, 143, 144, 145, 312, 313, 323 Logistics Centers, viii, 141 Louisiana, 199 low temperatures, 178 LPO intensity, 147, 148, 150, 151, 152, 154 LPO regulatory system, 148, 151 LSD, 34, 35, 36, 46 LTD, 113 lubricants, 299, 313, 315 lung cancer, 64

M machinery, 108, 109, 124, 126, 280, 316, 323 macromolecules, 306 magnesium, 61 majority, 92, 264, 387 man, 2, 319, 383 management, 145, 318, 319, 380, 385, 386, 387, 389, 394 manufacturing, 12, 55, 62, 316, 358, 360, 361 manure, 81, 86, 277, 288 market penetration, 303 market segment, 319, 321 marketing, 289, 316, 322, 335 Maryland, v mass, 45, 75, 78, 91, 111, 115, 132, 134, 136, 137, 138, 139, 168, 172, 175, 217, 218, 230, 231, 247, 248, 258, 272, 273, 274, 277, 284, 294, 296, 318, 326, 327, 328, 329, 348, 371 mass loss, 294, 296 mass spectrometry, 91

404

Index

material resources, 22 materials, xiii, 12, 13, 17, 19, 20, 21, 22, 55, 62, 63, 64, 68, 69, 70, 71, 72, 74, 75, 102, 105, 108, 115, 117, 119, 121, 122, 168, 209, 219, 247, 285, 286, 288, 293, 294, 299, 300, 301, 304, 305, 306, 307, 314, 316, 324, 334, 357, 358, 359, 360, 361, 363, 364, 365 matrix, 12, 17, 251, 252, 255, 294 matter, 1, 2, 3, 4, 5, 6, 8, 73, 85, 90, 94, 106, 124, 127, 168, 169, 175, 179, 214, 230, 231, 236, 273, 280, 383 measurement, 169, 170, 179, 195, 197, 206, 207, 293, 344 measurements, 25, 43, 44, 149, 150, 153, 247, 259, 339 meat, 209, 211 mechanical properties, 11, 20, 234, 238, 359, 360 media, 306, 348, 350 medical, xiii, xv, 229 medicine, 61, 63, 114, 316 melt, 11, 14, 19, 253 melting, 13, 14, 23, 183, 185, 360 melting temperature, 13, 14, 23, 183 membranes, 150, 306, 308 MES, viii, xv, 191, 192, 193, 194, 195, 196, 197, 198, 199, 201, 202, 203, 205, 206 Metabolic, x, 308, 325, 326 metabolic pathways, 325, 326, 327, 328, 329 metabolism, 280, 282, 326, 327, 328, 330 metabolites, xiii, 42, 329 metal ion, 258 metal ions, 258 metals, 69, 108, 212, 257, 258, 261, 262 meter, 98, 193, 203, 232 methane yield, 1, 4, 8, 94 methanol, 193, 194, 203, 204, 236, 348, 351, 352 methodology, 365 methyl ester sulfonate, 191, 197 methylene blue, 193, 203 Mexico, 261 mice, 61, 147, 148, 149, 150, 151, 152, 153, 154, 156, 281 microclimate, 57 micromycetes, 79 micronutrients, 82 microorganisms, 93, 252, 281, 299, 306, 337, 342 micropropagation, 223 microscope, 193, 203 migration, 64 mildew, 190 mineralization, 82, 86 miniature, 348 Ministry of Education, 8, 255

Minneapolis, 79 missions, 1, 68, 81, 83, 141, 143, 167, 168, 169, 175, 179 mitochondrial DNA, 224 mixing, 14, 72, 168, 236, 365, 369, 370 models, 272, 325, 326, 327, 328, 329 modernization, 109 modifications, 17, 167, 326 moisture, 11, 14, 16, 42, 44, 68, 71, 72, 74, 77, 95, 211, 238, 244 moisture content, 14, 68, 71, 74, 77, 238 molasses, 102, 113 molecular biology, 105 molecular mobility, 19 molecular weight, 198, 236, 258 molecules, 13, 42, 194, 236, 280, 281, 306, 335 monoecious hemp, 241, 242, 249 monosodium glutamate, 113 monounsaturated fatty acids, 353 morphogenesis, 42, 44, 47 morphology, 11, 12, 314 Moscow, xi, xii, xiii, xv, 147, 155, 156, 157 motivation, 9 moulding, 11, 14, 294, 358, 360, 361 mucus, 224, 226 mulberry, xv, 59, 60, 61, 62, 63, 64, 65 multiplication, 89 Myanmar, 364 mycorrhiza, 91

