Pub - Renewable Resources and Biotechnology For Material PDF

MATERIALS SCIENCE AND TECHNOLOGIES
RENEWABLE RESOURCES
AND BIOTECHNOLOGY
FOR MATERIAL APPLICATIONS
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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.
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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 1

Anja Hartmann, Karen Zeise and Maendy Fritz
Chapter 2 Enzyme Modified Grain by-Product Reinforced Polypropylene
Composites 11
Abdullah Al Mamun, Andrzej K. Bledzki and Jürgen Volk
Chapter 3 Biomass as Combustion Fuel – Experiences and Findings 21
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 31
H. Burczyk, R. Kaniewski, W. Konczewicz,
N. Kryszak and J. Turowski
Chapter 5 IWN-11 the New Biostymulator for Increasing Crops Resistance to
Drought Stress 41
Krzysztof Heller, Małgorzata Byczyńska
and Zenon Woźnica
Chapter 6 Yellow Natural Dyestuffs and their Light Fastness Properties and
UV Protection on Natural Fabrics 49
Katarzyna Schmidt-Przewoźna and Jakub Kowalinski
Chapter 7 Multipurpose White Mulberry (Morus Alba L.) 59
Malgorzata Lochynska and Grzegorz Oleszak
Chapter 8 Agricultural Residues as a Renewable Source of Bio-Energy with
Special Focus on Cereal Straws 67
Narra S., C. Glaser, H.-J. Gusovius,
C. Stollberg and P. Ay
viii Contents
Chapter 9 Prevention of Disposal of Greenhouse Gas from Digested Residues

by Optimal Use of the Nitrogen Fertilizer Potential 81
Ute Bauermeister
Chapter 10 Vetiveria Zizanioides Grass a Useful Tool 89
G. Bach and R. Hommel
Chapter 11 State of the Art of the Renewable Resources in Poland 97
R. Kozłowski, K. Seidler-Lozykowska,
M. Mackiewicz-Talarczyk, P. Baraniecki,
J. Mankowski, Cz. Ogurkowski and I. Pniewska
Chapter 12 Energy Efficiency of Four Crop Species 123
Jerzy Pudelko, Jerzy Mankowski and Jacek Kolodziej
Chapter 13 Enzymatic Biomass Hydrolysis 131
Viktor Antonov, Josef Marousek, Jan Marek,
Stanislav Kuzel and Tomas Rosenberg
Chapter 14 Sustainable Logistics Centers 141
Hartmut Zadek and Robert Schulz
Chapter 15 Relation between the Cell-Free DNA Content and the Lipid
Peroxidation in the Blood Plasma of Mice under Damaging Action 147
Lyudmila N. Shishkina, Mikhail A. Klimovich,
Mikhail V. Kozlov and Margarita A. Smotryaeva
Chapter 16 Mechanism of Stable Radical Generation in Lignin under the Action
of Nitrogen Dioxide 157
E.Ya.Davydov, I.S. Gaponova, S.M. Lomakin, G.B. Pariiskii,
T.V. Pokholok and G.E. Zaikov
Chapter 17 Spray Formation of Alternative Diesel Fuels under Engine-Like
Conditions 167
Dennis Backofen, Michael Könnig,
Helmut Tschöke and Jürgen Schmidt
Chapter 18 DNA Fingerprinting and Characterisation of Genetic Variation of
Different Clones of Urtica Dioica L. via RAPD and RAPD-Derived
SCAR Markers 181
Bettina Biskupek-Korell, Sabrina Becker, Jasmin Dufrenne,
Patricia Rauscher and Carolin Schneider
Chapter 19 IFT Performance of MES Surfactant from Palm Olein for EOR
Application 191
Erliza Hambali, Mira Rivai, Putu Suarsana, Sugiharjo,
Edi Zulchaidir and Hermansyah Handoko
Chapter 20 The Development Process of Jatropha Methyl Ester Sulfonic Acid
(MESA) to Enhance Oil Recovery 201
Siti Mujdalipah, Mira Rivai, Erliza Hambali, Ani Suryani,
Hermansyah Handoko and Edi Zulchaidir
Contents ix
Chapter 21 Rapeseed Proteins – Recent Results on Extraction and Application 209

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 223
Carolin Schneider, Sven Wartenberg, Jasmin Dufrenne
and Bettina Biskupek-Korell
Chapter 23 Survey of Jatropha Curcas Energy Potential for an Efficient
Production of Biodiesel as Renewable Energy 229
Sékou Traoré, Amadou Diarra, Macki Traoré
and Sékou Magassouba
Chapter 24 Genetic Resources of Cannabis Sativa L. in the Collection of the
Gene Bank at Infandmp in Poznan 241
Magdalena Chudy and Grażyna Mańkowska
Chapter 25 Production of Biodegradable Carrier Materials for the
Immobilization of Microorganisms for the Treatment of Water 251
Alvaro E. Gonzalias, A. Werner and Hans-Joachim Radusch
Chapter 26 The Changes in the Protein Profile in Response to Cadmium Stress
in Flax (Linum Usitatissimum L.) 257
Milena Szalata, Szalata Marlena and Wielgus Karolina
Chapter 27 Regional Value Added of Bioenergy Activities A Material Flow
Approach 263
Ruth Offermann, Thilo Seidenberger and Daniela Thrän
Chapter 28 Simple Kinetics of Methane Formation Using Model Substrates: A
Short Discussion 271
Robert Reinhard Pätz and Jan-Henryk Richter-Listewnik
Chapter 29 Biosafety of Transgenic Potatoes Producing the Biopolymer
Cyanophycin 279
Christoph Unger, Maja Hühns and Inge Broer
Chapter 30 Provision Pathways for Biomethane 283
Diana Weigl, Katja Oehmichen, Michael Seiffert,
Franziska Müller-Langer and Frank Scholwin
Chapter 31 Flammability of Polymers Reinforced with Lignocelullosic Raw
Materials 293
Maria Władyka-Przybylak and Krzysztof Bujnowicz
Chapter 32 Feedstocks and (Bio) Technologies for Biorefineries 299
Joachim Venus
x Contents
Chapter 33 Non-Food Crops as a Feed Stock for Modern Bio-Based Industry 311
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 325
Mohammad R. Hajirezaei, Mohammad R. Ghaffari,
Björn H. Junker, Johannes Müller, Björn Usadel
Michael Leps, Rainer Lemke and Falk Schreiber
Chapter 35 Bioactive Substances from the Balsam Poplar 333
S. Ludewig, S. Gille, D. Orzessek and C. Griehl
Chapter 36 The Potential of Microalgae to Produce Lipids for Biofuels 347
C.Griehl, H. Polhardt, D. Müller and S. Bieler
Chapter 37 Biomaterials for Consumer Products 357
Peter Gerth, Thomas Bagusch, Melanie Poschke
and Johann Zimprich
Chapter 38 Use of Rice Spelt as Component in Building Material 363
Stefanie Bachmann, Grete Bach and Rainer Loth
Chapter 39 Criteria System of Sustainable Agriculture (CSSA - KSNL) 371
Gerhard Breitschuh, Hand Eckert, Ines Matthes and
Jürfgen Strümpfel,
In cooperation with: Günter Bachmann, Landwirt Martin Herold,
Thorsten Breitschuh and Ulrich Gernand
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-von-
Guericke-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
 Biotechnology
 In vitro culture
 Smart breeding
2. Renewable Resources for Material Applications
 Biobased composites (Natural fibres, Nano particles etc.)

 Biobased polymers
 Biobased additives
3. Processing, Characterisation and Application of Secondary Plant Metabolites
 Biomass conversion technologies

 Biorefineries
 Cosmetics and pharmaceuticals
4. Biomass and Efficient Energy Generation
 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 Carl-

Gustav-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-Guericke-
Universität 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 "Otto-
von-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-von-
Guericke-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 scientists: ―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
In: Renewable Resources and Biotechnology… ISBN: 978-1-61209-521-9
Editors: G.E. Zaikov, D.P. Pudel and G. Spychalski © 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).
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
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].
35 30
t/ha %
Dry matter content

Dry matter yield
25
20
20
15
15
10
10
a a b b a b
5 5
Dry matter yield Dry matter content
0 0
)
n.
l)
e
t)
in
ge
ns
ua
ee
da
ra
ra
ne
(d
w
su
(g
(fo
(s
da
or
or
S.
or
ol
or
su
ol
ol
c
ol
x
c
bi
S.
c
bi
c
bi
.
bi
ol
S.
S.
S.
ic
S.
b
S.
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.
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
t/ha
Herkules
Dry matter yield
20 Zerberus
Latte Lussi
Green Grazer Wotan
Mithril Grazex II
18 Grazer N Bovital Maja
Inka
16
S. bicolor (forage) S. bicolor x S. sudanense
0
0 22 24 26 28 30 32 % 36
Dry matter content
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
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 Maize Reference
S. bicolor (forage) S. bicolor (forage)
t/ha S. bic x S. sud
t/ha S. bic x S. sud
Dry matter yield
Dry matter yield

20 20
15 15
10 10
100 80 99 84 75 100 98 102 101 93
5 5
Straubing Aholfing
0 0
H e
a
W 1
an
ka
H e
a
W 1
an
ka
KS aiz
aj
KS aiz
aj
In
In
83
ot
83
ot
M
M
M
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
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 7,9 1,2 28,6 0,2 12,0
(forage) ± 1,2 ± 0,2 ± 2,8 ± 1,0 ± 2,8
S. bicolor 7,5 1,1 25,6 0,1 18,6
(sweet) ± 0,7 ± 0,1 ± 1,6 ± 0,3 ± 1,3
S. bicolor 9,1 1,5 26,6 2,0 11,4
(dual) ± 2,2 ± 0,2 ± 2,4 ± 3,0 ± 1,3
S. bicolor 10,1 1,6 24,8 8,0 5,4
(grain) ± 0,8 ± 0,4 ± 3,0 ± 10,5 ± 3,6
S. bicolor x S. 8,3 1,4 29,0 2,8 11,4
sudanense ± 0,8 ± 0,3 ± 2,6 ± 4,0 ± 4,2
9,2 1,5 30,2 3,0 12,8
S. sudanense
± 0,6 ± 0,2 ± 2,9 ± 2,6 ± 5,3
8000 350
Methane yield Methane production
m³/ha Nl/kg oDM
6000
Methane production
Methane yield
250
5000
200
4000
150
3000
2000 100
a ac bc b ac ac
1000 50
0 0
)
d.
e
)
t)
in
ge
al
ns
ee
su
du
ra
ra
ne
sw
(g
S.
r(
fo
da
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r(
lo
or
su
lo
co
ol
lo
.
co
c
co
ic
bi
S.
bi
b
bi
bi
S.
S.
S.
S.
S.
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.
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
authors gratefully thank the technicians Alois Aigner, Josef Sennebogen, Benno Sötz and
Michael Kandler for their excellent work and high motivation.
REFERENCES
[1] Adam, L. (2008): Geeignete Rohstoffpflanzen zur Biogaserzeugung – Neue
Fruchtarten: Sudangras und Zuckerhirse. Bauernblatt Schleswig-Holstein, vol. 34, iss.
62, p. 28-31.
[2] Berenji, J.; Dahlberg, J. (2004): Perspectives of Sorghum in Europe. J. Agronomy and
Crop Science, vol. 190, p. 332-338.
[3] Böhmel, C. Jäger, F. (2007): Sorghum eine Ergänzung zu Mais?. Mais, vol. 34, iss. 4,
p. 138-142.
[4] DLG (1997): Futterwerttabellen Wiederkäuer. 7. Auflage. DLG-Verlag, Frankfurt a.
M., Germany.
[5] 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.
[6] FNR (2010): Anbau nachwachsender Rohstoffe in Deutschland. Gülzow: FNR. URL :
http://www.fnr.de/ (13.09.2010).
[7] Jäger, F. (2009): Vielfalt im Fermenter – Sorghum als sinnvolle Ergänzung im
Energiepflanzenanbau? Neue Landwirtschaft. vol. 3, p. 98-101.
[8] 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.
[9] 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.
[10] 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.
[11] 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.
[12] Schattauer, A.; Weiland, P. (2009): Grundlagen der anaeroben Fermentation. In:
Handreichung Biogasgewinnung und –nutzung. Fachagentur Nachwachsende Rohstoffe
e.V. (Hrsg.), Gülzow, Germany; p. 30.
[13] 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.
[14] VDLUFA (1976): Die chemische Untersuchung von Futtermitteln. Methodenhandbuch.
Band III, 4. Ergänzung 1997. VDLUFA-Verlag, Darmstadt, Germany.
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 by-
products 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 bio-
treatments 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
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 heat-
cooling 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.
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.

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 in-
plane 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.
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 FT-
IR 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
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.
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] Suddel B. C., Evans W. J.: 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.
[2] 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).
[3] World production data sheet, Foreign Agricultural Service, United States department of
Agriculture, USA, www.fas.usda.gov.
[4] 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.
[5] 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.
[6] Puglia D., Biagiotti J., Kenny L. M.: Journal of Natural Fibers 2004, 1, No. 3, 23.
[7] International Rice Research Institute The Rice Plant and How it Grows Retrieved,
January 29, 2008.
[8] Duke, J.A. Handbook of Edible Weeds, CRC Press, Boca Raton, FL, 1992.
[9] 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)
[10] Aehle, W., Enzyme in industry; production and application, ―industrial enzymes‖,
Wiley-VCH verlag, Germany, 2004.
[11] 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.
[12] 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.
[13] 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.
[14] 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.
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: ifnzeitz@t-online.de.
†
Kosel GmbHDorfstrasse 3, D-02991 Lauta, Phone: ++49-(0)35722 – 3 69 10, e-mail: post@kosel-gmbh.de.
‡
BEM Umweltservice GmbH Martin-Luther-Strasse 26, D-71636 Ludwigsburg. Phone: ++49-(0)7141-702 98-0, e-
mail: info@bem-umweltservice.de.
22 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/
Use of biomass Potential Potential Potential

Mio t
2003 2010 2020 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 max. diameter suitable suitable

of fuel of particles dosation systems 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 grate combustion, BFB
dosage
bales < 50 mm pneumatic dosage worm Dust combustion

conveyer 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 < 120 mm dosage in pieces grate combustion, station. Fluid

bed Stoker dosage
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
Compost Wooden Rapeseed Sunflower

Value Wood Miscanthus Bio mix
remnants dust expeller husk
Heat value
18,46 18,32 19,16 17,62 17,52 17,03 17,37
MJ/kg
Ash content
0,45 2,87 0,52 2,87 4,06 6,95 1,50
% OS
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.
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
Compost Wooden Rapeseed Sunflower

Value Wood Miscanthus Biomix
remnants dust expeller 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 Legacy Rel. to Emission limits

combustion O2
unit
Vol.-% CO Summ…-C NOx Dust
g/Nm³ g/Nm³ g/Nm³ g/Nm³
Combustion of natural wood
50 - <150 kW 1. BImSchV 13 2 - - 150
150 - <500 kW 1. BImSchV 13 1 - - 150
500 - <1000 kW 1. BImSchV 13 0,5 - - 150
Combustion of straw and comparable biomass
15 - < 100 kW 1. BImSchV 13 4 - - 150
100 kW - < 1 MW TA Luft 11 0,25 50 500 50
3. COMBUSTION STUDIES WITH ENERGY CROPS AND BIO RESIDUES

In preparation of combustion experiments with different biomass-fuels we checked their
behaviour in lab scale runs.
In a special laboratory combustion unit (Combustor 02/Gamab) the parts of organic and
inorganic carbon in different biomasses were determined. An example is given in Figure 1
with remnants from composting.
Figure 1. Parts of organic and anorganic carbon in composting remnants.
Analogous measurements were done with energy crops and crop residues (Figure 2).
Figure 2. Parts of organic (upper part) and inorganic (lower part) carbon in 4 - Miscanthus 5 - Biomix 6
- Rapeseed residue 8 - sunflower 9 – grain.
These experiments verify the analytical data given above. High amount of ash has to be
expected. We calculated the Theune-Number, characterising the tendency of ash production
for brown coal and for composting remnants – they are nearly identical. A similar behaviour
is to be expected for energy crops.
Table 5. Theune-Number for coal and composting remnants
Theune-number at T 815 °C 1000 °C

Brown coal 1,85 --
Composting remnants 2,198 1,150
The weight changes of composting remnants with increasing temperature were developed
in thermo balance experiments. With rising temperature weight loss in a first phase of the
reaction is caused by drying, in the second phase aliphatic C-C-bonding are ruptured with gas
generation, aromatic bonds are ruptured and gasified only at high temperatures, some
carbocyclic carbon doesn‘t break down at all but remains as coke and coke like substances
(Figure 3)
Figure 3. Thermogram of composting remnants.
Similar thermograms are got with the other bio substances also.
To check the combustion behaviour of selected energy crops and bio residues combustion
runs were performed in a 35 kW as well as in a 150 kW vessel of the type LawaMatic ®
(Figure 4).
Figure 4. Scheme of the LawaMatic® vessel.