N Na2SO4, 193, 203 NaCl, 193, 203, 259, 261, 337 nanocomposites, 166 narcotic, 248, 322 National Research Council, 95 natural fillers, 293, 297 natural gas, 142, 143, 283, 284, 285, 287, 291, 347 natural resources, 271, 373 natural science, 387 nature conservation, 390 Nd, 64 necrosis, 223 negative experiences, 368 net investment, 387 Netherlands, 166, 286, 311, 314, 317 neutral, 68, 77, 142, 213, 223, 280, 348, 349, 384 neutral lipids, 348 New Zealand, 53, 57, 122 niche market, 320 Nigeria, 364 nitric oxide, 237, 337, 344

Index nitrification, 83, 84, 85, 86, 87, 251, 252, 256 nitrite, 337 nitrogen, xiv, xv, 23, 32, 35, 36, 38, 81, 82, 84, 85, 86, 87, 124, 126, 157, 158, 160, 163, 164, 165, 168, 169, 170, 178, 179, 308, 319, 326, 328, 330, 336, 337, 338, 344, 385 nitrogen compounds, 87 nitrogen dioxide, xv, 157, 158, 160, 164, 165 nitrogen gas, 170, 178, 179 nitrous oxide, 81, 82, 83, 84 North America, 12, 334 nucleic acid, 42 numerical analysis, 180 nursing, 358 nutrient, xii, 1, 2, 4, 6, 8, 81, 86, 223, 299, 306 nutrients, 2, 3, 6, 33, 82, 83, 86, 89, 280, 306, 339 nutrition, 12, 105, 209, 210, 221

O oil production, 96, 99, 198, 232, 348 oilseed, xv, 97, 98, 99, 100, 101, 209, 210 oligomers, 258 operating costs, 142, 308 operating range, 219 operations, 77, 145, 232, 306, 318 opportunities, 89, 105, 115, 312, 313, 321, 379 optical measuring devices, 167 optical parameters, 172 optimization, 28, 168, 169, 229, 271, 305, 326, 361 oral presentations, xii, xiii, xiv, xv organic matter, 231 organism, 148 organs, 42, 328 osmosis, 320 overpopulation, 119 overproduction, 61, 99 overtime, 323 ownership, 104 oxidation, 61, 148, 168, 204, 276, 353 oxidation products, 148 oxygen, 60, 158, 220, 237, 293 oxygen consumption, 293

P Pacific, 95, 122, 309 paints, 100, 101 Pakistan, 364 palm oil, 192, 349 PAN, 39 parallel, 162, 170

405

Parliament, 309 participants, xii, 375 particle size distribution, 68, 69, 70, 71, 74, 75 pastures, 98 pathogens, 65, 243, 336 pathways, 283, 284, 285, 287, 288, 325, 326, 327, 328, 329 PCR, ix, xiii, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 223, 224, 225, 226 peptides, 257, 258, 259, 260, 261 peripheral blood, 148 peroxidation, 65, 147, 148, 155, 156 peroxynitrite, 65 Peru, 52 pesticide, 39 pests, 3, 12, 61, 63, 64 petroleum, 101, 193, 198, 202, 203, 300, 316, 317 Petroleum, 198 pH, 13, 33, 44, 82, 133, 136, 149, 193, 194, 203, 205, 206, 207, 212, 213, 259, 305, 336, 377 pharmaceutical, 59, 63, 105, 113, 114, 115, 280, 304, 314, 315, 316, 317, 334, 335 pharmaceuticals, xii, 100, 212, 311, 312, 313, 334 pharmaceutics, 335 PHB, 251, 253, 255 phenol, 157, 165, 193, 203, 316 phenolic compounds, 185, 281 phenolphthalein, 193, 203 phenoxyl radicals, 160 Philadelphia, 121 Philippines, 95 phloem, 62 phosphate, 13, 193, 202, 351, 352 phospholipids, 348 phosphorous, 61 phosphorus, 124 photometric analysis, 337 photosynthesis, 42, 231, 232, 326, 329, 330, 348 physical and mechanical properties, 234 physical characteristics, 44, 69 physical properties, 238 physicochemical characteristics, 193 physicochemical properties, 204, 205, 206, 207 physics, 166 Physiological, 53 phytochelatins, 257, 258, 261, 262 phytoremediation, 90, 257, 258, 261 pigmentation, 64 pitch, 206 plant growth, 82, 83, 85, 241, 329, 331, 339 plant protection, 33, 41, 106, 114, 119, 387 plasmid, 224 plastics, 12, 211, 357, 358, 360, 363, 364

406

Index

platform, 281 plausibility, 268 Poland, v, viii, xi, xii, xiii, xiv, xv, 39, 41, 42, 49, 59, 60, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 128, 129, 241, 242, 243, 249, 257, 293, 311, 313, 314, 320, 321, 322, 323, 324 polarity, 12, 197 policy, 128, 244, 320, 321, 322, 325, 380, 383, 387, 389, 390 policy makers, 322 politics, 264, 390 pollutants, 158, 258 pollution, 63, 64, 90, 119, 158, 229, 257, 258, 318, 373, 377 poly(3-hydroxybutyrate), xiii polyhydroxybutyrate, 281 polymer, 19, 166, 199, 251, 254, 279, 280, 282, 293, 304 polymer nanocomposites, 166 polymerase, 183, 184, 189 polymerase chain reaction, 189 polymers, xii, 158, 166, 251, 252, 255, 293, 304, 308, 313 polymorphism, 189, 248 polyoxyethylensorbitanmonooleate, 148 polypeptide, 211 polypeptides, 258 polyphenols, 185, 188 polypropylene, 11, 14, 18, 19, 20, 293, 294 polyunsaturated fat, 353 polyunsaturated fatty acids, 353 population, 12, 189, 299, 336 potassium, 24, 51, 61, 113, 124, 126, 193, 203 potato, 111, 277, 280, 281 potential benefits, 12 power generation, 68, 300 power plants, xiii, 69, 77, 143, 334 precipitation, 2, 308, 336 preeclampsia, 154 pregnancy, 148, 154 preparation, xv, 14, 22, 24, 42, 132, 133, 136, 137, 138, 139, 224, 232, 287, 306, 317, 336, 337 preservation, 357, 377, 380, 382 President, v, 280 President Clinton, 280 pressure gradient, 71 pressure groups, 394 prevention, xiv, 61, 81, 84, 86, 358 primary products, 264, 333 processing, xv, 12, 13, 71, 98, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 145, 182, 204, 209,

210, 212, 213, 216, 220, 221, 229, 230, 238, 242, 302, 306, 307, 314, 316, 318, 319, 320, 321, 322, 350, 363, 364 producers, 98, 102, 118, 122, 132, 142, 266, 267, 315, 358 production costs, 181, 264, 288, 289, 290, 291, 350, 355 production function, 385 production quota, 112 production technology, 286 professionalism, v profit, 283, 318 profitability, 109, 112, 127, 128, 301, 318, 377 project, 5, 38, 77, 81, 82, 87, 89, 90, 95, 102, 116, 143, 169, 170, 174, 176, 178, 181, 218, 220, 223, 281, 288, 303, 311, 312, 317, 326, 328, 329, 333, 335, 337, 340, 364, 370, 384 proliferation, 61 proline, 42 promoter, 280 propagation, 181, 223, 279 propane, 290 prosperity, v protection, 1, 33, 41, 50, 53, 55, 57, 90, 95, 97, 102, 106, 114, 119, 122, 181, 243, 284, 334, 357, 372, 376, 377, 378, 379, 387, 394 protein components, 262 protein synthesis, 42 proteins, xiii, 13, 42, 155, 209, 210, 211, 212, 213, 214, 215, 216, 220, 221, 257, 258, 259, 260, 261, 271, 275, 276, 277, 302, 329 public awareness, 320 public opinion, 279 publishing, v pulp, 31, 39, 106, 113, 158, 320 purification, 37, 185, 193, 194, 203, 285, 309, 355 purity, 149, 210, 215, 243, 307 PVP, 182, 183, 185, 186 pyrolysis, 70, 79, 288, 302, 348