The input fuel is transported to the grate by a worm conveyer from below. Primary and
secondary air is dispensed from the top and from below to generate the desired temperature
profile in the vessel.
Fumes are leaving the vessel viathe chimney and were analysed. Also fine dust was
sampled and weighed. Ash from the grate was selected and also analysed.
The experiments were started with wood pellets, which burned with good fire (Figure 5).
Figure 5. Flame with wood pellets.
With composting remnants, which were hauled as bulk material, we were not successful
in the vessel used – dosage was not continuous, material bridges occurred, the fire was
extinguished soon (Figure 6).
Figure 6. Three phases of burning of composting remnants in the LawaMatic® vessel.
Testing the material in an industrial power plant with a simple grain there were no
problems with burning. So a problematic residue from composting could be used as biomass
fuel bringing money, instead of giving them to expensive incineration.
With rapeseed residues from the oil mill and with biomix pellets we got sufficient
combustion results testing them in a modified LawaMatic® vessel with reconstructed
dispersion unit (conditions Table 6)
As an example the measured emissions during burning of composting remnants are given
in Table 7. They are not always below the accepted limits here. The high values could be
corrected by a higher dosage of secondary air in further experiments, NO and dusts were in
the limit.
Table 6. Burning conditions with different fuels
Bur-
Nr. Biomass Fuel T-Air T-Gas O2 CO Lambda CO2 Eta
ning
(°C) (°C) (°C) ppm (%) (%)
1 Wood pellets 21,6 118 5,9 73 1,39 14,6 95,7 good
2 Composting remnants 22,2 85 11 1069 6,73 3 86,3 bad
3 Biomix pellets 22,7 131 9,3 163 1,8 11,3 93,7 good
4 Rapeseed remnants pell. 22,6 119 11,9 123 2,3 8,8 92,8 good
Table 7. Emission from the 150 kW unit (composting remnants)
Zeit Temperature O2 (Vol.-%) CO NO Dust

Gas (°C) (mg/Nm³) (mg/Nm³) (MgNm³)
19.03.2008 11:02 172 14,7 226,56 88,38
19.03.2008 11:16 118 19,3 396,18 23,65
19.03.2008 11:34 77 20,3 833,03 4,98
19.03.2008 11:46 64 20,3 1045,64 4,98
19.03.2008 12:32 57 21 376,43 3,73 20
100 bis < 1.000 kW TA Luft 11 250 500 50
We did not mention a high slag formation during combustion, despite the fact, that the
ash generated had a high content of slag forming components (Si, Ca). Table 8 shows the
content of selected elements in the ash of some biomass fuels.
Table 8. Content of Si, Ca, K in ,4
Content in % Wood Composting remnants Miscanthus

Si 22,4 30,2 20,7
Ca 22,1 13,4 4,7
K 4,3 3,1 16,4
We will continue our research work now with the technical optimization of the
combustion process for all biomass fuels tested having in mind the results of the presented
here first burning tests.
REFERENCES
[1] Erhard Schuster: Wald und Holz – Daten aus der Geschichte der Nutzung und
Bewirtschaftung des Waldes, der Verwendung des Holzes und wichtiger Randgebiete, 2
Bände, Kessel Verlag, Remagen, 2006.
[2] El Bassam, N. Renewable energy – Potential energy crops for Europe and Mediterran
region, FAO, Rome, 1996.
[3] M. Kaltschmitt, H. Hartmann, Energie aus Biomasse, Grundlagen, Technik, Verfahren,
Springer Verlag, 2001.
[4] 2003 data from Eurostat; projections for 2010, 2020 and 2030 from European
Environmental Agency, "How much biomass can Europe use without harming the
environment", briefing 2/2005.
[5] I. Odernberger: Nutzung fester Biomassen in Verbrennungsanlagen unter besonderer
Berücksichtigung des Verhaltens aschebildender Elemente; Schriftenreihe „Therm.
Biomassenutzung― Bd. 1; dbv -Verlag TU Graz (1997)
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
Institute of Natural Fibres and Medicinal Plants, 60-630 Poznań
INTRODUCTION
Industrial hemp is considered the raw material for fibre which is used in textile industry
and in production of commodities used in other industries: construction, automotive,
composite as well as pulp and paper [2, 3, 4]. Hemp seed is used in bakery products and other
applications in food industry mainly as valuable hempseed oil [1].
Recently, it has been observed that industrial hemp can also be a valuable source of
essential oils which find many applications in cosmetic and food industries [9, 10].
Formation and role of essential oils in plants are investigated by many scientists
worldwide. Up to now, this research has not been truly successful [7].
It is known, however, that essential oils are synthesized in trichomes covering hemp
inflorescence. The essential oil is produced by the upper cells of the trichome head, secreted
inside and stored between the epidermis and walls of secreting cells. In time of the essential
oil secretion, epidermis is lifted and a transparent vesicle is formed where the essential oil is
collected [7, 9].
Observations made so far show that the number of trichomes covering the plant
inflorescence and connected with this essential oil content depends on many genetic,
agronomic and natural factors [8, 11]. Learning how these factors influence the efficiency of
essential oils is an objective of this study. Therefore, one can expect that the higher is the
number of trichomes covering the hemp inflorescence, and the higher are yield of panicles,
the higher amounts of essential can be harvested from the unit of area.
*
Institute of Natural Fibres and Medicinal Plants, 60-630 Poznań, ul. Wojska Polskiego 71b.
32 H. Burczyk, R. Kaniewski, W. Konczewicz et al.
MATERIALS AND METHODS

To learn the effect of selected factors on the content and efficiency of essential oils in
industrial hemp, a field experiments and laboratory tests have been carried out at the Institute
of Natural Fibres and Medicinal Plants in Poznań, in 2004-2008.
The content of essential oils in hemp panicles was tested in the INFMP chemical
laboratory according to the method described in Polish Pharma-copoeia. The method lies in
distilling the essential oil by water steam. The results were calculated for 100 g of raw
material expressing the content of essential oils in volume-weight percents.
Samples of fresh inflorescence (1 kg each) were taken each year by the same method, in
similar plant development stage from the field experiments conducted at the Experimental
Station in Pętkowo and in Agricultural and Seed Producing Company KOW-MAR in Błaszki
[4, 5] according to the following scheme.
1. Hemp cultivars: Białobrzeskie, Beniko, Silesia and Tygra (only in 2008). Samples
were taken according to the following schedule:
1st time – full bloom of inflorescence (>50%)
2nd time – full seed maturity (>50%).
2. New prospective lines from KOW-Mar in Błaszki – different genotypes marked as
IWN-104, 204, 304, 507, 608.
Samples were taken at full bloom stage of inflorescence (>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.
Table 1. Essential oils content in panicle depending on harvest dates and cultivar of
hemp [ %]
Harvest Cultivar 2004 2005 2006 2007 2008 Average Average for
time harvest dates
Białobrzeskie 0,87 0,37 0,30 0,41 0,46 0,48

Date 1 Beniko 0,71 0,35 0,23 0,39 0,32 0,40 0,41
Silesia – 0,44 0,33 0,27 0,38 0,39* 0,36
Tygra
Białobrzeskie 0,44 0,29 0,34 0,42 0,40 0,37

Date 2 Beniko 0,33 0,31 0,31 0,43 0,51 0,38 0,38
Silesia – 0,58 0,21 0,28 0,37 0,44* 0,38
Tygra
Average 0,56 0,31 0,29 0,40 0,42
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 harvest dates 0,01
Table 4. Essential oils content in panicle depending on sowing rate of hemp [ %]
kg∙ha-1
5 10 15 20 30 40 50 60 Average
Years
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 sowing rate 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 harvest time of day 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
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

2004 2005 2006 2007 Average
fertilization in kg∙ha-1
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 the nitrogen level of fertilization 0,01
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].
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 Środa 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).
Photo 1. Distiller for steam distillation of hemp essential oil in Experimental Farm Pętkowo N / Środa
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.
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 gęstości siewu i
terminu zbioru konopi włóknistych na wydajność biomasy oraz elementów składowych
plonu. Pamiętnik Puławski (w druku).
[6] Łubkowski Z., 1968, Metodyka doświadczalnictwa rolniczego. PNR i L, Warszawa.
[7] Kołdowski M., Wysocka - Rumińska A., Tałałaj S., Wiszniewski J., 1955, Rośliny
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.
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 ,
khel@inf.poznan.pl.
†
Agricultural University of Poznań, ul. Mazowiecka 45/46, 60-623 Poznań, woznica@au.poznan.pl.
42 Krzysztof Heller, Małgorzata Byczyńska and Zenon Woźnica
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

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:
 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
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.
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)
Yield (dt/ha)
Treatment Dose (kg, l/ha)
Total Roots Leaves
Untreated 1219,0 852,4 366,6
ASA * 0,4 1090,0 734,5 355,8
Asahi SL * 0,6 1269,0 856,2 412,7
IWN-11 * 1,0 1160,0 814,9 345,2
IWN-11 * 2,0 1146,0 808,7 337,6
ASA ** 0,4 1289,0 873,8 415,1
Asahi SL ** 0,6 1397,0 1028,1 369,2
IWN-11 ** 1,0 1320,0 929,0 389,1
IWN-11 ** 2,0 1412,0 1032,5 379,0
LSD 277,2 209,7 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)
Yield (dt/ha)
Treatment Dose (kg, l/ha)
Total Roots Leaves
Untreated 1050,0 815,0 235,0
ASA * 0,4 1050,0 675,0 285,0
Asahi SL * 0,6 1140,0 845,0 295,0
IWN-11 * 1,0 1097,5 800,0 297,5
IWN-11 * 2,0 1275,0 1000,0 275,0
ASA ** 0,4 1297,5 870,0 427,5
Asahi SL ** 0,6 1090,0 800,0 290,0
IWN-11 ** 1,0 1432,5 1045,0 387,5
IWN-11 ** 2,0 1365,0 1030,0 335,0
LSD 64,06 62,99 31,44
* growth stage of sugar beet (BBCH 14).
** growth stage of sugar beet (BBCH 18).
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] 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 24-
28. CD
[2] Gupta U.S.2007: Physiology of Stressed Crops. Vol. V, Membrane System. Ed.Science
Publishers, Georgia, USA: 403
[3] 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
[4] 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.
[5] Kozłowska M. 2007: Fizjologia roślin. Wyd. PWRiL. Poznań: ss.544
[6] 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.
[7] Singh S.K. 2005. Plant Physiology. Ed. Campus Books Int. New Delhi, pp. 342
[8] Widtsoe J. A. 2007. Dry Farming for Sustainable Agriculture. Ed. Agrobios, India:361
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 PN-
ISO 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.
Table 1. Light and washing fastness naturally dyed linen and silk samples
No Fabric Natural dyestuff mordant color Wash Light

fastness fastness
1 Silk French marigold Tagets alum Green 5 4-5
Shantung species (kwiaty bordo) yellow
2 Linen French marigold Tagets alum Green 3 change of 5
species yeallow shade
3 Linen French marigold Tagets alum yeallo 4 5-6
species (yellow flowers)
4 Knitted Kamala Mallotus alum Gold 5 3
silk philippinensis yellow
5 Knitted Weld Reseda luteolaL. alum Light 4 5
silk khaki
6 linen Cuth Accacia catechu Copper brown 3 5
sulphate
7 Silk Dyer‘s chamomile Anthemis Alum = Green 3 7
Shantung tinctoria L copper yellow
sulphate
8 Linen French marigold Tagets alum yellow 3 4
9 Silk French marigold Tagets alum yellow 3 3
10 Silk Safflower Cartamus tintorius Citric Peru 5 3
L acid brown
11 Silk Dyer‘s chamomile Anthemis Citric beige 3 change of 4
tinctoria L acid shade
12 Linen French marigold Tagets ferrous gray 4 chamge 2
species (kwiaty bordo) suplhate of shade
13 Silk French marigold Tagets Ferrous beige 4 5
species (kwiaty bordo) suplhate
14 Knitted Annato Bixa orelana Washing Dark 4 2-3
silk soda orange
15 Linen Annato Bixa orelana Washing orange 4 5
soda
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
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 UVR protection category UPF Ratings

15-24 Good but insufficient 15,20
protection
25-39 Very good protection 25,30,35
40-50, 50+ Excellent protection 40,45,50,50+
Table 3. The results of UPF on linen and silk samples dyed by natural
red dyestuffs. [4,5]
No Samples Natural Mordant Color UVA UVB UPF

Dyes
Linen No mordant White 10,069 5,455 10
hemp No mordant white 11,696 8,4 5
1 linen Tumeric Pre-mordant yellow 4,456 3,036 25
2 hemp Tumeric Pre-mordant yello 3,713 3,567 20
3 linen Tumeric No mordant Light 3,543 3,305 20
yellow
4 hemp Tumeric No mordant Sun 6,592 5,470 15
yellow
5 linen Tumeric Pre-mordant+copper Olive 2,907 2,011 30
sulphate yellow
6 hemp Tumeric Pre-mordant+copper Dark 3,758 3,614 25
sulphate yellow
7 linen Tumeric Pre-mordant+soda Sahara 3,302 2,53 30
yeallow
8 hemp Tumeric Pre-mordant+soda beige 2,789 2,477 25
9 linen Tumeric Pre-mordant+citric Sun 4,022 3,364 30
acid yellow
10 hemp Tumeric Pre-mordant+citric Bright 5,33 5,08 15
acid yellow
11 linen Tumeric Pre-mordant+ferrous olive 4,235 2,614 30
suplhate
12 hemp Tumeric Pre-mordant+ferrous Olive 2,772 2,532 30
sulphate brown
13 linen Dyers No mordant Old gold 1,646 1,979 30
Coreopsis
14 Linen Dyers Soda Old gold 1,854 2,137 40
Coreopsis
15 linen Dyers Citric acid gold 2,237 2,939 35
Coreopsis
16 linen Dyers Copper sulphate Old gold 0,967 1,255 50
Coreopsis
17 linen Dyers Ferrous sulphate Dark 0,881 1,117 50
Coreopsis brown
18 Linen Common No mordant brown 1,816 2,178 35
knotweed
19 linen Henna No mordant rust 1,739 2,438 35
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:
Excellent UVR Protection We Obtained on Samples
1. Dyers Coreopsis (linen ) – soda 50+

2. Dyers Coreopsis (linen ) - copper sulphate 50+
3. Dyers Coreopsis (linen ) - ferrous sulphate 50+
4. Dyers Coreopsis (linen ) – Soda 40
Very Good Protection We Obtained on Samples
 Henna (linen) - no mordant 35

 Common knotweed (linen) -no mordant 35
 Dyers Coreopsis (linen) – citric acid 35
 Dyers Coreopsis (linen) - no mordant: 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‖.
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.

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] Sun protective clothing – Evaluation and classification – Australian / New Zealand
Standard.
rd
[2] M.T. Pailthorpe: ―Sun Protection and Apparel Textiles‖ – The 3 Asian Textile
Conference, (1995).
[3] J. Rupp., A Bohringer., A Yonenaga., J. Hilden: „Textiles for protection against
harmful ultraviolet radiation― – International Textile Bulletin no 6, November (2001).
nd
[4] M. Zimniewska: ―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).
[5] 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).
[6] M.S. Reisch: ―New –wave sunscreens‖, ―Chemical@Enginiering News (April, 2).
Chapter 7
MULTIPURPOSE WHITE MULBERRY

(MORUS ALBA L.)
Malgorzata Lochynska1*and Grzegorz Oleszak2†

1
Department of Silkworms Breeding and Mulberry Cultivation
2
Experimental Farm in Petkowo, Institute of Natural Fibres
and Medicinal Plants, Petkowo, Środa 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: cardamina@inf.poznan.pl.
†
Experimental Farm in Petkowo, Institute of Natural Fibres and Medicinal Plants, Petkowo, 63-000 Środa
Wielkopolska, Poland. e-mail: g.oleszak@inf.pznan.pl.
60 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 oil-
bearing 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 1-
deoxynojirimycin (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
Multipurpose White Mulberry (Morus Alba L.) 61
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
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.
(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,
 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 wartości energetycznych konopi
wloknistych, kukurydzy i sorga z roślinami 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
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Antioxidant flavanol glycosides in mulberry (Morus alba L.) leaves isolated based on
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pochodzenia roślinnego. Motrol., 8a: 232–237 (in Polish).
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ganic substrates used. Microbios., 69 (279): 113-118.
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extractives from leaves of Morus alba on human and rat small intestinal disaccharidase
activity. British Journal of Nutrition, 95: 933-938.
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of streptozotocin-induced diabetic rats. Journal of Ethnopharmacology, 100: 333-338.
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some genotypem of mulberry (Morus alba). International Journal of Food Science and
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44: 1687-1690.
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 / narra@tu-cottbus.de.
†
E-mail: Is-at@tu-cottbus.de.
‡
E-mail: hjgusovius@atb-potsdam.de.
#
E-mail: christian.stolberg@hs-wismar.de.
^
E-mail: Is-at@tu-cottbus.de.
68 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
Agricultural Residues as a Renewable Source of Bio-Energy with Special Focus… 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 DIN 51 ÖNORM M

DINplus * Agro * Agro+
Pellets 731 7135
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 17.5 - 19.5 > 18 > 18 ≥ 14.7 ≥ 15.5

value MJ/kg MJ/kg MJ/kg MJ/kg MJ/kg
Water
≤ 12 % ≤ 10 % ≤ 10 % ≤ 15 % ≤ 11 %
content
Ash content < 1.5 % < 0.5 % < 0.5 % ≤7% ≤5%
> 540 > 540 > 540 ≥ 650 ≥ 650

Density
kg/m³ kg/m³ kg/m³ kg/m³ kg/m³
Binders / Not
≤2% ≤2% No limit No limit
Additives allowed
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.

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.

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

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. Twin-
screw 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
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.
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
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.
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 C H O N S Cl
Volume % of dry mass
Rye straw 46.6 6.0 42.1 0.55 0.085 0.19
Wheat straw 45.6 5.8 42.4 0.48 0.082 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.
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.
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.
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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
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Kaliyan N, R V Morey (2006a). Factors Affecting Strength and Durability of Den-sified
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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
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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.
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Mani S, L G Tabil, S Sokhansanj (2006b). Effects of Compressive Force, Particle Size and
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Produktionsprozess, Eigenschaften, Feuerungstechnik, Öko-logie und Wirtschaftlichkeit,
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1055 – 1066.
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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: info@gns-halle.de, www.GNS-Halle.de and BM - Verfahrenstechnologie, Weinbergweg 23,
D-06120 Halle/Saale. phone: +049 345 5583-705, e-mail: info@bm-processtechnology.com.
82 Ute Bauermeister
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-
Prevention of Disposal of Greenhouse Gas from Digested Residues… 83
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. without application of digested residue

B. spraying untreated digested residues on the soil surface
C. spraying ammonia-stripped digested residues on the soil surface
D. incorporating untreated digested residues in the soil
E. incorporating digested residues with added nitrification inhibitor in the soil
The realization of the tests and analysis of the emissions were performed by the Martin-
Luther-University Halle-Wittenberg and the UFZ – Centre of Environmental Research Halle-
Leipzig. 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).
84 Ute Bauermeister
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.
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).
86 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.
Figure 7. N-Nutrient loop for a biogas plant with 500 kW electricity.
REFERENCES
[1] 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.
[2] Mosier A. R.: Exchange of gaseous nitrogen compounds between agricultural systems
and the atmosphere. Plant Soil 228, 2001, 17-27.
[3] Herbst, F., Gans, W., Martin-Luther-Universität Halle-Wittenberg – Naturwissens-
chaftliche 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 Applikatio-
nsmethoden. research-project, sponsored by the FNR, Fachagentur Nachwachsende
Rohstoffe e.V., Gülzow (FKZ: 22025207, 2008 – 2010).
[4] 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 Nachwach-
sende Rohstoffe e.V., Gülzow, 2009.
[5] 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.
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.
90 G. Bach and R. Hommel
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 2006 2008

Month May August April
Site
Farm land A 3400 20000

Recultivation
Sites
P1 1040
P2 600
P3 525
P4 537
P5 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%.
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
05.07.07
Farm land Thai 9% Not cropped
Texas 56% Not cropped
Japan 13% Not cropped
Australia 25% Not cropped
3% Cropped, <20 cm
Open mining P1 Japan 78% Not cropped
Open mining, October 2008

Japan 2006 61% Not cropped
Japan 2007 30% Not cropped
Australia 2007 68 Not cropped
Texas 2007 52%
The first winter was connected with a remarkable outfall of plants cultivated on farm
land, table 2 summarises the data. The winter 2007 to 2008 resulted in complete loss
independent on plants had been cropped or not.
During this winter the temperature was not dramatically below 0 °C – it was a mild
winter. Only some selected plants grow up during 2008 (table 3). Only two of the survivals
from the farm land came from the plantation in 2007, the majority was from 2006 dominated
by the clones Australia and Texas, respectively.
These plants showed an untypical shoot development. The new shoots did not come from
the old growth centre. The new initiation point was shifted and the biomass formation was
relatively low, both in respect to roots and to shoots.
Table 3. Characterisation of surviving vetiver plants in farm land, October 2008
Clon plants shoots length of leaves per plant [cm]

min average max min average max
Australia 8 1 1,9 4 28 70,3 123

2006/2008
Japan 1 7 124
2006/2008
Texas 6 1 1,5 2 33 49 75
2006/2008
Australia 2 6 7 8 199 258 316
2007/2008
In contrast to the farm land, survival rates in the recultivation areas were good.
These plants had been taken off in early winter 2008 and kept for possible selection of
winter resistant plants this year.
OIL PRODUCTION
Vertiver is known to produce a special essential oil that is accumulated in the root
system. It is composted of over 120 different compounds that are mostly derivatives of
isoprenoids. It‘s composition differs from site to site. Recent studies have shown that the
formation of some of the components is done with the aid of soil microorganisms [5]. So it
was of interest, to know the yields of the respective oil were, and how its composition will be
under conditions chosen. The oil contents are summarised in Table 4. The application of dried
roots resulted in lower oil yields. There is no common figure in respect to content of essential
oil in dependence of time between plantation and harvesting. Remarkable low contents had
been obtained with the clone Japan even from the open mining site. The clone Japan had the
highest content grown on farm land and within the same vegetation period. It is known, that
the temperature plays an important role in formation of essential vetiver oil [Massado et al.
2006] [6].
The qualification of components of the essential oil revealed more than 100 compounds
(Figure 1). In all chromatograms one main component, 9,10-dehydro-Isolongifolen (20 %),
was present.
[3S-(3.alpha.,3a.alpha.,6.alpha.,8a.alpha.)]-octahydro-7,7-dimethyl-8-methylen-1H-3a,6-
methanoazulen-3-carboxylic acid could be identified (> 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
Japan 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
Thai clone 2006/2006 0,03 0,6
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
Clone
Australia 2007 Japan 2007 Texas 2007 Japan 2006 (straw, open
farm land f.l. f.l. mining)
Fresh matter [dt/ha] 33 22 24 2,9
Dry matter [dt/ha] 9,74 6,43 6,75 1,25
Yield biogas [lN/kg odm] 460 390 500 130
Methan content in biogas [%] 60 65 60 58
Biogas yield [m³N/ha a] 450 251 338 16
Methane yield [m³N/ha a] 270 163 203 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.
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] 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.
[2] 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.
[3] Hommel R., Bach G., Mülker Ch. and Schmidt M. 2008, Narossa 2008 Abstracts.
[4] European Pharmacopoeia, third ed., 1997 Council of Europe, Strasbourg, 121 pp.
[5] 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.
[6] 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.
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 non-
food 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:
maria.talarczyk@escorena.net.
1
Data from Polish Chamber of Flax and Hemp.
2
Central Statistical Office 2008.
98 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)
State of the Art of the Renewable Resources in Poland 99
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 Cultivation area [‗000 ha] Yield [t/ha] Production [‗000 t]

1990 500.4 2.41 1,206.0
1991 467.8 2.23 1,042.7
1992 417.1 1.82 758.2
1993 348.5 1.71 594.4
1994 370.3 2.04 755.7
1995 606.4 2.27 1,376.6
1996 282.6 1.59 449.3
1997 317.4 1.87 594.9
1998 466.0 2.36 1,099.1
1999 545.3 2.08 1,131.9
2000 436.8 2.19 958.1
2001 443.2 2.40 1,063.6
2002 439.0 2.17 952.7
2003 426.3 1.86 793.0
2004 538.2 2.77 1,492.6
2005 569.0 2.63 810.0
2006 658.0 2.65 962.0
2007 825.0 2.67 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
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
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
2005 2006 2007 2008 2009 2010 By value type:

Directive provisions – 2.00% 2.75% 3.50% 4.25% 5.00% 5.75% Energy
percentage of
biocomponents
Bioethanol 3.20% 4.41% 5.61% 6.81% 8.01% 9.21% Volume
ETBE (as per bioethanol) 6.82% 9.37% 11.93% 14.49% 17.04% 19.60% Volume
Ester 2.12% 2.92% 3.71% 4.51% 5.30% 6.10% 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.
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 2005 2006 2007 2008 2009 2010
Demand in thousand cubic metres for:
Bioethanol 218 300 382 464 545 627
Or ETBE 464 638 812 986 1160 1334
Diesel fuel – consumption in thousand cubic metres 2005 2006 2007 2008 2009 2010
Demand in thousand cubic metres for:
Esters 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 envir-
onmental 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., India: ―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.
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 2007 2008

est.
1/ 6/ 5/ 6/ 1/ 1/ 10/ o
AUSTRIA 132 171 142 109 134 129 0,02 /
BELARUS 70 000o 67 900 70 900 79 000 78 500 75 200 78 5001/ o
/
BELGIUM 16 9903/ 15 3155/ 19 3065/ 19 8231/ 18 6701/ 16 3541/ 14 6301/ 12 030/
BULGARIA 210 470 150o 70 o
/ o
/ o
/ o
/
CHINA 100 0006/ 80 0006/ 133 0006/ 200 0006/ 130 000 118 500 110 000 o
/
130 0007/
CZECH 7 095 5 885 6 003 5 500 4 3181/ 2 73611/ 82410/ o
/
REPUBLIC
DENMARK 191/ 01/ 01/ o
/ o
/ o
/ o
/ o
/
EGYPT 7 6499/ 8 9369/ 13 0109/ 17 1389/ 5 8479/ o
/ 20 0001/ o
/
o o o
ESTONIA 89 35 17 0 Fibrous Flax / / /
0, Linseed
91ha
FINLAND 3656/ 2025/ 975/ 675/ 571/ 171/ o
/ o
/
FRANCE 67 9703/ 68 4161/ 76 4395/x 60 0811/ 81 5081/ 76 4971/ 75 5231/ 67 0001/
GERMANY 2001/ 2006/ 2246/ 1806/ 381/ 30 51 o
/
IRELAND 03/ o
/ o
/ o
/ o
/ o
/ o
/ o
/
ITALY 13/ 05/ 205/ 80 181/ o
/ o
/ o
/
o o o
LATVIA / / / 1 6546/ 2 0721/ 1 420 22010/ o
/
LITHUANIA 9 600 9 346 9 4441/ 5 494 3 5991/ 1 0571/ 9501/ o
/
NETHERLANDS 4 4151/ 4 0005/ 4 6155/ 4 5171/ 4 6911/ 4 36611/,1/ 3 4581/ 2 5001/
POLAND 4 5204/ 5 1004/ 6 0004/ 6 3454/ 6 8434/ 4 22511/ 2 05610/ 19914/
PORTUGAL 03/ 01/ o
/ o
/ o
/ o
/ o
/ o
/
RUMANIA 3006/ 3006/ o
/ o
/ o
/ o
/ 10710/ o
/
RUSSIA 127 340 110 820 118 060 112 300 95 450 86 000 75 000 81 000
127 3616/ 100 0006/ 104 0006/
o o o o o o
SLOVAK REP. / / / / / / 6710/ o
/
SPAIN 3421/ 605/ 25/ o
/ o
/ o
/ o
/ o
/
SWEDEN 323/ 251/ 01/ 301/ o
/ o
/ 34 o
/
UKRAINE 28 280 28 200 32 4808/ 38 2208/ 25 5308/ 16 164 12 0008/ o
/
UNITED 4 4301/ 1565/ 1755/ 1 8201/ 1961/ 211/ o
/ o
/
KINGDOM
Source: 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: albert.daenekindt@vlasverbond.be
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: hempflax@inf.poznan.pl, data provided by the Ministry of Agriculture
and Rural Development.
5/
54ème Congrès CELC – Berlin, Réunion d'information Générale / Section commune Culture-
Teillage
6/
CELC/MASTERS OF LINEN, 15, rue du Louvre, 75001 Paris, France, tel.: +33(0)1 42 21 06 83,
e-mail: info@mastersoflinen.com
7/
Research Institute of Industrial Crops of Heilong Academy of Agricultural Sciences, Harbin,
China, 150086, tel.: (86)0451-55261351, E-mail: wuguangwenflax@163.com
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, e-
mail: profelhariri@aim.com; acc. to Agricultural Economics Bulletins of the Central Administrati-
on 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
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).
Table 5. Fibrous flax cultivation area [thous. ha]
Year Flax cultivation area

1989 34.3
1990 29.6
1991 9.7
1992 5.5
1993 4.0
1994 8.1
1995 13.8
1996 5.9
1997 3.7
1998 2.4
1999 3.1
2000 4.1
2001 5.2
2002 5.1
2003 3.0
2004 5.7
2005 6.5*
2006 4.2*
2007 2.1*
2008 1.99
2009 est. 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
 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.
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.
Table 7. The production of linen yarn and fabrics in Poland in 2008
Linen Company Linen yarn production [tons] Linen fabric

production [mio m2]
LAMBRECHT 1,200
SAFILIN 3,000
MADEX 540 0.4
ORZEL 160 1.8
ZYRARDOW ----------- 1.5
Source: the Linen Companies listed in the table.
Table 8. Data on export and import of fibre and linen products in Poland in 2003
Specification Export in thous. USD Import in thous. USD

Scutched flax fibre 2,137 10,661
Linen tow and waste products 2,255 3,789
Linen yarn total 24,840 9,877
Linen fabrics total 21,302 31,552
Source: Ministry of Economy, Labour and Social Policy.
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
 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 1,205 862 762 517
for food and non-
food)
-of which for starch 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).
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.
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
1999 2000 2001 2002 2003 2004

Production of spirit [mio liters 100]o 167 173 181 210 220 240
In which: potato spirit [mio liters 10,1 13,9 11,0 7,5 7,0 7,0
100o]
The share of potato spirit in total 6,0 8,0 6,1 3,6 3,2 2,9
spirit production [%]
Processing of potato [thous. tons] 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 2001 2002 2003 2004

Production of chips 28.5 36.4 37.3 40.0 45.0
Productio n of ―French fries‖ 1007.7 126.4 151.1 121,0* 150.0
Source: CSO.
* the market did not show the decreased demand for ―French fries‖.
** forecast.
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
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.
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 2000 2005 2006 2007

Cultivation area [thous.ha] 333 286 262 247
Yields from 1 ha [dt] 411 416 438 513
Harvest [thous.t] 12,236 11,912 11,475 12,682
a
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.
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 Cultivated area [ha]

Thyme (Thymus vulgaris L.) 3000
Valerian (Valeriana officinalis L.) 2500
Chamomile (Chamomilla recutita (L.) Rausch.) 2000
Peppermint (Mentha piperita) 2000
Milk thistle (Sylibum marianum Gaertn.) 1500
Caraway (Carum carvi L.) 1500
Savory (Safureja hortensis L.) 400
Lemon balm (Melissa officinalis L.) 300
Lovage (Levisticum officinalis Koch.) 300
Sage (Salvia officinalis L.) 300
Evening primrose (Oenothera paradoxa) 250
Purple coneflower (Echinacea purpurea Moench.) 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.)
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 human-
friendly 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
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.
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
Major agricultural raw materials applied for production of ethanol [thous.tons]

Year Raw material
Rye Potatoes Molasses
[thous.t] Share of total rye [thous.t] Share of total potatoes [thous.t]
production [%] production [%]
1995 664.8 10.6 649.0 2.6 0.0
1996 680.0 12.0 640.0 2.4 31.0
1997 630.5 11.9 370.6 1.8 50.0
1998 522.0 9.2 216.0 0.8 76.0
1999 378.0 7.3 112.0 0.6 107.0
2000 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 Yield of Production of The consumption of

in Poland [t/ha] ethanol [dm3/t] ethanol [dm3/t] raw material for 100
dm3 ethanol
Corn 6.1 390 2,379 256
Wheat 3.5 340 1,190 294
Potatoes 19.0 140 2,660 714
Sugar beet 45.0 100 4,500 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 1994 1995 1996 1997 1998 1999 2000 2001 2002
Raw spirit 210 245 278 240.6 208 167.2 173.3 181 210
Ethanol- 27 63 100.9 110.6 99.8 88.5 51.5 69.4 82.8
dehydrated spirit
Source: Ministry of Agriculture and Rural Development. Department of Land Economy and Rural
Infrastructure (MRRW) 2003.
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.
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 Raw Materials

Over 1,000 tons Matricariae flos, Menthae piperitae folium, Valerianae radix
Carduri mariae fructus
500-1,000 tons Hippocastani semen, Thymi herba, Hypernic herba, Oenotherae semen
200-500 tons Tilae flos, Mellisae folium, Boraginis semen
100-200 tons Aroniae fructus, Frangulae cortex, Sambuci fructus, Betulae folium, Quercus
cortex, Salicis cortex, Hippocastani cortex,
Urticae folium, Cynarae herba, Visci albi herba
50-100 tons Equiseti herba, Sambuci flos, Crataegi inflorescentia, Taraxaci radix, Salviae
folium, Millefolii folium, Farfarae folium, Violae tricoloris herba
10-50 tons Agropyri rhizome, Calami rhizome, Levistici radix, Calendulae anthodium,
Chelidonii herba, Phaseoli pericarpium, Althaesae radix, Arnicae anthodium,
Galegae herba, Origani herba, Angelicae radix
5-10 tons Unilae radix, Euphrasiae herba, Cichorii radix, Meliloti herba, Cnici herba,
Anthemidis anthodium, Convallariae herba, Malvae arboeae flos
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 – Producer: PPH ―HIMAL‖ Lodz

 Biosept 33 SL – Producer: Cintamani Poland – Piaseczno.
 Polyversum – Producer: Biopartner S. C. Poznan.
 Adpros 850 SL – Producer: Varichem T. Ostrowski- Huta Zabikowo.

 Olejan 85 EC – Producer: 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 Shape (form) Producer The components of plant origin

Geriavit Capsules PHARMATION (CH) Ginseng extractum sic.
Amol Liquid ROLAND (D) Oleum Citronellae, Oleum Myristicae,
Oleum Caryophylli, Oleum Cinnamomii,
Oleum Citri, Oleum Mentae, Oleum
Lavandulae
Sylimarol Pills HERBAPOL Poznan (PL) Silybi mariani fructus extractum siccum
Raphacholin Pills HERBAPOL Wroclaw Raphani radix recens extractum sic.,
C (PL) Cynarae herba extractum sic.
Bilobil Capsules KRKA (SLO) Ginkgo bilobae extractum siccum
Wyciag ze Tablets VITAMEX (S) Eauiseti extractum sic., Urticae extractum
Skrzypu sic.
Polnego z
Witaminami
Urosept Pills HERBAPOL Poznan (PL) Extractum sic.: Petroselini radix, Phaseoli
pericarpium, Betulae folium, Chamomilae
anthodium, Vitis idaeae folium
Oeparol Capsules AGROPHARM (PL) Oenothera paradoxa oleum
Tablets Tablets LABOFAM (PL) Melissae folium, Leonuri cardiacae herba,
Uspokajajace Valerianae radix, Lupuli strobili, Menthae
piperitae folium, Lavandulae, flos
Poldanen Tablets coated HERBAPOL Poznnan Pygeu africani cortes extractum sic.
(PL)
Alax Pills HERBAPOL Poznnan Alona, Glycyrrhizae radiz, Frangulae
(PL) cortex extr., Atropa radix
Sirupus Syrup HASCO-LEK (PL) Plantaginis extractum fluidum
Plantaginis
Tadenan 50 Capsules DEBAT (Foumier Group) Pygei africani cortex extractum sic.
(F)
Cardiol C Drops HERBAPOL Wroclaw Tinc. Convallariae titr., Tinc Valerianae,
(PL) Tinc. Crataegi, Colae extractum fluidum
Boldaloin Tablets HERBAPOL Wroclaw Aloe extractum sic., Boldinum
(PL)
Kalms Tablets G.R.LANE HEALTH Lupuli strob. pulvis, Valerianae extractum
PRODUCTS (GB) sic., Gentianae extractum sic
Esberitox N Tablets SCHAPER AND Thujae herba extractum sic., Echinaceae
BRUMMER (D) rad. extractum sic., Baptisiae extractum sic.
Echinacea Tablets RATIOPHARM (D) Echimaceae angustifoliae radix extractum
sic.
Pyrosal Syrup HERBAPOL Wroclaw Extractum fluidum: Farfarae folium,
(PL) Sambuci flos, Tiliae inflorescentia, Salicis
cortex; Fructus Ribis Concentratum
Melisana Liquid KLOSTERFRAU (D) Extractum: Melissae fol., Inulae rad.,
Angelicae rad., Zingiberis rhiz., Piperis
nigri fruct., Gentianae rad., Myristicae
sem., Phaseoli pericar., Cinchonae cort.,
Casiae flos, Cardamomi fruct.
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, Olsztyn: ―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‘; ‗The Sugar market‘; ‗The Cereals market‘; '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
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 Copper-
Producing 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.
Chapter 12
ENERGY EFFICIENCY OF FOUR CROP SPECIES
Jerzy Pudelko*, Jerzy Mankowski and Jacek Kolodziej

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.
124 Jerzy Pudelko, Jerzy Mankowski and Jacek Kolodziej
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 follows: 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

Energy Efficiency of Four Crop Species 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
80
[GJ/ha]
60
47,37
38,50
40
20
0
spring barley winter wheat winter sugar beet
rapeseed
Figure 1. Average energetic value of tested crop yields.
100
90,67
90 85,38
80
70
60
[GJ/ha]
50
40
29,69
30 26,78
20
10
0
spring barley winter wheat winter sugar beet
rapeseed
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
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
Energy inputs for yield Crop energetic value

Crop Years Net energy [GJ/ha]
obtaining [GJ/ha] [GJ/ha]
2006 13.8 34.20 21.02
Spring barley 2007 11.17 42.56 31.38
2008 10.81 38.76 27.94
2006 17.51 45.60 28.08
Winter wheat 2007 19.19 47.88 28.68
2008 16.32 48.64 32.31
2006 21.58 106.80 85.21
Winter
2007 17.24 109.47 92.22
rapeseed
2008 17.53 112.14 94.60
2006 26.46 100.44 73.97
Surgar beet 2007 13.28 109.62 96.34
2008 15.48 101.34 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 2006 2007 2008 Mean

Spring barley 1,36 1,70 1,55 1,53
Winter wheat 1,82 1,91 1,94 1,89
Winter rapeseed 4,27 4,37 4,48 4,37
Sugar beet 4,01 4,38 4,05 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
200
[GJ/ha]
150
109 103
100
50
0
winter rapeseed sugar beet 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
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] 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.
[2] Grzybek A., Gradziuk P., Kowalczyk K. (2001): Sloma energetyczne paliwo. Akademia
Rolnicza w Lublinie. Warszawa.
[3] Kolodziej J. (2009): Efektywnosc energetyczna konopi w zaleznosci od czynnikow
agrotechnicznych. Praca doktorska. Uniwersytet Przyrodniczy. Poznan.
[4] Panczyszyn T. (2008): Polityka energetyczna Polski do 2030 roku. Czysta Energia
9/2008.
[5] 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.
[6] Wos A. (pr. zbiorowa) (1998): Encyklopedia Agrobiznesu. Fundacja Innowacja.
Wyzsza Szkola Spoleczno Ekonomiczna. Warszawa.
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.
132 Viktor Antonov, Josef Marousek, Jan Marek et al.
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 Microbial producers Main activity

Texazym APN Aspergillus oryzae cellulase
Inosample CLC Trichoderma reesei cellulase
Inosample APC Trichoderma sp. cellulase
Inosample HMP Trichoderma reesei hemicellulase
Inosample HMC Humicola insolens hemicellulase
Inosample ASD Bacillus licheniformis alpha‐amylase
Texazym BIO‐TK Trichoderma sp. cellulose, hemicellulase
Enzymatic Biomass Hydrolysis 133
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) 10% solid fytomass is mixed and the pH is adjusted to the optimal pH of the enzyme
2) when the pH is unchanging, the enzyme is added
3) sample mixed and is tempered in optimal enzyme reaction temperature for 6 hours
4) 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
Alkalic thermopressure preparation:
1) 10% solid fytomass material mixed with 300 ml H2O and (3, 6, 9, 12%) NaOH
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.