Q quality control, 71, 315 quality standards, 70 quercetin, 60, 61 quotas, 110

R radiation, 53, 55, 57, 90, 106, 143, 147, 148, 149, 154, 155, 156, 231, 232 Radiation, 154, 155, 156

Index radiation damage, 155 radical reactions, 158, 160 radicals, 157, 158, 160, 161, 162, 163, 164, 165 rape, 1, 43, 46, 47, 62, 97, 98, 99, 100, 101, 102, 173, 214 rape seed, 173 rapeseed, protein, 209 raw materials, xiii, 21, 68, 69, 70, 72, 74, 75, 102, 115, 117, 119, 168, 209, 286, 300, 301, 304, 305, 307, 314, 316, 318, 324, 334, 357, 358, 365 reactant, 192, 193, 194, 202 reactants, 193 reaction mechanism, 155 reaction temperature, 133, 203, 206 reactions, 12, 42, 57, 75, 157, 158, 160, 165, 166, 182, 183, 184, 185, 273, 274, 384, 387 reactive groups, 158 recombination, 161 recommendations, 313, 314, 315, 317, 318, 323 recovery, xv, 191, 192, 193, 194, 198, 201, 202, 207, 229, 230, 234, 238 recovery process, 207 recycling, 285 reforms, 104 regenerate, 230, 231, 237 regional economic impacts, 263 regionalization, 318 regions of the world, 241, 242 regression, 148, 153, 154 regulations, 106, 209, 212 reinforcement, 12, 17, 360 remediation, 262 renewable energy, 68, 90, 118, 121, 128, 230, 264, 348 renewable fuel, 102, 113 repair, 155 reparation, 224, 317 repellent, 39 repetitions, 266, 336 reproduction, 380, 383 requirements, 1, 62, 99, 102, 106, 210, 211, 213, 217, 300, 305, 306, 314, 317, 320, 358, 360, 375, 393 research institutions, 249 researchers, 317 reserves, 99, 374, 389 residuals, 299 residues, xii, xiv, 11, 12, 21, 22, 23, 24, 25, 26, 27, 67, 68, 70, 81, 82, 83, 84, 85, 86, 257, 259, 280, 285, 286, 288, 300, 302, 316 resins, 308, 311, 312, 313, 314, 315, 316 resistance, 11, 19, 20, 41, 42, 43, 47, 49, 61, 77, 89, 90, 101, 190, 192, 194, 319, 358, 359, 360, 366

407

resolution, 14, 71, 74, 170, 171, 182, 327, 393 resource utilization, 308 resources, xiii, xv, 12, 19, 22, 68, 86, 97, 102, 108, 121, 123, 128, 131, 143, 229, 233, 238, 241, 243, 244, 245, 264, 266, 267, 271, 279, 284, 299, 300, 302, 303, 305, 306, 311, 316, 317, 319, 333, 334, 357, 361, 373, 380 respiration, 230, 231, 237 response, 42, 147, 151, 259, 261, 347 restoration, 258 restrictions, 187, 287 restructuring, 104 rice husk, 12, 13, 14, 15, 16, 17, 18, 19 risk, 2, 24, 68, 75, 82, 322, 323, 363, 364, 383, 385, 392 risk assessment, 392 risks, 243, 322, 373, 378, 383 RNA, xiii, 185, 189, 190 rodents, 63, 64 Romania, v, 311, 313, 314 room temperature, 13, 14, 161 root, 2, 46, 65, 86, 90, 91, 93, 96, 226, 258 root system, 90, 93 roots, 44, 60, 61, 86, 90, 91, 92, 93, 94, 116, 189, 258, 262 rotations, 2, 73, 326 roughness, 12 routes, 283, 285, 286, 287, 291, 312, 313, 314, 315, 326 rowing, 106, 209, 257 rubber, 236 rules, 123, 303, 329 rural areas, xii, 116, 119, 263, 264, 287, 302, 357, 380, 388, 394 Russia, v, xi, xii, xiii, xv, 147, 149, 154, 157, 247 rye straw, 68