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).
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 HMC APN BS TK APC
enzymes only 80 46 84 186 74 63 48 87 115 82
enzymes after
12
thermopressure 91 221 148 138 322 201 184 113 201 143
4
preparation
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 mg glucose equivalents / g dry mass

BIO-TK 115
thermopressure preparation 91
BIO-TK after thermopressure preparation 201
3% acid thermopressure 131
3% alkal. thermopressure 115
6% alkal. Thermopressure 117
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 mg glucose equivalents / 1 g dry mass

Type of enzyme used no ASD AO HMP SLAD CLC HMC APN BS TK APC
enzymes only 46 36 35 41 26 16 15 16 20 14
enzymes after
thermopressure 33 95 114 125 118 137 96 87 117 136 127
preparation
Figure 7. Corn silage saccharification.

Table 5. Comparison of different pretreatment of corn silage, i.e. different types of

CORN SILAGE mg glucose equivalents / g dry mass

BIO-TK 20
Figure 8. Comparison of different pretreatment of corn silage, i.e. different types of thermopressure
Table 6. Beet tuber saccharification
BEET TUBER mg glucose equivalents / 1 g dry mass

Type of enzyme
no ASD AO HMP SLAD CLC HMC APN BS TK APC
used
enzymes only 164 175 295 215 277 332 265 201 386 271
enzymes after
thermopressure 184 180 202 346 246 277 410 299 226 392 295
preparation
Figure 9. Beet Tuber saccharification.
Table 7. Comparison of different pretreatment of beet tuber, i.e. different types of

BEET TUBER mg glucose equivalents / g dry mass

BIO-TK 386
Figure 10. Comparison of different pretreatment of beet tuber, i.e. different types of thermopressure
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.
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: Zadek@ovgu.de.
†
E-mail: Robert.Schulz@ovgu.de.
142 Hartmut Zadek and Robert Schulz
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
Sustainable Logistics Centers 143
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
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.
Sustainable Logistics Centers 145
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] Logistik fuer Unternehmen: Alternative Antriebstechnik im Praxistest. In: Logistik fuer
Unternehmen, issue 1/2-2010, pp. 18-19. Springer-VDI-Verlag, Duesseldorf (2010).
[2] 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.
[3] Logistik fuer Unternehmen: Brennstoffzellen-Projekt demonstriert den CO2-freien
Materialtransport. In: Logistik fuer Unternehmen, issue 3/4-2010, pp. 24-25. Springer-
VDI-Verlag, Duesseldorf (2010).
[4] Brexel, F.: Energieeffizienz und Nachhaltigkeit - Zukunft der modernen Logistikim
mobilie. (2009). http://www.straubing-sand.de/fileserver/straubingsand/files/ 31.pdf.
[5] Kramm, M.: Der Energieausweis fuer Distributionszentren. In: Tagungsband
Energieeffizienz im Lager, Cologne (2008).
[6] 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).
[7] Scharf, A.: Klimaschutz erfordert Umdenken in der Antriebstechnik. In: VDI
nachrichten, issue 45/2009, p. 9. VDI-Verlag, Düsseldorf (2009).
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: shishkina@sky.chph.ras.ru.
148 Lyudmila N. Shishkina, Mikhail A. Klimovich, Mikhail V. Kozlov et al.
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 cell-
free 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 low-
toxicity 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
Relation between the Cell-Free DNA Content and the Lipid Peroxidation… 149
(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 electric-
Electron 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 (TBA-
reactive 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
error of average mean to average mean for group expressed as a percentage. The significance
of differences were evaluated by Student t- criterion.

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 TBA-
reactive 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 September October October (the November December

(beginning) end of month)
[TBA-RS],
0.076 ± 0.007 0.0765±0.004 0.172± 0.005x 0.148±0.014x 0.082 ± 0.013
nmol/mg of
(n* = 4) (n = 2) (n = 2) (n = 6) (n = 10)
protein
[DNA], 0.037± 0.003 0.0415 ± 0.0075 0.060±0.009xxx 0.0615±0.008xx
μg/mg of protein (n = 10) (n = 4) (n = 6) (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.
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 cell-
free 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 cell-
free 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 cell-
free 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 X-
ray 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].
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.
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 [TBA-RS], nmol/mg of [DNA], g/mg of protein

protein
Mice Balb/c The intact age control (n* = 4) 0.021  0.006 0.078  0.016
(males) Administration of 0.3% Tween
80 in 10% water acetone (n = 2) 0.046  0.012 0.0325  0.0055xx
Mice SHK The intact age control 0.092  0.007 (n = 5) 0.102  0.008 (n = 9)
(females) The black liquor in drinking
water at the concentration of
0.005% 0.146  0.029 (n = 5) 0.144 0.012 (n = 3)x
0.015 % 0.143  0.029 (n = 5) 0.116  0.016 (n = 5)
0.05 % 0.075  0.007 (n = 5) 0.096  0.025 (n = 5)
Mice SHK The intact age control (n = 6) 0.1915  0.028 0.072  0.008
(females) [27] X-rays at the dose of 16 (n = 3) 0.42  0.20 0.117  0.043
* Number of replicate measurements. x – Significant differences from the control mice (p < 0.02). xx
-
Significant differences from the control mice (p < 0.06).
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)– X-
rays irradiation at the low doses under changing the dose rate. c) – X-rays irradiation at the dose of 16
cGy.
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).
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303-310, 2002
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[14] L.N. Shishkina, E.B. Burlakova, ―The value of antioxidant properties of lipids in
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Cells in vitro and in vivo by the Method of Alkali Elution‖, Biological Bulletin, no. 3,
pp. 276-281, 1996 (in Russian).
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Reports, vol. 10, no. 6, pp. 830-837, 1991 (in Russian).
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antioxidants‖, Achievements of Chemistry, vol. 54, no. 9, pp. 1540-1658, 1985 (in
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1456-1462, 2000.
[25] M.V. Kozlov, V.V. Urnysheva, L.N. Shishkina, ―Interconnection of Parameters of
Regulation System of Lipid Peroxidation and Morphophysiological Parameters of
Mouse Liver‖, Journal of Evolutionary and Biochemical Physiology, vol. 44, no. 4, pp.
470-475, 2008.
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Products‖. In: Itogi nauki I tekhniki. Moscow: VINITI Akad. Nauk SSSR. Ser.
Biofizika, 1986 (in Russian).
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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.
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Antioxidative Status in Development of the Consequences of Biological Action Under
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no. 2. pp. 162-7 (in Russian).
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Sciences, vol. 64, pp. 425-431, 1994.
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. Chembio@sky.chph.ras.ru.
158 E.Ya.Davydov, I.S. Gaponova, S.M. Lomakin et al.
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).
Mechanism of Stable Radical Generation in Lignin… 159
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.
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).
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.
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.
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.
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.

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 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.
3. The formation of stable iminoxyl radicals in the presence of NO2 is the typical
process for lignin.
REFERENCES
[1] N.M. Emanuel, G.E. Zaikov, V.A. Kritsman ―Chemical kinetics and chain reactions.
Historical aspects‖, Nova Science Publishers, New York, 1992, 625 pp.
[2] ―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.
[3] ―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.
[4] 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.
[5] G.E. Zaikov ―Chemical and biochemical physics. New frontiers‖, Nova Science
Publishers, New York, 2006, 272 pp.
[6] ―Resent advances in polymer nanocomposites: synthesis and classification‖, Ed. by S.
Thomas, G.E. Zaikov, S.V. Valsaraj, A.P. Meera, Brill Academic Publishers, Leiden-
Boston (The Netherlands - USA), 2010, 436 pp.
[7] ―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.
[8] G.E. Zaikov, S.K. Rakovsky ―Ozonation of organic and polymeric compounds‖,
Smithers, Shawbury, Shrewsbury, Shropshire, UK, 2009, 414 pp.
Chapter 17
SPRAY FORMATION OF ALTERNATIVE DIESEL FUELS

UNDER ENGINE-LIKE CONDITIONS
Dennis Backofen1, Michael Könnig2,

Helmut Tschöke1 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, CO2 reduction, alternative

fuels, optical measuring devices, spray formation
168 Dennis Backofen, Michael Könnig, Helmut Tschöke et al.
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 (CO2-
emissions) 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 Fischer-
Tropsch 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
Spray Formation of Alternative Diesel Fuels under Engine-Like Conditions 169
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:
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 Otto-
von-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.

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 Rapeseed methyl ester

specifications (RME)2
Density (at 15°C) kg/m³ 834 883
Kinematic viscosity
mm²/s 2.9 3.50 – 5
(at 40 °C)
Surface tension (at 20 °C) N/m 0.028 (25 °C) not spec.
Boiling point/range °C 176 °C – 364 °C 330 °C – 360 °C
Ester content % 0 > 99,0
1
= Shell Global Solutions GmbH, 2009.
2
= According to Werkszertifikat Bio-Ölwerk Magdeburg, 2009.
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.
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.
25
1500 bar
2000 bar
Injected Fuel Volume [mm³]
20 2500 bar 1.18 ms

3000 bar
1.01 ms
0.93 ms 1.5 ms
15
3 0.86 ms
12 mm
10
5
Nozzle: Sac-hole, 1 hole
Fuel: Reference fuel
0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Control Duration [ms]
Figure 2. Injected fuel volume.
For discussing the spray image, the penetration, the spray angle, the maximum diameter
and the volume were selected for this paper:
Spray
angle
Max.
+ Volume
diameter
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.
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
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 Control duration: 1.5ms

11000 TChamber: 20 °C
PChamber: 20 bar
10000
B0
Sprayvolume [mm³]
9000
B100
8000 1500 bar
7000 2000 bar
2500 bar
6000
3000 bar
5000
4000
3000
2000
1000
0
1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0
Time after start of control [ms]

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.
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.
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
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.
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
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.: Höchstd-
ruckeinspritzung 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 Benzindirekt-
einspritzung V in Berlin, expert-Verlag, 2006
[6] Fischer, S.: Untersuchung des Effekts einer Höchstdruckeinspritzung auf die Ruß/NOx-
Emissionen 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.
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
182 Bettina Biskupek-Korell, Sabrina Becker, Jasmin Dufrenne et al.
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).

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. Q-
Solution (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`) Tm °C

KB-8657d GGT GAC GCA G 34
KB-8659d TGG GGG ACT C 34
Tm = Melting temperature [4(G+C) + 2(A+T)].
Table 2. Temperature profile for RAPD PCR with Urtica dioica genomic DNA; primers
KB-8657d and KB-8659d, 45 cycles
Step Time Temperature °C

Initial denaturisation 2:00 min 95
Denaturisation 30 sec 95
Annealing 45 sec 35
Extension 1:30 sec 72
Final extension 10:00 min 72
Storing ∞ 4
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

PCR with Control Primer Pair EU+/EU-
The 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, trouble-
shooting 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.
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 KB-
8657d (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
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
clone
B1 B12 B13 B28 Z6 Z10
RAPD marker Product bp
KB-8657d 1900 - - - - x -
1200 - x - - - -
750 x - - - - -
580 - x - x - x
KB-8659d 950 - - - x - -
Genotyping via SCAR Markers
The innate restrictions of RAPD fingerprinting, as well as problems with limited inter-
laboratory 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 B12-
KB8657d-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.
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
clone
SCAR marker B1 B12 B13 B28 Z6 Z10
Product bp
B12-KB8657d-1200-1.0-l +1.1-r 350 - x x x x x
B12-KB8657d-1200-1.0-l +2.0-r 950 - - - x - x
B1-KB8657d-390-3.0-l +3.4-r 750 x x - x x x
1350 - - - - - x
B28-KB8659d-950-6.0-l + 6.1-r 910 - 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 B1-
KB8657d-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
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
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: PCR-
Nachweis 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 Thaila-
nd jasmine rice using RAPD and SCAR methods. Eur. Food Res. Technol. , 229, 515–
521.
Chapter 19
IFT PERFORMANCE OF MES SURFACTANT FROM

PALM OLEIN FOR EOR APPLICATION *
Erliza Hambali1, Mira Rivai1, Putu Suarsana2, Sugiharjo3,

Edi Zulchaidir4 and Hermansyah Handoko5
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.
192 Erliza Hambali, Mira Rivai, Putu Suarsana et al.
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 Percentage (%)

Lauric (C12:0) 0.1 – 0.5
Myristic (C14:0) 0.9 – 1.4
Palmitic (C16:0) 37.9 – 41.7
Palmitoleic (C16:1) 0.1 – 0.4
Stearic (18:0) 4.0 – 4.8
Oleic (18:1) 40.7 – 43.9
Linoleic (C18:2) 10.4 – 13.4
Linolenic (C18:3) 0.1 – 0.6
Arachydate (C20:0) 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.
IFT Performance of MES Surfactant from Palm Olein for EOR Application 193
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
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%.

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
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
Input IFT at different MES concentration

No Sample Steam Temperature (oC) (dyne/cm)
0.1% 0.3% 0.5% 1.0%
1 EH 1 0% 80 0.0327 0.0269 0.0236 0.0201
2 EH 2 0% 90 0.0222 0.0172 0.0188 0.0067
3 EH 3 0% 100 0.0620 0.0356 0.0307 0.0276
4 EH 4 75% 80 0.0248 0.0467 0.0036 0.0053
5 EH 5 75% 90 0.0453 0.0046 0.0063 0.0043
6 EH 6 75% 100 0.0224 0.0048 0.0078 0.0458
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%
EH 1
3.27 x 10-2 2.69 x 10-2 2.36 x 10-2 2.01 x 10-2
EH 2
2.22 x 10-2 1.72 x 10-2 1.88 x 10-2 6.7 x 10-2
EH 3
6.20 x 10-2 3.56 x 10-2 3.07 x 10-2 2.76 x 10-2
EH 4
2.48 x 10-2 4.67 x 10-2 3.6 x 10-3 5.3 x 10-3
EH 5
4.53 x 10-2 4.6 x 10-3 6.3 x 10-3 4.3 x 10-3
EH 6
2.24 x 10-2 4.8 x 10-3 7.8 x 10-3 4.58 x 10-2

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.
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.
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%.
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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.
Chapter 20
THE DEVELOPMENT PROCESS OF JATROPHA

METHYL ESTER SULFONIC ACID (MESA) TO
ENHANCE OIL RECOVERY *
Siti Mujdalipah1, 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.
202 Siti Mujdalipah, Mira Rivai, Erliza Hambali et al.
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.
The Development Process of Jatropha Methyl Ester Sulfonic Acid (MESA)… 203
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%.

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 Value

Water content (%) 8.90
Ash content (%) 4.62
Oil content (%) 39.87
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 sized-
impurities 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 Unit Value

Water content % 0.25
Ash content % 0.042
FFA % 10.98
Acid number mg KOH/g fat 20.94
Iodine number mg iodine/g fat 99.34
Saponification number mg KOH/g fat 197.6
Density g/cm3 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 300-
500 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.
Table 3. Results of the analysis of jatropha-based methyl ester

physicochemical properties
No Property Unit Value

1 Water content % 1.33 – 2.29
2 Acid value mg KOH/g ME 0.16 – 0.41
3 Iodine number mg Iodine/g ME 98.33
4 Total glycerol %-(w/w) 0.12 – 0.27
5 Unsaponified value % 0.39
6 Ester content %, counted 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 Parameter Unit Value (average)

1 Acid number mg KOH/g MESA 19.81
2 Active ingredient % 30.41
3 IFT Value dyne/cm 0.73
4 pH 1.15
5 Iodine number Mg iodine/g MESA 33.53
6 Surface tension dyne/cm 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
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 Physicochemical properties Unit Value (average)

1 Acid number mg KOH/g ME 1.34
2 Active ingedient % 29
3 IFT value (1% MES in water formation) dyne/cm 8.08 x 10-2
4 Iodine number Mg iodine/g MES 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)

Surfactant rate (%) 15,000 ppm 30,000 ppm
0.1 8.76 x 10-01 1.67 x 10-00
0.3 8.14 x 10-02 1.45 x 10-01
0.5 8.67 x 10-02 1.14 x 10-01
1 8.08 x 10-02 1.40 x 10-01
a) b) 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).
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] Akzo Nobel Surfactants. 2006. Enhanced Oil Recovery (EOR) Chemicals and
Formulations.Akzo Nobel Surface Chemistry LLC. www.surfactants.
[2] Lee, C and P. Berger, 2010. Surfactant Injection Projects-Field Cases. Oil Chem.
Technologies, Inc.www.oil-chem.com.
[3] 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.
[4] 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.
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
210 Frank Pudel
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].

Rapeseed Proteins – Recent Results on Extraction and Application 211
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,
212 Frank Pudel
 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.
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).
214 Frank Pudel
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.

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.

216 Frank Pudel
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.
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).

218 Frank Pudel
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.
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 Glucosi- Phytic Sinapic

nolates Acid Acid
% µmol/g g/kg g/kg
AOCS EG 1864/90L SAA A004 LC/MS

Ba 10b-09 (LUFA) (ÖHMI) (LUFA)
Commercial Press Cake 23,7 18,2 16,9 11,5
(Oil mill)
Commercial Meal 13,6 7,0 16,7 8,0

(Oil mill)
Gentle Desolventized Meal 33,4 20,3 13,9
(Fluidized Bed, 75° C)
Gentle Desolventized Meal 32,0 18,6 14,4 13,6
(Fluidized Bed, 95° C)
“Air Desolventized” Meal 30,4 20,5 16,8 13,7
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.
220 Frank Pudel
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 pre-
desolventizing 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 by-
products 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
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] FEDIOL. [Online] 2010. http://www.fediol.be/6/index.php.
[2] Becker, K. W. Current trends in meal desolventizing. JAOCS. 2 1983, pp. 216-219.
[3] 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 Verfah-
rens 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].
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*1, Sven Wartenberg1, Jasmin Dufrenne2

and Bettina Biskupek-Korell2
1
Institut für Pflanzenkultur e.K., Solkau 2, 29465 Schnega, Germany
2
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: schneider@pflanzenkultur.de.
224 Carolin Schneider, Sven Wartenberg, Jasmin Dufrenne et al.
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.

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 DNA: 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.

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
PCR-Based Detection of Different Endophytic Bacteria Appearing… 225
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.
226 Carolin Schneider, Sven Wartenberg, Jasmin Dufrenne et al.
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. Micro-
bacterium 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 Alphaprot-
eobacteria, too, and other non-culturable bacteria.
Figure 3. Electrophoresis of PCR with specific primer pairs BacZ6-1-Prod.228 L+R and BacB30-
1Prod.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 BacB30-
1-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, BacZ6-3-
Prod.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
PCR-Based Detection of Different Endophytic Bacteria Appearing… 227
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 Wisconsin-
Madison, 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.
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
Polytechnic Institute, University of Conakry, Conakry; Guinea
2
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 petro-
centric society to alternative energy resources becomes ever more apparent. The extraction of
230 Sékou Traoré, Amadou Diarra, Macki Traoré et al.
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 physico-
chemical 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.
Survey of Jatropha Curcas Energy Potential for an Efficient Production… 231
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 carbon-
containing 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:

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 (80-
90%).
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]
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.

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 30-
40 wt.%, which makes it a high oil content seed.
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.
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 (C12:0-
C15:0) [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 fuel-
air 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.
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.
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
[1] Yamamoto, H.K., Yamaji, K. and Fujino, J.; 1999: ‗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. 335-
345.
[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
[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.
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), fах: +48 61 84 17 830, E-mail: sekretar@inf.poznan.pl.
242 Magdalena Chudy and Grażyna Mańkowska
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.
Genetic Resources of Cannabis Sativa L. in the Collection of the Gene Bank… 243
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
24 2 11 2
2
27
14
2
3
24
15
2
25 9
2
1
11
Bulgaria China Finland France India
Yugoslavia Poland Slovakia Netherland Romania
Russia Ukraine Thailand Hungary Italy
Cuba Turkey Breeding lines
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.
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.

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.
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.

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.

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 quality; 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).
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 Dioecious/ Beginning of Full maturity date

monoecios flowering date
Zenit monoecious 13.07 20.09
Juso 11 monoecious 8.07 15.09
Futura 77 monoecious 4.08 2.10
Chamaeleon dioecious 28.07 2.10
Diana dioecious 1.07 5.09
Uniko B dioecious 4.08. 8.10
Silistrenskie dioecious 10.07 20.09
Fedora 17 monoecious 7.07 15.09
Finola dioecious 30.05 20.08
Juso 15 monoecious 8.07 15.09
K - 195 dioecious 10.07 20.09
Dnieprowska monoecious 2.07 10.09
Fibrimon 24 monoecious 12.07 20.09
Zolotonoska 26 monoecious 20.07 25.09
He Bei dioecious 1.09 29.09
Gigantheus dioecious 16.08 29.09
Białobrzeskie monoecious 15.07 20.09
At the Department of Biotechnology at INFandMP the following studies are carried out:
the studies on polymorphism of the gene synthesizing THCA (1 – tetrahydro-
cannabinolicacid) 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
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 Average fiber kontent (%) Cultivar Average fiber kontent (%)
Zenit 23,51 Juso 15 28,36
Juso 11 26,05 K - 195 16,48
Futura 77 20,49 Dnieprowska 19,11
Chamaeleon 27,06 Fibrimon 24 22,28
Diana 20,74 Zolotonoska 26 22,54
Uniko B 29,16 Gigantheus 17,62
Silistrenskie 17,27 He Bei 12,00
Fedora 17 21,82 Białobrzeskie 26,60
Finola 12,28
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 Możliwościach 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.
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.: 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 Zdolności Kiełkowania, Prace Ikwn, Rocznik
Xxix, Poznań: 99-106,
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: alvaro.gonzalias-
mosquera@iw.uni-halle.de.
†
www.kunststofftechnik.uni-halle.de.
252 Alvaro E. Gonzalias, A. Werner and Hans-Joachim Radusch
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
Production of Biodegradable Carrier Materials for the Immobilization… 253
temperature was selected so that the poorly biodegradable component (polycaprolactone-

PCL) 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 PHBPCL 30_70 WB

Material PHB 70%: PCL 30 %
Diameter 4.00 ± 0.106 mm
Height 3.78 ± 0.316 mm
Density 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.

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.

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 Age DNA-amount Denitrification

PCL 6500 1 Jahr 1,8 mg/g 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.

Production of Biodegradable Carrier Materials for the Immobilization… 255
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.
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.
Chapter 26
THE CHANGES IN THE PROTEIN PROFILE IN

RESPONSE TO CADMIUM STRESS IN FLAX
(LINUM USITATISSIMUM 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.
258 Milena Szalata, Szalata Marlena and Wielgus Karolina
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) (Li-
Chan 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, 2009; Barałkiewicz et al., 2009; 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
The Changes in the Protein Profile in Response to Cadmium Stress in Flax… 259
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). Anion-
exchange 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.

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 1-
step (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 (Cys-
rich) 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.
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 (0-
30%) 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 (0-
30%, 30-50% and 50-80%) and dialysis.
The Changes in the Protein Profile in Response to Cadmium Stress in Flax… 261
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 plant-
growth 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.
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 Cadmium-
Binding 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.
Chapter 27
REGIONAL VALUE ADDED OF BIOENERGY

ACTIVITIES A MATERIAL FLOW APPROACH
Ruth Offermann, Thilo Seidenberger1

and Daniela Thrän1,2
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: ruth.offermann@dbfz.de, tel.: +49(0)341-2434-453.
264 Ruth Offermann, Thilo Seidenberger and Daniela Thrän
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. Long-
distance 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).
Regional Value Added of Bioenergy Activities… 265
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 bottom-
up 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)
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
Analysis of regional Regional specification of Validation of value added
standardised regional
development concepts value added chains chains (regional survey)
value added chains
Quantitative analysis of value added chains

Status quo assessment (12/2009)
Identification of plant
Survey of plant operators and Identification of regional
operators and fuel producers
fuel producers biomass flows
(regional survey)
Intermediate survey (12/2010)

Analysis of changes
Final survey (2012)
Valuation of regional biomass flows

Survey of selected regions concerning
Secondary data (national statistics, etc.)
financial figures
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
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
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
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] 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.
[2] Agentur für Erneuerbare Energien e.V.: Regionale Wertschöpfung durch die Nutzung
Erneuerbarer Energien: Hintergrundinformationen. (http://www.unendlich-viel-
energie.de/de/wirtschaft/detailansicht/article/16/ regionale-wertschoepfung.html), Berl-
in, 2009.
[3] Samuelson, P.A. und Nordhaus, W.D.: Volkswirtschaftslehre. (ISBN: 3636031120,
9783636031129), mi-Fachverl., 2007.
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 two-
step 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
272 Robert Reinhard Pätz and Jan-Henryk Richter-Listewnik
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 first-
order rate in integrated form yields eq. 2
s(t) = s0 e-kt (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) = s0 - s0 e-kt (3)
c(t) = s0(1- e-kt) (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.
Simple Kinetics of Methane Formation Using Model Substrates 273
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…

4.1. Carbohydrates as Substrate
Carbohydrates are hydrolysed to mono- and disaccharides and immediately acidified.

Some of possible acidification reactions are
Acidification (examples)
Glucose ... Acidic acid AA + (2) CO2 + H2 (6)

180 kg 2* 22,4 m3
Glucose … Butyric acid BS+ CO2 + H2 (7)
Glucose … Lactic acid LA  Propionic acid PA (8)
BS, PS ... AA + CO2 + H2 (9)
CO2 + H2 … AA (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.
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).
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 k-value in h-1

Glucose 0,011
Amylose 0,029
Microcrystalline cellulose 0,035
Potato starch, raw 0,023
Yeast extract 0,030
Pepton 0,047
Sunflower oil 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
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] LINKE, B. , P. MÄHNERT. Biogasgewinnung aus Rindergülle und nachwachsenden
Rohstoffen. Agrartechnische Forschung 11(2005) Heft 5, S. 125 -132.
[2] CHEN, Y.R. Kinetic analysis of anaerobic digestion of pig manure and its design
implication; Agricultural Wastes 8(1983) , 65 – 81.
[3] 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.
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
280 Christoph Unger, Maja Hühns and Inge Broer
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, 3-
aminotetrahydrofurane, 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-α-
aspartic 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
Biosafety of Transgenic Potatoes Producing the Biopolymer Cyanophycin 281
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.
282 Christoph Unger, Maja Hühns and Inge Broer
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.
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: michael.seiffert@dbfz.de.
284 Diana Weigl, Katja Oehmichen, Michael Seiffert et al.
Keywords: Biomethane pathways, biogas, Bio-SNG, biomass fermentation, biomass

gasification, transportation biofuels, LCA, costs
ABBREVIATIONS
EEC end energy consumption
PEC primary energy consumption
CIS Commonwealth of Independent States
EU European Union (Belgia, France, Germany, Italy, Luxembourg, Netherlans)
DE Germany
SRC short rotation coppice
STP standard temperature pressure
w/o without
PSA pressure swing adsorption
PWW pressure water wash
FICFB fast internally circulating fluidised bed
AER absorption enhanced reforming
EF entrained flow
FM fresh mass
Ar 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
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
Development of natural gas use per sector
3.000 EEC-private households
2.500 EEC-trade and commerce
EEC-transport
in DE in PJ/a
2.000
EEC-industry
1.500
District heating
1.000 PEC-electricity
500 Target biomethane
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
Biomethane
Biogas substrates Solid biofuels
production (e.g.energy crops, residues) (e.g. woody, herbaceous)
Bio-chemical anaerobic Thermo-chemical gasification

digestion (e.g. fluidised bed, pyrolysis/
(e.g. wet fermentation) torrefaction + entrained flow)
Gas clean-/conditioning
(e.g. oil / alkaline wash, activated
coal, ZnO)
CHP Synthesis: Methanation

(heat / electricity) (e.g. fluidised, fixed bed)
Raw biogas Raw Bio-SNG
Biogas upgrading Bio-SNG upgrading

(e.g. acid / MEA wash, TEG
(e.g. PSA, water wash, amine) drying, H2 membrane)
Biomethane Biomethane
Biomethane feed in and distribution (Energetic) biomethane use

Feed in Transfer- Fuel station Electricity /
G260 /G262 / Distribution Mobility Heat
station DIN 51624 CHP
G685
© DBFZ, 2009 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 Biogas Bio-SNG

Suitable raw materials Biogas substrates (liquid, pasty) Solid biofuels, predominantly wood (e. g.
energy crops, residues residues, energy crops), in the future probably
also herbaceous biomass possible
Raw materials demand approx. 7,000 - 17,000 t per year approx. 3,500 - 4,500 t per year and MW CH4, th
(fresh) and MW CH4, th
Transport distance raw 5 to 30 km for residues, energy Unimodal up to 150 km, adapted logistic
materials crops up to approx. 100 km concepts required; harbour locations unlimited
Area specific biomethane 3,000 to 4,500 m³ N / (ha a) (e. g. 3,500 to 5,000 m³ N / (ha a) (e. g. SRC -
yield maize silage) willow); lower for forest wood
State of technology Biogas plants and gas treatment Gasification systems available for fossil fuels,
technology commercial available 1st demonstration plant in 2008, commercial
availability in the years to come
RandD demand Further development of process Demonstration: overall chain, plant
integration; need for increased availability and reliability, use of approved
efficiency system components
Installed overall plant Germany: > 240 MW CH4, th Germany: -
capacity Germany / EU EU: approx. 430 MW CH4,th EU: 1 MW CH4,th (Austria), plans in
(Main regions: Sweden, Switzerland and Sweden
Netherlands, Switzerland)
(Expected) plant capacities up to 20 MW CH4,th up to 300 MW CH4,th
(up to 16 mn m³ N/a) (up to 240 mn m³ N/a)
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
280
Biogas base
240
(natural gas quality) in bn m³STP/a
Technical biomethane potentials
CIS
200
EU+3
Bio-SNG base
160 EU+10
Biogas base EU-15 (ohne DE)
120
DE
80
40
0
Forestry wood
Forestry wood
Energy crops
Energy crops
Industrial wood
Industrial wood
Energy crops
Energy crops
Manure
Manure
(all options)
(all options)
(only SRC)
(only SRC)
residues
residues
residues
residues
2005 2020
Total: Total:
approx. 300 bn m³STP/a approx. 484 bn m³STP/a
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
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 Medium term

Status 2005 Short term (ST) Long term (LT)
(H gas quality) (MT)
Based on Biogas
Raw materials Manure and Mix of energy Mix of energy Mix of energy
mix of energy crops and manure crops, biowaste crops, biowaste
crops 47 / 48 kt FM/a and manure and manure
46 / 27 kt FM/a 77 / 74 kt FM/a 73 / 71 kt FM/a
Processes 2,5 MW 5 MW 10 MW 10 MW
(main PSA / PWW PWW / Amine PWW / Amine + PWW / Amine +
differences) combustion of combustion of
ferm. residues ferm. residues
Based on Bio-SNG
Raw materials - forestry wood forestry wood Forestry wood
residues, residues, res.,
98 kt ar/a willow, (straw) willow, straw
330 / 328 kt ar/a 1.673 kt ar/a
Processes - 22 MW 75 MW 380 MW
(main FICFB gasifier, FICFB gasifier FICFB gasifier
differences) Activated coal / 77 MW 293 MW
ZnO alkaline gas AER gasifier EF gasifier with
cleaning 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
(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.
SNG-LT-293MW
Biomethane /
SNG -MT-77MW
SNG
SNG -MT-75MW
SNG-ST-22MW
BG -LT- 10MW
Biomethane /
Biomass treatment
Biomass conversion
Biogas
BG-MT-10MW
Biomethane upgrading
Building
BG- ST-5 MW Auxiliaries
BG -Status -2,5 MW
0 300 600 900 1.200 1.500 1.800 2.100 2.400

Specific total capital investment in EUR/kWbiomethane
Figure 4. Specific total capital investments of different biomethane concepts.

price spread biomethane household
8,5 Capital
SNG- LT- 293MW
Biomethane /
Operation
9,3 Raw material
SNG
SNG - MT-77MW Revenues by-products

9,0 Propane
SNG -MT-75MW Total
11,6
SNG - ST-22MW
BG -LT-10MW 8,1
Biomethane /
Biogas
7,9
BG-MT-10MW
11,3
BG - ST-5MW
BG - Status- 2,5MW 13,6
-2 - 2 4 6 8 10 12 14 16 18 20
Biomethane production costs free plant in EURct/kWhbiomethane
Figure 5. Biomethane production costs.

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.
Fossil reference EU RED 302

Raw material
SNG-LT-293MW 102 Raw material supply
Biomethane /
Conversion
Bio-SNG
SNG - MT-77MW 114 Distribution methane

106 Credit manure handling
SNG -MT-75MW Total
SNG- ST-22MW 126
BG -LT-10MW 108
Biomethane /
Biogas
BG -MT-10MW 109
BG -ST-5MW 143
BG-Status -2,5MW 106
-150 -100 -50 0 50 100 150 200 250 300 350
Specific GHG emissions (WTT) in g CO2-Eq./kWhbiomethane
Figure 6. Specific GHG emissions of developed concepts.

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.
100 100 100
90 90 90
FUEL HEAT ELECTRICITY
80 80 78 80
74
70 66 70 70
62
60 60 60
50 48 49 50 50
42
40
38 38 38 40 40 40
36 35 34
31 30 33 29
30 30 27 29 29 30 29
25
20 22 22 23 23 22
20 19 20 20
10 10 10
0 0 0
Gasoline/Diesel
CCPP natural gas

Biomethane from liquid manure
BG-Status-2,5MW
BG-ST-5MW
BG-MT-10MW
BG-LT-10MW
SNG-ST-22MW
SNG-MT-75MW
SNG-MT-77MW
SNG-LT-293MW
Heating-mix Oil/Gas
Useful heat natural gas
BG-Status-2,5MW
BG-ST-5MW
BG-MT-10MW
BG-LT-10MW
SNG-ST-22MW
SNG-MT-75MW
SNG-MT-77MW
SNG-LT-293MW
BG-Status-2,5MW
BG-ST-5MW
BG-MT-10MW
BG-LT-10MW
SNG-ST-22MW
SNG-MT-75MW
SNG-MT-77MW
SNG-LT-293MW
Ethanol from sugar beet
Ethanol from wheat

Rape Diesel
Electricity mix Germany 2008

fossil Default Biomethane / Biomethane / fossil Biomethane / Biomethane / fossil Biomethane / Biomethane /
values EU Biogas Bio-SNG Biogas Bio-SNG Biogas Bio-SNG
RED
Use as fuel Use for heat production 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.
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] 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.
[2] Thrän, D., Seifert, M., Müller-Langer, F., Plättner, A., Vogel, A.: Möglichkeiten einer
europäischen Biogaseinspeisungsstrategie. Teilbericht. January 2007.
[3] 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.
[4] 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.
[5] 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.
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.
294 Maria Władyka-Przybylak and Krzysztof Bujnowicz
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.

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.
Flammability of Polymers Reinforced with Lignocelullosic Raw Materials 295
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.
296 Maria Władyka-Przybylak and Krzysztof Bujnowicz
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
Flammability of Polymers Reinforced with Lignocelullosic Raw Materials 297
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] A.K. Błędzki, J. Gassan, Prog. Polym. Sci., 24 (1999) 221.
[2] K. Błędzki, A. A. Mamun, O. Faruk, eXPRESS Polymer Letters, 1 (2007) 755.
[3] 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.
[4] A.P. Mouritz, Z. Mathys, A.G. Gibson, Composites: Part A, 37 (2006) 1040.
[5] Sz. Matko, A. Toldy, S. Keszei, P. Anna, Gy. Bertalan, Gy. Marosi, Polym. Degrad.
Stab., 88 (2005) 138.
[6] L.B. Manfredi, E.S. Rodríguez, M. Władyka-Przybylak, A. Vázquez, Polym. Degrad.
Stab., 91 (2006) 255.
[7] ISO 5660-1:2002 Fire Tests - Reaction to Fire. Part 1: Rate of Heat Release from
Building Products (Cone Calorimeter Method), 2002.
[8] 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.
Chapter 32
FEEDSTOCKS AND (BIO) TECHNOLOGIES

FOR BIOREFINERIES
Joachim Venus1*
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, D-
14469 Potsdam. Fon/Fax: +49(0331)5699-112/-849; e-mail: jvenus@atb-potsdam.de.
300 Joachim Venus
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].

Feedstocks and (Bio) Technologies for Biorefineries 301
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).
302 Joachim Venus
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 thermo-
chemical (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
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.
304 Joachim Venus
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 Raw materials: starchy materials (e.g. cereals)
information : Products: Lactates, lactic acid, starter cultures
Stirred vessels (up to 1000 L), 450-L-fermentor, power, steam, water,
Services :
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, jvenus@atb-potsdam.de

Web : www.atb-potsdam.de

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.
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 products:
 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.

306 Joachim Venus
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
state. The main parameters of a continuous mode lactic acid fermentation is shown in the
following Figure 7.
150 1,E+11
biomass
lactic acid
glucose
cell number
biomass, substrate, product
cell number [cfu/ml]

100 1,E+10
[g/L]
50 1,E+09
0 1,E+08
20 30 40 50 60 70 80 90 100 110 120 130 140
time [hours]
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.