S safety, 102, 171, 238, 279, 280, 319 salinity, 191, 192, 194, 195, 199, 202, 206, 207 salinity levels, 202, 206, 207 salts, 299, 304, 306 samplings, 358 saponin, 113 saturation, 198, 259, 260, 261 savings, 144, 168 scaling, 289 science, 20, 249, 320, 322, 387 scientific progress, 300 scope, 5, 243, 290, 313, 314, 327, 358, 385, 387 sea level, 2 seasonality, 231

408

Index

second generation, 168 secondary data, 266 secretion, 31 security, 284, 348, 384 seed, 3, 12, 31, 32, 33, 99, 100, 173, 203, 204, 210, 214, 229, 230, 233, 234, 236, 238, 239, 241, 242, 244, 245, 247, 248, 253, 259, 261, 349, 377 seedlings, 257, 259, 260 self-image, 55 semen, 119 sensing, 350 sensitivity, 147, 148, 154, 323 sequencing, 184, 188, 224 shape, 67, 68, 70, 71, 72, 73, 74, 175, 195, 217, 390 shoot, 45, 90, 92 shoots, 60, 92, 116, 258 shortage, 12, 42, 43, 167, 210, 320, 333 showing, 259, 328 shrubs, 334 silica, 23, 364 silk, 49, 51, 52, 53, 54, 55, 56 silkworm, 60, 61, 62 silver, 261 simulation, 325, 329 skilled personnel, 316 skin, 55, 57, 61, 204, 334, 335 sludge, 253, 271 social development, 229 social environment, 389 social interests, 383 social structure, 385 society, 115, 118, 119, 229, 314, 318, 373, 374, 394 sodium, 13, 24, 113, 135, 149, 193, 203, 224, 308 sodium hydroxide, 14, 135 software, 144, 145, 169, 172, 184, 224, 314, 329 soil erosion, 371 soil pollution, 90 soil type, 336 solidification, 236 solubility, 44, 47, 212, 213, 215, 342 solution, 44, 86, 134, 142, 145, 148, 149, 185, 191, 195, 203, 215, 257, 259, 260, 261, 297, 307, 308, 316, 337, 387, 392 solvents, 203, 304, 313 somatic embryogenesis, 223, 224, 227 Sorghum, vii, xiv, 1, 2, 3, 4, 5, 9 South Dakota, 78 sowing, 6, 8, 32, 33, 35, 36, 37, 38, 39, 99, 106, 108, 112, 124, 241, 243, 244, 245, 320 Spain, 90, 102 specialists, 322 specialization, 319 specialty crop, 313

species, xiv, 3, 49, 50, 52, 59, 60, 114, 115, 116, 118, 119, 121, 123, 124, 127, 147, 151, 153, 181, 186, 187, 223, 224, 225, 226, 227, 241, 261, 329, 334, 336, 337, 338, 339, 340, 341, 344, 351, 352, 353, 354, 377, 387 specifications, 171, 265, 266, 268, 388 spectroscopy, 158 spin, 157 spleen, 150 spray formation, xiv, 167 Spring, 43, 44, 45, 126 spring barley, 45, 123, 124, 125, 126, 127, 128 Sri Lanka, 364 stability, 67, 90, 182, 184, 185, 194, 238, 256, 322, 366, 368, 369, 370 stabilization, 77, 90, 210, 215 stable radicals, 157, 158, 161, 165 stakeholders, 312, 314, 321 standard deviation, 4, 14, 18, 377 standardization, 37 starch, 3, 6, 13, 97, 109, 110, 111, 112, 122, 193, 203, 231, 276, 277, 301, 302, 306, 325, 326 state, xiv, 6, 61, 74, 75, 76, 104, 128, 148, 165, 189, 243, 266, 307, 385 states, 74, 304, 318, 376, 388 sterile, 184, 337 sterols, 348 stinging nettle, 181, 223 stoichiometry, 326 stomach, 334 stomata, 42, 330 storage, 69, 70, 73, 74, 77, 82, 144, 145, 204, 212, 220, 231, 244, 245, 277, 280, 318, 325, 326, 327, 353 storage and retrieval vehicles (SRVs), 144 straw pellets, 68, 72, 73, 74, 75, 77 stress, 1, 41, 42, 45, 47, 214, 258, 259, 261, 262, 312, 389 stretching, 16, 163 structure, 16, 42, 53, 54, 102, 104, 109, 110, 112, 167, 169, 197, 211, 231, 244, 264, 280, 320, 370, 380 substitutes, 316 substitution, 279, 299, 306 substrate, 1, 42, 63, 83, 86, 252, 254, 255, 271, 272, 273, 274, 275, 276, 277, 278, 285, 306 substrates, xiv, 65, 86, 220, 267, 271, 272, 273, 275, 277, 283, 285, 286, 306 sucrose, 325 sugar beet, 44, 46, 47, 97, 98, 113, 117, 123, 124, 125, 126, 127, 128, 313 sugar mills, 113 sulfate, 86, 193