308 Joachim Venus
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, bio-
based 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.
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 1-
60021-224-7).
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.; Blagoev, M.; Kishi, A., CEH Marketing Reasearch Report ―Lactic Acis, Its
Salts And Esters‖. SRI Consulting: 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.
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 crops-
to-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: khel@inf.poznan.pl.
312 Maria Mackiewicz-Talarczyk, Krzysztof Heller, Przemyslaw Baraniecki et al.
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 21st
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-to-
products 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.
Non-Food Crops as a Feed Stock for Modern Bio-Based Industry 313
 WP3 will explore the potential and feasibility of the European industry to make high-
value 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 EU-
27, 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).
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,
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]
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 are:
fundamental and applicative research regarding technological development in biotechnology,
medicine, pharmaceutical areas, production at small scale that sustains the scientific research
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 entitled: ―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,
 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.
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 bio-
convertible 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
 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 bio-
fractionisation or biorefining concept, which implies innovation in industry, priorities in
demand. These require investment and policy changes. [3]
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
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.
 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
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] Myrsini Christou, CRES, Greece: Description of work, FP7-KBBE-2007-2B
„Crops2Industry‟ Coordination Action. November 2008.
[2] 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.
[3] 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.
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 Leps4, 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
326 Mohammad R. Hajirezaei, Mohammad R. Ghaffari, Björn H. Junker et al.
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 modeling: 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?
Multiscale Metabolic Modeling of Cereals 327
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.
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 plant-
physiological 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.ipk-
gatersleben.de).
Figure 3. Interactive visualization of stoichiometric networks in VANTED (FBA-SimVis Add-on )

showing specific fluxes.
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.ipk-
gatersleben.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.
Figure 4. Pearson coefficients of metabolic traits against yield components in Leaves (A) and seeds (B).
Significant correlations are shown by one asterisk (p<0.05) or two asterisks (p<0.01).
REFERENCES
Braune H, Müller J, Diepenbrock W (2009). Integrating effects of leaf nitrogen, age, rank,
and growth temperature into the photosynthesis-stomatal conductance model LEAFC3-N
parameterised for barley (Hordeum vulgare L.). Ecological Modelling 220: 1599-1612.
Weise S, Colmsee C, Grafahrend-Belau E, Junker BH, Klukas C, Lange M, Scholz U,
Schreiber F (2009). An integration and analysis pipeline for systems biology in crop plant
metabolism. LNBI, 5647: 196-203.
Grafahrend-Belau E, Schreiber F, Koschützki D, Junker BH (2009). Flux Balance Analysis of

Barley Seeds: A Computational Approach to Study Systemic Properties of Central
Metabolism. Plant Physiology 149: 585-598.
Junker BH, Klukas C, Schreiber F (2006). VANTED: A system for advanced data analysis
and visualization in the context of biological networks. BMC Bioinformatics 7: 109.1-13.
Rhonda C. Meyer, Matthias Steinfath, Jan Lisec, Martina Becher, Hanna Witucka-Wall, Otto
Toe rjek, Oliver Fiehn, Anne Eckardt, Lothar Willmitzer, Joachim Selbig, and Thomas
Altmann (2007). The metabolic signature related to high plant growth rate in
Arabidopsis thaliana. PNAS 104 (11): 4759–4764.
Ronan Sulpice, Eva-Theresa Pyl, Hirofumi Ishihara, Sandra Trenkamp, Matthias Steinfath,
Hanna Witucka-Wall, Yves Gibon, Bjoe rn Usadel, Fabien Poree, Maria Conceicaeo
Piques, Maria Von Korff, Marie Caroline Steinhauser, Joost J. B. Keurentjes, Manuela
Guenther, Melanie Hoehne, Joachim Selbig, Alisdair R. Fernie, Thomas Altmann, and
Mark Stitt (2009). Starch as a major integrator in the regulation of plant growth. PNAS
106 (25): 10348–10353.
Chapter 35
BIOACTIVE SUBSTANCES
FROM THE BALSAM POPLAR
S. Ludewig1, S. Gille2, D. Orzessek2 and C. Griehl1*

1
Anhalt University of Applied Sciences, Department of Applied Biosciences
and Process Technology, Köthen, Germany
2
Anhalt University of Applied Sciences, Department of Agriculture,
Ecotrophology and Landscaping, Bernburg-Strenzfeld, Germany
ABSTRACT
The cooperative project ‗Extraction and application of bioactive substances from the
balsam poplar – product extraction from renewable primary products‘ sought to combine
material and energetic utilisation of the balsam poplar.
Experiments were carried out on different sorts of balsam poplar. The biological
effects of differences in fertilising, spacing and harvesting time were also investigated.
The results demonstrate the potential for energetic and material utilisation of the balsam
poplar. The extracts of poplar buds are particularly suitable for application in natural
cosmetics due to their considerable antimicrobial effects.
Keywords: balsam poplar, bioactive substances, renewable resources, short rotation

forestry, agar diffusion test, DPPH-Assay, NO-Assay
INTRODUCTION
The mounting shortage of fossil fuels makes renewable resources an ever more
significant player in the field of energy production. With regard to this, the balsam poplar, an
extremely fast-growing tree, is of considerable importance [Pearce et al., 2002]. A high
biomass yield is possible on short rotation plantations; the yield can be made available to
*
Contact: c.griehl@bwp.hs-anhalt.de.
334 S. Ludewig, S. Gille, D. Orzessek et al.
combined heat and power plants (CHPs) in the form of woodcut or pellets [Strom – Lömpke,
2006]. Additionally, balsam poplar contains bioactive substances, which can be used in the
pharmaceutical and cosmetics industries.
AIM AND BACKGROUND

The cultivation of fast-growing trees and shrubs contributes to an increase in the
production of energy on the basis of the combustion and gasification of wood chips. Thus, a
new major market for energy production from alternative sources opens up. Balsam poplars
are cultivated as renewable resources for the energy production in CHPs [Bronowski, 2004].
They quickly generate a considerable amount of biomass [LWF, 2009; Burger, 2007] and
yield 10-15 t/ha p.a. [Thüringer Landesanstalt, 2007; Scholz, 2006].
Table 1. Bioactive substances in balsam poplar [Trinkaus, 2001]
substance effect application

1-salicin analgesic, slight ache, rheumatic troubles, ague
anti-inflammatory,
antipyretic
2-bisabolol anti-inflammatory, stomach and intestinal discomfort,
antifungal, irritation of the skin
antibacterial
3-phenolic acids analgesic, slight ache, rheumatic troubles
(e.g. anti-inflammatory,
coffee acid) antimicrobial,
anticancerogenic
4-eucalyptol anti-inflammatory, respiratory diseases
(1,8-cineol) expectorant
5-crysin raising testosterone level, muscle-growth, enhancing virility, breast
enhancing virility, cancer, anti-aging
anti-inflammatory,
antioxidant
6-farnesol enhancing skin elasticity, skin care, anti-aging, deodorant, foot spray,
wrinkle reduction, acne, bad skin
antibacterial
7-populin various effects raw materials or intermediates for scents
8-acetophenon and pharmaceuticals
9-caryophyllen
10-curcumen
11-cinnamic acid
12-humulen
bud extract sun protection
Balsam poplars (Populus L.) are native to South East Asia and North America and grow
in the northern temperate hemisphere. They are deciduous trees and grow on moderately dry
to wet-dry, fertile, loamy, sandy soils [Thüringer Landesanstalt, 2007; Weih, 2002]. The
species Populus L. belongs to the family of Salicaceae, which are, according to the current
cladistic analysis, classified as Malpighiales along with 36 other families. Following the
Bioactive Substances from the Balsam Poplar 335
generally accepted classification by Eckenwalder, there are 29 species of Populus in 6

sections [Eckenwalder, 1996].
The balsam poplar contains various substances which are used in pharmaceutics and
cosmetics [Pan et al., 2006]. These bioactive substances are mainly constituents of the buds
[Warnant, 2004; Dembitsky, 2005]. There are numerous examples for their use, such as in
analgesics, skin-care products, deodorants, acne remedies or remedies for enhancing virility
and muscle growth (Tab 1, Figure 1). The anti-inflammatory substances also find application
as ointments [Trinkaus, 2001]. A selective application is based on the analysis of the effects
of all principal substances contained in the balsam poplar.
Figure 1. Chemical structures of substances from balsam poplar.
The extracted agents need to be integrated into the vegetation, harvest and utilisation
cycles of the balsam poplar as a renewable resource so as to ensure successful marketing and
sale of the obtained pharmacologic and cosmetic substances.
By combing the cosmetic and pharmacologic use of biologically active substances with
the energetic use of balsam poplar a highly-efficient utilisation of the plant could be achieved.
Consequently, it was firstly the aim of this project to make innovative use of the cellular
and molecular properties of the balsam poplar, with particular regard to the functional bio
molecules in bud extracts, bark and leaves and secondly to develop methods for obtaining
new valuable products from the balsam poplar. The obtained extracts were analysed in terms
of their pharmaceutical and cosmetic properties using different bioassays.
EXPERIMENTAL PART
balsam poplar species investigated:
 Androscoggin (P. trichocarpa x P. maximowiczii) from Bernburg – Strenzfeld

 Muhle Larssen (P. trichocarpa) from Bernburg – Strenzfeld
 Max 5 (P. nigra x P. maximowiczii) from firm Grünbau GbR in Langeneichstädt
 Soil conditions at Bernburg-Strenzfeld (Central German arid zone):
 field capacity: 220 m2
 loess-black earth / loess pararendzina
 soil type: mainly loamy with 2-3 % humus
 pH: 7.5
 nitrogen: 0.16%
 annual mean precipitation: 488 mm (decreasing)
 annual mean temperature: 9.3°C (increasing) [Deutscher Wetterdienst, 2008]
Planting and harvesting:
 cuttings of the balsam poplar clone Muhle Larssen and Androscoggin were arranged
in a 4-part block (0.5ha) with 4 repetitions
 growth periods of two or three years
 fertilisation: - without fertilisation
o fertilisation of 50kg nitrogen/ha
 planting space: - 0.5 m spacing with 12,500 plants/ha
o 1m spacing with 5,000 plants/ha
 the planting bed was cultivated using plough and grubber with mechanical weeding
to prepare the soil for planting unrooted cuttings
 population was cultivated (fortnightly) with rotary hoe and hoe
 one irrigation was necessary during the first month of cultivation
Extraction and analysis of bioactive substances:
 between March and October, samples were hand-picked on a monthly base

 the individual plant parts (buds, leaves, bark) were weighed (6g per sample of humid
biomass)
 extraction of each sample: - 70% ethanol (84ml) over 24 hours
 filtration
 evaporation until dry
 sample preparation for bioassays: dissolving of 100mg dry biomass in 1ml 70%
ethanol
Agar diffusion test (DIN 58940-3: ‗Susceptibility testing of microbial pathogens to

antimicrobial agents‘):
 test organisms: gram-positive bacteria (Bacillus subtilis, Micrococcus luteus,

Staphylococcus capitis), gram-negative bacteria (E. coli, Proteus myxofa-ciens),
yeast (Candida glabrata)
 3-day-old precultures were used for vaccination  3-day incubation  separation of
microorganisms using a sterile salt solution (0.9% NaCl)  application on
isosensitivagar (Difco)  using 1mg crude extract per sample  24-hour incubation
(38°C)  evaluation of the test
DPPH test (test of antioxidant activity using the 2.2-Diphenyl-1-pikrylhydrazyl-radical

DPPH):
 the DPPH radical has a violet colour which is ascribed to the unpaired electron of the
nitrogen atom  colour changes from violet to yellow if the radical bonds with the
hydrogen atom of a radical scavenger  reduced DPPH-H  the anti-oxidant effect
of isolated substances or plant extracts can be quantified by means of a photometric
analysis of the absorption reduction at 517 nm
NO-Assay (nitric oxide assay kit, cell line HaCat, Calbiochem):
 inflammatory cells discharge nitric oxide  indirectly measuring of NO across

nitrate and nitrite with Griess – reagent  accumulation of an Azo – complex with
purple colour  absorptiometry at 540 nm

As part of the cooperative project at Bernburg-Strenzfeld tests were carried out to assess
the material and energetic utilization of balsam poplar. It was the aim of these analyses to
obtain constituents from buds, bark and leaves that could be used for commercial exploitation
in the chemical and cosmetic industries.
So as to prove the effectiveness of the ingredients, the different parts of the balsam poplar
were analysed. After harvesting and preparation of the individual plant parts, the substances
were extracted, cleaned and dried using different extraction procedures. The extracts thus
isolated were investigated for anti-inflammatory, antimicrobial and antioxidant properties
using bioassays.
Results of Planting and Harvesting
During the first harvest year (2009), the constituents of the balsam poplar were
investigated on a monthly base. In the course of these investigations the species Muhle
Larssen showed a substantially stronger and bushier growth than the species Androscoggin.
Measuring the growth height supported this observation.
Figure 2. Crop in April 2009.
Figure 3. Crop in July 2009.
The highest concentration of the relevant substances in buds and bark could be
ascertained at the beginning of the vegetation period (Figure 2). In summer, the plants (Figure
3) produced more biomass but contained less biological active substances.
The research showed that fertilisation had an effect on the substances contained in both
species. By fertilising with 50kg nitrogen/ha p.a. an increase in the growth height could be
achieved (Figure 4).
Figure 4. Growth height of plants with 0.5m and 1m plant spacing in 08/2009.
The measurements taken in August show that the annual fertilisation with 50kg N/ha had
a positive, yield-increasing effect on the species Muhle Larssen (Figure 4) but not on
Androscoggin. The research yielded inconclusive results with regard to the question whether
fewer plants per m² would increase the plant growth, provided that the soil contained
sufficient amounts of nutrients.
Figure 5. Growth height of plants with 0.5m and 1m plant spacing in 12/2009.
Measurements taken in December showed again that nitrate fertilisation had a positive,
yield-increasing effect only on the growth height of species Muhle Larssen (Figure 5). The
December measurements, on the other hand, show for both species an increase in growth
height with a reduced number of plants per m² (difference in height 60cm).
Buds of the balsam poplar could be harvested in March/April and September/October.
The highest bud yield of the species Androscoggin (AS) was at the end of March and the
beginning of April (Figure 6). By fertilisation the bud yield could be increased by 10 kg/plant
during the first year of harvest. Reduced plant spacing did not contribute to an increase in the
bud yield. The autumn buds of September/October had generated only half the yield of the
spring harvest (Androscoggin). Compared to the spring harvest, the species Muhle Larssen
(ML) generated double the amount of buds in autumn (Figure 6). Both Androscoggin and
Muhle Larssen showed an increase in the bud yield after nitrate fertilisation. Plant spacing did
not influence the bud yield in either species. In March/April, Androscoggin produced a bud
yield of 60g/plant (300kg/ha), whereas Muhle Larssen produced a bud yield of 78g/plant
(390kg/ha) in October.
Figure 6. Bud yields, plant spacing at 0.5m and 1m.

The bark was removed from the wood by mechanical means during the first harvest year
and sealed air-tight in ethanol for transport and analysis. The bark could not be removed from
the wood in September and October.
Figure 7. Bark yield, plant spacing at 0.5m and 1m.
Research showed that the bark yield could be raised by an extended plant spacing (Figure
7). The bark yield increase was particularly evident with Muhle Larssen. Irrespective of
fertilising and plant spacing, Muhle Larssen produced a greater bark yield than Androscoggin.
Initial research demonstrated that fertilisation had next to no effect on the bark yield. Plant
spacing and the species selection were of considerably greater influence. The bark yield was
at its peak in September/October (Muhle Larssen 1100g/plant (5.5 t/ha); Androscoggin
700g/plant (3.5t/ha) at 1m plant spacing).
Figure 8. Wood yield, plant spacing at 0.5m and 1m.
In view of the dual utilisation of the balsam poplar in the cooperative project, the wood
yield was also investigated (Figure 8). The yield values represent an average yield per plant
and are given for barkless wood. Regardless of harvest time, the wood contained considerable
amount water and had to be dried prior to thermal use.
It appears that the influence of fertilisation on the wood yield increases towards the end
of the vegetation period. This statement needs to be backed up by the results of additional
harvest years, though. The wood yield of both Muhle Larssen and Androscoggin were more
or less similar. Muhle Larssen generated wood yields of between 3000 and 6000g/plant (15-
30t/ha, plant spacing 1m) and Androscoggin of between 4200 and 6000g/plant (21-30 t/ha,
plant spacing 1m). With reduced plant spacing, Muhle Larssen yielded larger amounts of
biomass than Androscoggin.
Based on previous investigations, a plant spacing of 1m and fertilisation once a year
proved to be elements of the optimal planting method. There was no discernable difference
between Muhle Larssen and Androscoggin with regard to the yield of bioactive substances,
but Muhle Larssen produced a higher amount of biomass.
Results of Optimal Harvest Time and Most Effective Extraction Method
The Agar diffusion test was carried out for determining the optimum harvest time as well
as the most effective extraction method. For this purpose, fresh material extracts could be
obtained from bark, buds and leaves of the species Max 5 (company: Grünbau GbR) and of
the species Androscoggin and Muhle Larssen (Bernburg-Strenzfeld). About 850 samples
were extracted and analysed by means of bioassays over the course of the year. The relevant
results are summarised in following result passage so as to aid clarity.
Figure 9. Carrying out the Agar diffusion test.
Figure 10. Sample plates.
Ethanol was used as solvent to extract plant material in order to ensure further use for
cosmetic and pharmacological products and their application in dermatology. 30% and 70%
ethanol was tested for its efficiency as a solvent. The extracts were screened for their
antimicrobial properties using the Agar diffusion test (DIN 58940-3).
Figure 11 compares the inhibiting effects of the extracts. Only microorganisms with an
inhibition are listed.
Figure 11. Comparison of 30% and 70% ethanol as solvent.
The results of the assay demonstrate a considerably higher antimicrobial efficacy of 70%
ethanol as extracting substance. This may be due to a better solubility of the inhibitor or the
extraction of other inhibitors. The extractions with 70% ethanol showed three inhibitors per
extract, whereas extractions with 30% ethanol from Muhle Larssen showed only one
inhibition, and with Androscoggin two inhibitions. The antibiotic Ampicillin was used to
enable comparison with the extracts. The extracts obtained with 70% ethanol were
significantly more effective on the microorganisms Micrococcus luteus and E. coli than the
antibiotic.
Figure 12. Inhibition areola.

Additional solving methods using shaker and ultrasound were compared.

The results (Figure 13) showed that with ultrasound an essentially higher grade of
disruption could be reached, because a higher antimicrobial effect could be proved. It is
therefore advisable to carry out further extractions of plant parts using 70% ethanol as solvent
and ultrasound as extraction method.
Figure 13. Comparison of the extraction methods shaker and ultrasound.
Further investigations so as to determine the optimum harvest time were performed using
extracts of Max 5 from plant parts taken at different times of the harvest. The Agar diffusion
test was also applied.
Figure 14. Comparison of different harvest times.

The results (Figure 14) showed that the days about the 20th of March constituted the
optimum harvest time for the year 2009, as this was the time of the highest antimicrobial
effect of the bud extracts. At the end of March, the buds had not yet opened up. It is thus
advisable to harvest the buds before they open up that is to say in the second half of March,
depending on weather conditions and bud development.
Another focus was on identifying the anti-inflammatory activity of poplar extracts. The
test was based on the measurement of nitric oxide (NO), which is secreted by inflammatory
cells. With this assay an anti-inflammatory effect was expected to exist at a 15 to 20%
reduction of NO concentration. This applied only to samples originating from the bark of the
tested poplar clones. It can therefore be assumed that anti-inflammatory substances are only
contained in the bark of poplars, as no anti-inflammatory effects could be detected in bud and
leaf extracts.
CONCLUSION
The research confirms an effect of fertilisation on the substances contained in both
species (Androscoggin, Muhle Larssen). Fertilisation with 50kg nitrogen/ha p.a. led to an
increase of the antimicrobial effect and of growth height, whereas differences in plant spacing
did not influence parameters decisively. The optimum harvest time was determined during the
first year of experiments. The highest concentration of active substances was achieved in
March, just before the trees came into leaf, making this the optimum harvest time.
The antibiotic influence of the different parts of the poplar was ascertained by means of
the Agar diffusion test. The antimicrobial influence of extracts from poplar buds was
particularly high. The growth of Micrococcus luteus, E. coli, Candida glabrata, Proteus
myxofaciens and Bacillus subtilis was inhibited. A further focus was on detecting anti-
inflammatory activity by measuring the nitric monoxide content (NO). The anti-inflammatory
effects applied solely to the extracts from poplar bark.
These investigations prove that both poplar species are suitable for the combination of
material and energetic use of balsam poplar.
The results demonstrate the potential of balsam poplar, in particular with regard to an
application of bud extracts in natural cosmetics.
The investigations were also carried out in 2010 and confirmed the results of the previous
year.
REFERENCES
Bronowski, Christine And Kaulfuß, Peter – „Energieholzproduktion In Der Landwirtschaft―;
Bornimer Agrartechnische Berichte, Heft 35, 2004
Burger, Frank – „Potenziale Von Energiewäldern Auf Landwirtschaftlichen Flächen―; Afz-
Der Wald, 14: 749-750; 2007
Dembitsky, Valery M. – „Astonishing Diversity Of Natural Surfactants: 5. Biologically
Active Glycosides Of Aromatic Metabolites―, Department Of Organic Chemistry And
School Of Pharmacy, Hebrew University, 2005
Eckenwalder, James E. – ―Systematics And Evolution Of Populus‖ In: R.F. Stettler Et Al.:
Biology Of Populus And Its Implications For Management And Conservation. 1996, S. 7-
32.
Lwf Bayerische Landesanstalt Für Wald Und Forstwirtschaft – „Anbau Und Ernte Von
Energieholzplantagen Auf Landwirtschaftlichen Flächen―; Zentrum Wald, Forst, Holz
Weihenstephan, 2009.
Pan, Xuejun Et Al. – Organosolv Ethanol Lignin From Hybrid Poplar As A Radical
Scavenger: Relationship Between Lignin Structure, Extraction Conditions, And
Antioxidant Activity,J. Agric. Food Chem., 54: 5806-5813, 2006.
Pearce, David W. Et Al. – Gibberellins In Shoots And Developing Capsules Of Populus
Species―, Phytochemistry 59: 679–687, 2002.
Scholz, Volkard Et Al. – „Produktion Von Pappeln Und Weiden Auf Landwirtschaftlichen
Flächen―; Ktbl-Datensammlung Energiepflanzen, Juli 2006
Strom – Lömpke, Renate – Recherche Zu Forschungs-, Entwicklungs- Und Modell-Vorhaben
Mit Beiträgen Zur Innovativen Nutzung Und Erhaltung Von Agrarbiodiversität―, Agentur
Für Landentwicklung Und Landerlebnis, 2006.
Thüringer Landesanstalt – Anbautelegramm Für Energieholz (Populus Und Salix)―;
Thüringer Ministerium Für Landwirtschaft, Naturschutz Und Umwelt, Juni 2007.
Trinkaus, Peter – „Vielfalt Der Nutzungsmöglichkeiten Von Balsam-Pappeln―, Ländlicher
Raum, 2001.
Warnant, Paul Et Al. – „Screening Of Poplar Biomass For Bio-Active Compounds: A Simple
Method To Assess Antioxidant Activity‖, Bioresource Technology 93: 43–48, 2004.
Weih, Martin – ―Kurzumtriebsplantagen In Schweden‖; Swedish University Of Agricultural
Sciences, 2002.
Chapter 36
THE POTENTIAL OF MICROALGAE TO PRODUCE

LIPIDS FOR BIOFUELS
C. Griehl, H. Polhardt, D. Müller and S. Bieler

Anhalt University of Applied Sciences, Department of Applied Biosciences
and Process Technology, 06366 Köthen, Germany
ABSTRACT
Microalgae have been suggested as a potential renewable source for fuel production
due of their high lipid content.
In view of this, different algae strains of the division Chlorophyta (Botryococcus sp.,
Scenedesmus sp., Chlorella sp., Muriella sp., Nannochloris sp. and Nannochloropsis sp.)
were investigated with regard to biomass productivity and lipid content. The fatty acid
composition was analysed using GC. The microalgae strains Scenedesmus sp. 1 and
Chlorella sp. 1 were found to have the highest biomass and lipid productivity.
Keywords: microalgae, biofuels, biodiesel, lipid extraction, fatty acid analysis, FAME,
GC
INTRODUCTION
The increase of the concentration of atmospheric carbon dioxide is considered to be one
of the main causes of global warming. It is mainly generated through the combustion of the
fossil fuels coal, crude oil and natural gas. In response to this, Germany aims at reducing its
emissions of the greenhouse gas CO2 until 2020 to 40% of the level of 1990.
In addition to rigorous energy saving, CO2 emissions and the dependence on fossil fuels –
a limited and increasingly expensive resource – can be further reduced by an expansion of the
‗Renewable Energy‘ base. In Germany, 10.1% of electricity is currently generated through
renewable means, a figure that is envisaged to rise to 18% by 2020. Renewable energy refers
to energy sources that are regarded as inexhaustible and include solar energy, water and wind
348 C. Griehl, H. Polhardt, D. Müller et al.
power, geothermal energy and bioenergy from biomass. With 70%, bioenergy currently has
the largest share in the renewable energy mix.
Worldwide, scientists are working on the development of new biofuel production
methods. Due to their high lipid content, microalgae have been suggested as a potential
renewable source for fuel production.
AIM AND BACKGROUND

Microalgae are fast growing aquatic plants, which have been exploiting the greenhouse
gas CO2 through photosynthesis for millions of years.
The ability of microalgae to form lipids, which can be converted to biodiesel, is
increasingly being investigated. Compared to energy crops such as rapeseed, microalgae have
the advantage of growing in water-based media while requiring considerably less water than
terrestrial plants. They can be cultivated in photobioreactors on low-value land and thus do
not compete with agricultural land for food production. Furthermore, their growth is non-
seasonal. The potential advantages of algae as biofuel and biomass feedstock are their
exceptional growth rates together with the accumulation of neutral lipids, generally ranging
between 20 and 50% of dry weight (Chisti, 2007, Mata et al., 2010). Based on calculations, an
annual oil production of about 60,000 litres per hectare microalgae is possible in mass
cultivations. This is 50 times higher than the oil yield from rapeseed.
In summary, microalgae are veritable miniature biochemical factories because of the
photosynthetic production of biofuels and the provision of biomass feedstock for the
production of high-value products and generating electricity.
Unlike conventional fossil fuels, biofuels reduce the environmental impact and offer
greater energy security (Cassedy, 2000).
Plant oil usually contains lipids in the form of triacyglycerols (TAGs), free fatty acids,
phospholipids, sterols, water, odorants and other impurities. These prevent its direct use as
biodiesel thus making a chemical modification necessary (transesterification, pyrolysis and
emulsification).
Fatty acid methyl esters (FAMEs), which can be used as a substitute for fossil-derived
diesel fuel, can be synthesised from TAGs via a straightforward transesterification reaction in
the presence of acid/base and methanol (Figure 1).
Figure 1. Transesterification process.
Realistically, biodiesel out of oil crops, waste cooking oil or animal fat can only satisfy a
small portion of the existing demand for fuels. Table 1 illustrates that microalgae appear to be
the ideal candidates for a biodiesel source because of their high lipid content and high
The Potential of Microalgae to Produce Lipids for Biofuels 349
biomass productivity. In addition, the production of biofuels from renewable algal biomass
has the potential of being carbon neutral.
Table 1. Comparison of different biodiesel feedstocks with microalgae (Mata et al.,

2010)
seed oil content land use biodiesel

oil yield [L
plant source [% oil by wt in [m² year / kg productivity [kg
oil / ha year]
biomass] biodiesel] biodiesel / ha year]
corn 44 172 66 152
cannabis sativa 33 363 31 321
soybean 18 636 18 562
jatropha 28 741 15 656
camelina 42 915 12 809
rapeseed 41 974 12 862
sunflower 40 1,070 11 946
castor 48 1,307 9 1,156
palm oil 36 5,366 2 4,747
Microalgae (low oil
30 58,700 0.2 51,927
content)
Microalgae (medium
50 97,800 0.1 86,515
oil content)
Microalgae (high lipid
70 136,900 0.1 121,104
content)
Algae produce more biomass p.a. than other feedstocks and can accumulate more than
70% lipids in their biomass. Table 2 provides an overview of the oil content of some
microalgae (Chisti, 2007).
However, the lipid content of algae is highly dependent on the extraction method used. In
the literature, therefore, different values are often given for lipid content and lipid
composition of algae.
Table 2. Oil content of some microalgae (Chisti, 2007)
oil content
microalgae
[% of dry wt)
Botryococcus braunii 25 – 75
Chlorella sp. 28 – 32
Crypthecodinium cohnii 20
Cylindrotheca sp. 16 – 37
Dunaliella primolecta 23
Isochrysis sp. 25 – 33
Monallanthus salina > 20
Nannochloris sp. 20 – 35
Nannochloropsis sp. 31 – 68
Neochloris oleoabundans 35 – 54
Nitzschia sp. 45 – 47
Phaeodactylum tricornutum 20 – 30
Schizochytrium sp. 50 – 77
Tetraselmis sueica 15 - 23
In view of their immense potential, the production of algae-based fuels is being

intensively explored. However, algal lipid production is at present still too expensive because
of the high production costs including down stream processing.
The aim of our investigation was firstly, to find fast-growing microalgae with high lipid
content. Secondly, so as to study the lipid content of the algae, different extraction methods
had to be examined with the aim of establishing a straightforward procedure for producing
lipids from microalgae. Further investigations need to focus on verifying, modifying and
optimising the downstream processes.
EXPERIMENTAL PART
The experiments were carried out in the algae biotechnology laboratory at the University
of Applied Sciences in Köthen/Saxony-Anhalt.
Algae Strain and Culture Conditions
The microalgae strains Scenedesmus sp., Chlorella sp., Muriella sp., Nannochloropsis
sp., Nannochloris sp. and Botryococcus sp. were obtained from the ‗Culture Collection of
Algae‘ department (Sammlung von Algenkulturen, SAG) at the University of Göttingen,
Germany. Nannochloropsis sp. was grown in an ESP medium; MiEB12 medium was used for
the growth of Botryococcus sp. (media of SAG department at the University of Göttingen).
Scenedesmus sp., Chlorella sp., Nannochloris sp. and Muriella sp. were grown in Setlik-
medium with the following composition (mg/l): KNO3 2020; KH2PO4 340; MgSO4*7H2O
990; Fe-EDTA 18.5; Ca(NO3)2*4H2O 10; H3BO3 3.09; MnSO4*4H2O 1.2; CoSO4*7H2O 1.4;
CuSO4*5H2O 1.24; ZnSO4*7H2O 1.43; (NH4)6Mo7O24*4H2O*4H2O 1.84.
The microalgae were cultivated in 0.5L bubble-column glass reactors at a constant
illumination of 150 µE*m-2*s-1 and at a temperature of 28°C. The cultures were aerated with
200 L/h ambient air enriched with 15% CO2.
The algae cells were harvested by centrifugation after a cultivation time of 14 days.
Determination of Cell Density and Biomass Concentration
The cell density was measured with a Coulter Counter by Beckman Coulter, using the
electrical sensing zone method.
For biomass determination 10-mL samples were taken, centrifuged in pre-weight tubes,
washed twice with distilled water and dried to weight constancy at 100°C.
Cell Disruption and Lipid Extraction
Cell disruption was performed with a vibration mill with 0.2g freeze-dried biomass and
0.4g sea sand. Different solvent systems and methods were used for the extraction of lipids.
Duplicate samples were analysed for each species with regard to total lipid content and fatty
acid composition. The former was determined gravimetrically after extraction. The extraction
procedure was performed using:
1) BlighandDyer (1959)
a)extraction with chloroform / methanol
b)extraction with water / methanol / chloroform
c)extraction with chloroform / methanol / 50mM phosphate buffer
2) Soxhlet preceded by acid digestion (20% HCl) and extraction with n-hexane
3) extraction with hexane-isopropanol
4) ultrasound modified (a previously used acid digestion instead of an alkaline digestion
- 2h cooking in 20% HCl), extraction with dichloromethane
5) extraction with chloroform / methanol according to Folch (1956)
Quantification of FAME By Gas Chromatography
The extracts were evaporated and solved in hexane. The transesterification of the samples
was performed with HCl / methanol (3M) for 1hour at 70°C. After the samples had been
cooled, hexane and water were added and the upper layer was removed. The fatty acid
composition was analysed with GC (Perkin Elmer, Clarus 400, Restek Stabilwax).

Growth Analysis
According to the screening studies of the different microalgae species, the microalgae
strains Scenedesmus sp.1 and Chlorella sp.1 reached the largest increase in biomass
concentration after 14 days of cultivation (Figure 2: Scenedesmus sp.1: 4.6 g/l; Chlorella
sp.1: 5.2 g/l).
6
biomass concentration
[g/l; dry weight]
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.
.1
.2
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.1
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Figure 2. Biomass concentration of the cultivated microalgae species after 14 days of cultivation.
Lipid Extraction
Different lipid extraction methods were performed with Scenedesmus sp. as a preliminary
analysis (Table 3).
Table 3. Lipid content of Scenedesmus sp. obtained through different

lipid extraction methods
Method Total lipid content Evaluation of extraction method
Bligh and Dyer A 30.48 % very complex, small losses of dried biomass
(chloroform-methanol extraction) during the extraction and washing process
Bligh and Dyer B 5.77% less complex, barely any losses of dried
(chloroform-methanol-water biomass
extraction)
Bligh and Dyer C 5.68% less complex, barely any losses of dried
(chloroform-methanol-phosphate biomass
buffer extraction)
Soxhlet, acid hydrolysis 29.60% acid hydrolysis and Soxhlet are very complex,
(hexane extraction) barely any losses of dried biomass
hexane-isopropanol extraction 25.19% less complex, barely any losses of dried

(Guckert et al., 1988) biomass
ultrasonic, acid hydrolysis 38.30% complex (acid hydrolysis), smaller losses of

dried biomass
Folch (modified) 7.31% less complex, smaller losses of dried biomass
According to the analysis, Soxhlet and BlighandDyer A achieved high lipid yields while
requiring a long extraction time. The ultrasonic and acid hydrolysis also involved complex
extraction steps and is therefore very time-consuming. The hexane-isopropanol-based method
was suitable and also provided satisfactory lipid yields.
60
50
lipid content [%; dry weight]
40
30
20
10
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.
.
.1
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Figure 3. Total lipid content of different microalgae species.

Consequently, the gravimetric determination of the total lipid content was performed for
all samples using the hexane-isopropanol extraction method (Figure 3).
The investigation of the lipid content showed that the microalga Scenedesmus sp.1
contained the highest lipid concentration with 52% of dry weight; Chlorella sp.1 achieved a
lipid content of 28%. These results are comparable to those mentioned in the literature
(Scenedesmus sp. 50-56% lipid content, Chlorella sp. 30% lipid content, Spolaore et al., 2006
and Xiong et al., 2007).
Analysis of the Fatty Acid Composition
The properties of algal biodiesel need to be consistent with the European standard for
‗Fatty acid methyl esters (FAME) for diesel engines‘ (EN14214:2008) if it is to be accepted
as a substitute for fossil fuels. A high degree of polyunsaturated fatty acids in algal oil makes
algal biodiesel susceptible to oxidation in storage, thus limiting utilisation (Chisti, 2007).
The selection of a suitable microalga species, which produces a high content of TAGs
depending on culture conditions and time of harvest, and the fatty acid composition are
crucial elements in microalgae-based biodiesel production. A high content of saturated and
monounsaturated fatty acids (typically with a chain length of C14-C22) is considered to be
optimal for biodiesel production.
Anhören
Umschrift
The fatty acid analysis was performed using gas chromatography. Based on the obtained
data, the total fatty acid methyl ester (FAME) concentration and the concentration of single
FAMEs was determined. Figure 4 shows the chromatogram of the FAME determination of
Chlorella sp.1.
Figure 4. Gas chromatography chromatogram of the FAME analysis of Chlorella sp.1.

Table 4 illustrates the fatty acid methyl ester (FAME) composition of cultivated
microalgae species. The results of the fatty acid analysis show that all tested microalgae
strains form a considerable quantity of fatty acids with chain lengths of 10-18 carbon atoms.
Many of the fatty acids contain two or three double bonds. Long-chain fatty acids were only
detected in low concentrations.
Myristic acid (C14:0) and stearic acid (C18:0) are the two fatty acids least represented in
all investigated microalgae species. Within an algae genus, the fatty acid composition varies
considerably. Chlorella sp.1 and Chlorella sp.2 show significant variations in the
concentrations of myristic acid (C14:0), palmitic acid (C16:0) and palmitoleic acid (C16:1).
The results of the different Scenedesmus species, however, are comparable and show only
minor variations. Scenedesmus sp.1 is insofar an exception within its genus, as the
concentration of α-linolenic acid (C18:3) is significantly higher than in the other Scenedesmus
species.
Table 4. FAME composition of the cultivated microalgae
microalgae species C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3

Botryococcus sp. 3.45 29.13 28.50 0.72 7.12 6.63 21.92
Scenedesmus sp.1 2.91 11.41 0.54 0.28 10.23 14.40 23.43
Scenedesmus sp.2 0.81 15.58 7.59 0.92 25.02 18.34 10.00
Scenedesmus sp.3 0.21 23.63 3.27 1.84 24.86 11.71 9.11
Scenedesmus sp.4 1.12 25.51 9.47 0.59 18.31 11.98 10.83
Scenedesmus sp.5 1.85 27.06 7.45 1.02 13.53 10.09 9.96
Chlorella sp.1 3.48 21.74 2.37 1.09 22.19 29.00 20.25
Chlorella sp.1 0.65 9.80 13.55 0.97 21.06 23.15 22.64
Muriella sp. 0.87 31.34 11.71 2.32 18.26 12.39 21.74
Nannochloris sp. 1.24 24.18 19.83 1.12 15.32 12.07 21.33
Nannochloropsis sp. 4.62 33.81 28.08 - 2.61 10.91 18.21
In summary, some of the investigated microalgae were found to be potential candidates

for biodiesel production due to their high lipid productivity and fatty acid composition.
CONCLUSION
According to screening studies of different microalgae genera, the microalgae species
Scenedesmus sp.1 and Chlorella sp.1 showed the largest increase in biomass concentration
after 14 days of cultivation (Scenedesmus sp.1: 4.6 g/l; Chlorella sp.1: 5.2 g/l). The analysis
of different lipid-extraction methods demonstrated the hexane-isopropanol method to be a
promising method for lipid extraction because of its easy application and short extraction
times. Regarding the lipid content, the microalgae Scenedesmus sp.1 was shown to contain
the highest concentration with 52% of dry weight, while Chlorella sp.1 achieved 28%. These
results are comparable with the ones in the literature. Consequently, a comparison of lipid
yields of the same algae species with data from the literature is difficult as lipid contents can
differ significantly depending on extraction method.
The results indicate algae to be able to accumulate high lipid contents, thus making them
promising candidates for biodiesel production. Algal biofuels have obvious potential for
contributing to environmental and economic sustainability. However, biodiesel production

from microalgae is currently inefficient due to the high production costs. Among others, an
optimisation of microalgae harvesting and oil extraction processes is necessary for achieving
a highly efficient microalgae production. A considerable investment in technological
development and technical expertise is still needed before algal biodiesel production becomes
economically viable.
REFERENCES
Bligh, E.G., Dyer,W.J.: A rapid method of total lipid extraction and purification. Can. J.
Physiol. Pharmacol. 37(8), 911–917 (1959).
Cassedy, E.S., 2000: Prospects for a sustainable energy future: a critical assessment.
Cambridge University Press (2000).
Chisti, Y.: Biodiesel from microalgae. Biotechnology Advances 25, 294–306 (2007).
Folch, J., Less, M., Sloane Stanley, G.H.: A simple method for the isolation and purification
of total lipids from animal tissue. J. Biol. Chem. 226, 497-509 (1957).
Guckert, J.B.,. Cooksey, K.E., Jackson, L.L., 1988: Lipid solvent systems are not equivalent
for analysis of lipid classes in the microeukaryotic green alga, Chlorella, Journal of
Microbiological Methods 8, 139-149 (1988).
Mata, T.M., Martins, A.A., Caetano, N.S, 2010: Microalgae for biodiesel production and
other applications: A review. Renewable and Sustainable Energy Reviews 14, 217–232
(2010).
Spolaore, P.: Commercial Applications of microalgae. Journal of Bioscience and
Bioengineering 101 (2), 87–96 (2006).
Xiong, W., Li, X., Xiang, J., Wu, Q.: High-density fermentation of microalga Chlorella
protothecoides in bioreactor for microbio-diesel production, Appl. Microbiol. Biotechnol.
78, 29–36 (2008).
Chapter 37
BIOMATERIALS FOR CONSUMER PRODUCTS
Peter Gerth, Thomas Bagusch, Melanie Poschke

and Johann Zimprich
University of Applied Sciences Magdeburg-Stendal
Centre of Competence Engineering Sciences/Renewable Materials
1. INTRODUCTION
The requirement for concrete efforts concerning the protection of environment and
climate, finite nature of fossil resources and rising prices of oil-based synthetics have caused
a bigger interest in biomaterials in recent years. Other reasons for that are the technical
advance as well as the improvement of the economy of use of these materials. Regarding the
trend of the rising prices of raw materials the potential of the utilisation of renewable raw
materials for the future is recognized.
Regarding the complicated present economic situation, possible concomitant effects like
the increase of independence of imports, the creation of alternatives for the income of
agriculture (long-range without subsidies from the EU in a degree usual today) regional added
value and the preservation of the attraction of rural areas have an increasing impact.
Since 2002 the University of Applied Sciences Magdeburg-Stendal set in an attractive
industry and research location and surrounded by highly cultivated and productive farmland
(Börde and Altmark) works on regional, national and international projects concerning the
use of renewable resources.
2. BIOMATERIALS FOR CONSUMER PRODUCTS