Index supervision, 390, 393 supplementation, 148 supply chain, 79, 266, 312, 313, 318, 320, 358 surface area, 194 surface chemistry, 12 surface properties, 13 surface tension, 44, 47, 175, 207 surfactant, xv, 44, 156, 191, 192, 193, 194, 195, 196, 197, 198, 199, 201, 202, 205, 206, 207 surfactants, 41, 191, 199, 207 surplus, 68, 98, 382 survival, 92, 279 survival rate, 92 susceptibility, 8 sustainability, 141, 142, 145, 168, 283, 284, 300, 308, 312, 313, 355, 372, 373, 374, 375, 383, 384, 385, 386, 388, 389, 390, 393 sustainable development, xii, 102, 123, 372, 373, 374, 383, 384, 385, 388, 389, 390, 393, 394 sustainable energy, 145, 264, 283, 355 sweat, 49 Sweden, 78, 104, 286, 287 Switzerland, xii, 286, 287 symptoms, 258, 259 synchronize, 171 synthesis, 42, 166, 232, 258, 279, 280, 302, 360 synthetic fuels, 168

T talc, 360 talent, v tanks, 142, 277 tannins, 62, 114 target, 184, 215, 314, 315, 392 tax incentive, 323 taxes, 264 taxonomy, 60 techniques, 71, 105, 189, 229, 230, 232, 237, 249, 320 technological progress, 380 technologies, xii, 79, 86, 105, 121, 141, 142, 143, 144, 145, 230, 238, 264, 285, 293, 300, 303, 312, 317, 320, 322 technology, 12, 20, 55, 86, 89, 102, 105, 141, 143, 144, 145, 191, 192, 198, 209, 213, 218, 221, 284, 286, 290, 302, 317, 318, 320, 322, 325, 358, 359 technology transfer, 302 temperature, 2, 11, 13, 14, 15, 18, 19, 23, 26, 27, 35, 42, 44, 51, 52, 72, 73, 92, 93, 133, 136, 161, 167, 169, 170, 171, 172, 173, 177, 178, 179, 180, 183, 184, 192, 193, 194, 196, 206, 214, 224, 233, 245, 253, 278, 284, 321, 330, 336, 350, 360, 368

409

tensile strength, 17 tension, 44, 47, 171, 175, 191, 192, 193, 194, 195, 196, 197, 198, 202, 205, 206, 207 test procedure, 389 testing, 3, 5, 27, 73, 76, 131, 142, 293, 336, 360, 361, 365, 369 testosterone, 334 textiles, 13, 53, 55, 103, 107, 211 Thailand, 78, 190, 364 thermal decomposition, 294 thermal energy, 143 thermal properties, 11, 359, 360 thermal resistance, 359, 360 thermal stability, 185 thermodynamics, 12 thermograms, 26 thermolysis, 161 thermoplastics, 13, 212, 293 time frame, 289 tissue, xiii, 106, 223, 226, 258, 323, 355 toluene, 193, 203 tones, 62, 63, 100, 110 tonic, 64 total energy, 231 total product, 102, 112, 117, 231 toxic effect, 44 toxicity, 147, 148, 151, 259, 261, 262, 281 toxicology, 281 TP53, 154 trade, 55, 112, 284 trainees, 393 training, 116, 379, 390 traits, 181, 238, 312, 313, 317, 318, 329, 330 transesterification, 193, 203, 204, 236, 348, 351 transformation, 104, 230, 231, 237 transformations, 82 transition temperature, 360 transmission, 53 transparency, 389, 390 transport, 42, 77, 142, 219, 229, 230, 234, 236, 258, 264, 287, 291, 301, 323, 340 transportation, 67, 69, 70, 73, 74, 77, 82, 236, 283, 284, 293, 323 treatment, 14, 15, 17, 19, 61, 79, 81, 82, 86, 100, 114, 133, 134, 135, 136, 158, 206, 216, 217, 218, 253, 255, 256, 261, 275, 286, 288, 289, 297, 302, 377 turbulence, 174, 175, 178 turnover, 111, 231

U UK, 14, 104, 143, 166, 311

410

Index

Ukraine, 104 Ultra-high pressure injection, 167 ultrasound, 320, 343, 351 unification, 374 uniform, 70, 72, 242, 381 United Nations, 78, 269, 394 universities, 132, 311 updating, 380 urban, 287 urban areas, 287 USA, 9, 19, 20, 47, 57, 78, 79, 90, 91, 121, 166, 221, 280 USSR, 157 UV, vii, 49, 53, 55, 57, 106, 149, 224, 359 UV light, 224 UV radiation, 53, 55, 57, 106 UV-radiation, 149

V vacuole, 258 vacuum, 14 validation, 267, 326, 327 valuation, 359, 373, 381, 383, 387 variables, 322, 386, 387 variations, 126, 197, 354 varieties, xiv, 70, 100, 121, 181, 189, 241, 242, 243, 244, 247, 249, 329 vegetable oil, 99, 101, 201, 232, 236, 237, 315 vegetation, 35, 86, 89, 93, 94, 245, 246, 247, 248, 335, 338, 340 vehicles, 142, 144, 168, 229, 230 velocity, 168, 174, 180, 195, 198, 216, 219 Venus, ix, xii, 299, 303, 304, 309 vessels, 61, 304, 306 vibration, 16, 350 Vietnam, 363, 364, 365, 367, 370 viruses, xiii, 61 viscoelastic properties, 11 viscose, 53, 320 viscosity, 171, 194, 198, 236 visualization, 197, 328, 329, 331

waste water, 90 wastewater, 253, 255, 256 water, 2, 4, 5, 6, 9, 12, 13, 14, 32, 37, 41, 42, 43, 44, 47, 51, 52, 69, 70, 72, 73, 75, 76, 77, 82, 86, 90, 91, 143, 144, 145, 148, 149, 151, 153, 191, 192, 193, 194, 195, 197, 198, 202, 203, 204, 206, 207, 210, 213, 217, 218, 220, 230, 231, 232, 251, 252, 256, 259, 264, 284, 299, 300, 304, 308, 318, 340, 347, 348, 350, 351, 352, 365, 376, 378, 384, 390 weakness, 372 weight changes, 26 weight gain, 16 weight loss, 15, 26 wettability, 12, 192, 193, 194, 198, 202 wetting, 12, 170 wheat straw, 68 white blood cells, 148, 155 winter rapeseed, 123, 124, 125, 127, 128 winter wheat, 45, 123, 124, 125, 127, 128 Wisconsin, 227 wood, 12, 14, 17, 18, 21, 22, 23, 24, 27, 39, 50, 62, 63, 68, 69, 73, 78, 79, 95, 118, 158, 266, 267, 283, 286, 288, 297, 316, 334, 340, 366 World Bank, 90, 95 World War I, 103 worldwide, 12, 31, 168, 209, 210, 229, 249, 279, 304, 305, 363

X X-irradiation, 148, 149

Y yarn, 55, 107, 108 yeast, 113, 275, 306, 337 Yeasts, 309 yield, 1, 2, 3, 4, 5, 6, 7, 8, 31, 36, 37, 41, 44, 45, 46, 62, 63, 85, 94, 99, 110, 113, 116, 118, 126, 127, 164, 165, 209, 212, 213, 214, 215, 221, 229, 233, 234, 237, 242, 247, 248, 271, 272, 273, 276, 286, 305, 306, 312, 319, 326, 329, 330, 333, 334, 339, 340, 341, 348, 349

W wage level, 379, 385, 387 wages, 380 Washington, v, 95 waste, 11, 12, 21, 22, 90, 108, 118, 144, 278, 285, 288, 318, 319, 348 waste heat, 144

Z zinc, 61 ZnO, 288