The automobile industry was the precursor in the natural fibre reinforced plastics in the
interior and still is the main applicant of biomaterials and an attractive partner for the relevant
implementation of RandD projects.
358 Peter Gerth, Thomas Bagusch, Melanie Poschke et al.
Additionally to the past niche applications market potentials in different areas developed.
One of the most attractive segments in our view is the area of basic commodity and consumer
products. In contrast to the automobile industry the following structural differences consist:
 SMEs as end producers

 shorter supply chains
 a faster product change
 a big variety of products
Next to the propensity for innovations, this branch also has high and special manifold
requirements for the quality. An increased scope of the applications of biomaterials was
limited till 2008 because of a limited availability to these materials and accordingly to that the
prices as well as deficits of properties which are manly limited to their fire behaviour and heat
resistance.
3. BIOMATERIALS AT THE UNIVERSITY OF APPLIED SCIENCES

MAGDEBURG-STENDAL
At the University of Applied Sciences Magdeburg-Stendal since 2002, intensive RandD
for the application of natural fibre reinforced plastics has been done. Hereby in the focus of
the research were Fibre-Matrix-Adhesion as well as the appearance and the prevention of
smell during the manufacturing process of biomaterials in hot pressing moulding processes.
Since 2006 these research is performed by the Centre of Competence Engineering
Sciences/Renewable Materials as part of the State Campaign of Excellence in Saxony-Anhalt.
Thereby natural fibre injection moulding, the use of biopolymers and WPC-materials are
under investigation.
Typical for all present projects in the area of application-oriented researches is that they
are performed generally in cooperation with enterprises and in due consideration of the whole
value chain. This way it can be ensured that relevant amounts of raw materials and semi-
finished-products are available and that the developed manufacturing processes can be
transferred into the manufacturing in a short time period.
The following table shows samplings with biomaterial compositions, which were
trimmed for the particular case of application.
Table 1. Realised product samples
Area Application
Automobile Industry Interior Door panels, covers
Medical and orthopaedic technology Parts of nursing beds; amputation appliance
Construction engineering Door mounting elements, spacer double glazed
windows
Communication technology Components of cell phones
Cosmetics Sharpener for eyeliner und lipliner
Biomaterials for Consumer Products 359
More requests, mainly from the automobile and the electronic industry and researches
which were mad in this context lead to the question that the reasons for the unsuccessful
establishment in the market are especially thermal properties and the fire behaviour. While
the needed properties like electric strength, surface resistance and creep resistance are
efficient for the practice in electro technology, fire behaviour and thermal resistance are
insufficient. Through intensive and successful development works the possible mechanical
properties are enough and no more other measures are needed.
The following table shows an overview of the valuation of the properties after the traffic
lights system (red: unsatisfactory aptitude, high need for development work; yellow:
sufficient aptitude, low need for development work; green: good to very good aptitude, no
need for development work).
Table 2. Valuation of the properties according the traffic lights system
Property level of biomaterials

NF/PP PLA
Electrical properties
 Electric strength
 Surface resistance
 Creep resistance
Mechanical properties
 Strength
 Stiffness
 Impact strength
Fire behavior
 UL-94
 Glow wire test
 LOI
Thermal properties
 HDT
 Vicat
Other properties
 UV-resistance
 Colour fastness
 Odour emission
4. RESEARCH FOR THE OPTIMIZATION OF BIOMATERIALS

The described property deficits of known and market relevant/available biomaterials on
the one hand and the interest of a housing manufacturer in the usage of new products lead to
the decision to do own works regarding the improvement of heat deflection temperatures of
PLA and the development materials that meet the classification of UL-94 V0 in fire
behaviour.
360 Peter Gerth, Thomas Bagusch, Melanie Poschke et al.
4.1. Heat Deflection Temperatures
4.1.1. PLA + PLA

Lactic acid is a chiral molecule that means that it exists in stereoisomer forms which act
like image and mirror image. From literature research it was known, that stereo-chemical
conditions between D- and L-isomers in the PLA have an impact on the properties. The
stereochemistry of the used lacitids defines the thermal properties of the polylactid. Poly-(L-
lactid) is with a crystallinity of about 35-40 % semi-crystallin, has a glass transition
temperature of 55-59 °C and a melting point at 175-185 °C. The poly (D,L-lactid) is
amorphous and has a heat deflection temperature around 50-60 °C. The racemat of poly-(L-
lactid) and poly(D-lactid) is semi-crystallin and has it‘s melting point at about 230 °C.
In the Centre of Competence there is no own researches about synthesis, dimerisation and
ring opening polymerisation of lactic acid. That‘s why the improvement thermal resistance of
PLA wasn‘t by this way was not investogated. Meanwhile there are PLA-verities which use
the possibility of this modification available on the market.
4.1.2. PLA + Natural Fibre

With this concept it was attempted to improve the mechanical properties trough fibre
reinforcement and simultaneously to increase the heat deflection temperature. This effect is
known for other thermoplastic bonded reinforced plastics (e.g. PP). As reinforcing fibres flax
and bamboos have been used. The manufacturing of injection-moulding compounds was done
in the twin screw extruder. After that the standard test specimen were made and examined.
The thermal properties were partly significantly improved while the strength and stiffness
also improved strongly as expected.
4.1.3. PLA + Inorganic Fillers

The known improvement of heat-deflection temperature (Vicat/HDT) by addition of
inorganic fillers (talc in the case of PP) was tried to attain by the addition of
diatomite/infusorial earth.
The materials were manufactured by compounding, folowed by injection moulding of the
test specimen and testing. The filling degrees were between 5% and 20%. The mechanical
properties remained nearly unchanged, the heat defection temperature could not be increased
significant.
4.2. Flame Protection of PLA
Concrete requirements for flame-protected PLA (UL-94: V0) were the result of the
cooperation with a manufacturer of cases for electronic devices. In March 2009 biomaterials
of the Competence Centre were tested successfully for this application in this enterprise.
Mechanical, optical and electrical properties lay in the area of the demanded specification.
For a follow-up of the use of PLA as an alternative material the evidence for the UL-94: V0
specification was required.
The selection of appropriate products was carried out according the advice of a
manufacturer of flame retardants. Four different types of halogen free flame retardants were
Biomaterials for Consumer Products 361
examined which were compounded at amounts of 30 % each. After the manufacturing of test
specimen they were tested in the vertical flame test according to UL-94 by an external testing
institute.
For all flame protected materials the UL-94: V0 classification could be verified. The
stiffness in tensile and flexural test stayed constant, strength and impact strength decreased.
For two of the tested flame retardants an optimization potential was reached which has to be
measured in future projects.
CONCLUSION
Based on the demands of the consumer domain at the University of Applied Sciences
Magdeburg-Stendal different injection moulding compounds have been developed in the last
two years and their properties were tested. Materials after these compositions are available for
potential users in a short term.
Especially in the last 12 Month with the start of the manufacturing respectively the
successful implementation of improved materials in the area of biomaterials has been reported
(e.g. heat-deflection resistant PLA; flame protected PLA; WPC for injection moulding).
In consideration to our researches the actual availability on the market is still not good
enough. The Centre of Competence Engineering Sciences/Renewable Materials is available
for interested parties as a contact and cooperation partner for projects for the usage of
renewable resources.
REFERENCES
[1] Carus, M.: Trends bei Biowerkstoffen, Biowerkstoff-Kongress, Stuttgart 2009.
[2] bioplastics Magazine 04/08 Vol. 3, pp. 18 – 20; bioplastics Magazine 01/09 Vol. 4, pp.
42 – 44.
[3] Müller, K. et. Al: ―Bio-Plastics and Bio-Composites for Houshold Applications‖,
bioplastics Magazine 02/10 Vol. 5, pp.
Chapter 38
USE OF RICE SPELT AS COMPONENT

IN BUILDING MATERIAL
Stefanie Bachmann1*, Grete Bach and Rainer Loth2†

1
ifn Forschungs- und Technologiezentrum, Elsteraue OT Tröglitz
2
POMA Maschinen- und Anlagenbau GmbH, Dürrröhrsdorf-Dittersbach
ABSTRACT
Rice spelt is an undesired by-product of rice processing with a very small application
spectrum up to now. So they are burned and the ashes are deposited in Asian countries
with a high risk for people‘s health. On the other hand side there is a high demand for
cheap light building materials in Asia. Rice spelt, rice spelt ash combined with natural
fibres and light plastics were tested as composite materials for bricks and building plates
but also as thermo insulating material. Recipes and processing for the production of
bricks and building plates on the base of concrete, loam and clay were developed, in
which rice spelt and rice spelt ash were included.
Keywords: rice spelt, rice spelt ash, light building materials
INTRODUCTION
Rice is the most important food for more than the half of the citizens of the world. In
2008 the worldwide production of rice was 685 m tons (Table 1).
20% of this amount, i.e. more than 137 m tons, are undesired by-products, such as rice
spelt, rice straw and polishing flour of rice production.
This problem also applies to Vietnam, which is the 5th largest producer of rice. Here the
amount of side products of the rice production sums up to 7.7 m tons.
*
ifn Forschungs- und Technologiezentrum, Dr.-Bergius-Straße 19, D-06729 Elsteraue OT Tröglitz.
†
POMA Maschinen- und Anlagenbau GmbH Lohmener Straße 13, D-01833 Dürrröhrsdorf-Dittersbach.
364 Stefanie Bachmann, Grete Bach and Rainer Loth
Table 1. Worldwide production of rice in 2008 /1/
Country Amount [t] Country Amount [t]

1 China 193,354,175 11 Pakistan 10,428,000
2 Indien 148,260,000 12 Vereinigte Staaten 9,239,630
3 Indonesien 60,251,072 13 Ägypten 7,253,373
4 Bangladesch 46,905,000 14 Kambodscha 7,175,473
5 Vietnam 38,725,100 15 Südkorea 6,919,250
6 Myanmar 30,500,000 16 Nepal 4,299,264
7 Thailand 30,466,918 17 Nigeria 4,179,000
8 Philippinen 16,815,500 18 Sri Lanka 3,875,000
9 Brasilien 12,100,138 19 Iran 3,500,000
10 Japan 11,028,750 World 685,013,374
In Vietnam the spelt are mostly deposited near the traditional rice mills (Figure 1).
Partially they are burnt in production processes to generate heat, otherwise the ashes must
be deposited. Ash deposits bear a high risk for people‘s health: fine ash particles are emitted
to the surrounding atmosphere and their high silica content is dangerous for the respiratory
tract and eyes of people living nearby.
Figure 1. Traditional rice mill in Vietnam and rice ash deposit near a farmhouse.
On the other hand side in Vietnam, similar to many other Asian countries, there is a high
demand for cheap light building materials for houses on the countryside. Rice spelt, rice spelt
ash together with natural fibres and light plastics could be an interesting composite material
for bricks and building plates but also as thermo insulating material.
AIM AND BACKGROUND

The development of suited compositions, the optimisation of recipes and the processing
for the production of bricks and building plates on the base of concrete, loam and clay where
rice spelt and rice spelt ash are included was the aim of a project, realised in co-operation
with the machine factory POMA, which is the owner of a rice mill in Vietnam. So the results
Use of Rice Spelt as Component in Building Material 365
of the research and development can be tested at the actual place with local raw materials,
using own engineering for the production of building materials.
Through building and testing experimental houses with the newly developed building
materials containing rice spelt or rice spelt ashes it would be possible to offer the new light
building materials to the local and governmental building authorities.
EXPERIMENTAL PART
1. Compositions for Light Building and Isolation Plates
In a set of optimisation experiments the possibility of producing stable plates with

different parts of composition components was tested. The following ingredients were used in
the given variation width:
rice spelt from a rice mill in Vietnam (25 – 45 wt%)

rice spelt ash from an Indian factory (10 – 35 wt%)
Portland composite concrete (CEM II/B-M(S-LL) 32,5 R (55 – 75 wt%)
a foaming agent from a German producer (Sika® Schaumbildner SB2) (1.2 – 12.5 wt%)
Natural filaments (bamboo, sugar cane) (0.17 – 2.5 wt%)
Styropor (PUR foam) (0.8 – 2.2 wt%)
water ad 100 wt%
Mixing was done by hand and the mixture was placed in special forms (Figure 2a) and
the plates were then air-dried.
Stable plates could be produced with rice spelt and with homogeneous distributed short
filament pieces (Figure 2b, c)
Figure 2. Plate form (a), Plate with rice spelt (b), Plate with bamboo filaments (c).
In all cases tested, stable plates could be produced only if the weight-proportion of rice
spelt to concrete was ≥ 1 : 1.6.
A good quality of plates could also be attained by mixing the concrete with a mixture of
rice spelt and small pieces (5 mm) of Styropor. In this case the density of the plates could be
lowered (Figure 3)
The addition of rice spelt ashes was not successful – even with very little amounts of ash
to the optimal recipe developed in systematic tests it was not possible to get stable plates.
Neither the addition of special binding additives nor the change of methodology of mixing
gave positive results.
Figure 3. Plates with rice spelt and Styropor.
Measuring the mechanical stability of plates with different additives data given in table 2
were obtained.
Table 2. The resistance to pressure of plate samples
Sample Pressure load Stability

[kN] [N/mm²]
Plate with rice spelt 2.4 0.44
Plate with rice spelt and bamboo 0.8 0.77
Plates with rice spelt and 1% Styropor 3.5 0.65
For comparison:
Plates with wood shaving 10 1.00
from the market
It is obvious that plates only with rice spelt are not very stable in comparison to
established plates with wood shavings. Adding different filaments or plastic particles
increases the stability data significantly, so that it seems possible to reach an equal strength
with optimised recipes.
The heat isolation of rice spelt plates was tested in a laboratory test – a good isolation
characteristic was obtained (Figure 4).
Figure 4. Heat isolation tests with plates with rice spelt addition.
So the tested mixtures could be used not only as light building material in form of plates
but also for isolating purposes either as plates or as a bulk good.
2. Compositions for Bricks
In a further set of experiments the possibility of adding rice spelt ashes to loam and clay
for the production of bricks was examined.
Different types of ashes were used in these experiments (Table 3)
Table 3. Different types of rice spelt ashes used in experiments
Ash from Ash from Ash from Ash from

rice spelt rice spelt rice spelt rice spelts
Parameters Symbol Unit
Vietnam Vietnam India India
sample 1 sample 2 sample 3
Loss of Ignition Ll Wt.% OS 15.00 5.00 4.00 2.00
Carbon C Wt.% OS 13.00 3.74 1.31 0.76
Sulfur S Wt.% OS 0.10 0.05 0.05 0.05
Silicon Si Wt.% OS 56 60. 62. 69
Mixtures with different content of ash (5, 10, 20, 30 wt.%) were prepared by hand and
placed into forms of 80 × 120 × 70 mm.
Partly the generated bricks were air dried for some weeks; partly they were fired at
different temperatures (600°C, 700°C, 800°C, 900°C) in a laboratory furnace.
All air-dried clay bricks with ash contents up to 30% were stable after drying (Figure 5).
Figure 5. Bricks with clay and ash-contents of 5%, 10%, 20%.
Firing at the mentioned temperatures was possible only for bricks with an ash content of
at least 10%, the bricks with lower amounts of ash exploded during firing.
The same procedure was done with clay-ash bricks adding rice spelt to the mixture
instead of parts of ash. stable bricks with a very low density were received compared with
usual bricks in all cases – during firing the rice spelt were burnt up, the density decreased.
With loam analogue experiments as with clay were done. Only the experiments to
produce bricks with 5% ash content were not repeated due to the negative experiences in the
experiments with clay bricks.
Similar results for air-dried and fired brick samples were received (Figure 6).
Figure 6. Loam bricks with a mixture of rice ash and rice spelt (a) and with ash only (b).
The bricks had some different stability dependent on the temperature of firing – higher
temperatures give higher stability. The addition of rice spelt to the loam-ash mixture resulted
in a decreasing density. All samples were measured by the Oberlausitzer Baustoff- und
Umweltlabor GmbH Löbau according to DIN 18134.
Table 4. Stability and density of different types of bricks
Stability Density
Sample
[N/mm²] [kg/m3]
Air-dried loam/ash bricks are more stable than air-dried clay/ash bricks
clay + 30% ash, air-dried 1.1 1,200
loam + 30% ash, air-dried 1.8 1,230
After firing clay/ash bricks are more stable than loam bricks
clay + 30% ash, 900 °C 3.3 1,230
loam + 30% ash, 900 °C 1.1 800
The higher the firing temperature the higher is the stability of clay/ash-bricks
clay + ash, air-dried 1.1 1,200
600 °C 3.0 1,170
700 °C 2.7 1,160
800 °C 3.1 1,210
900 °C 3.3 1,230
Bricks with only spelt have a low stability even after firing
loam + 30% spelt, air-dried 1.0 980
Clay + 30%spelt, 900 °C 0.8 800
From the results of a large series of tested brick-mixtures with loam and clay together
with rice spelt ashes and rice spelt the mixture of clay with 30 wt.% rice spelt ash after firing
at 900 °C showed the highest stability:
Pressure load – 28 kN Stability – 3.3 N/mm2 Raw density – 1,230 kg/m³
The air-dried analogue showed significantly lower data:

Pressure load – 8 kN Stability – 1.1 N/mm2.
Three other samples had a similar stability:
Table 5. Stability and Density of fired clay/ash bricks
Sample Pressure load Stability Raw Density

[kN] [N/mm2] [kg/m³]
ZT 16 clay/ash (600 °C) 23 3.0 1,170
ZT 18 clay/ash (800 °C) 24 3.1 1,210
ZT 19 clay/ash (900 °C) 28 3.3 1,230
Theses samples would be allowed to be used in protected walls according to the

European Norm for bricks EN 771 – 1, which demands densities up to 1,000 kg/m3 in such
cases. Bricks with higher densities are allowed to be used in building unprotected walls.
According to the stability data in Germany the DIN 105 deals bricks in 10 classification-
types:
Table 6. Stability classification according to DIN 105
Pressure Stability [N/mm²]

stability
classification mean lowest single value
2 2.5 2.0
4 5.0 4.0
6 7.5 6.0
8 10.0 8.0
12 15.0 12.0
20 25.0 20.0
28 35.0 28.0
36 45.0 36.0
48 60.0 48.0
60 75.0 60.0
Our data are at the lowest end of this classification, which is caused – as we think – by
the sub-optimal conditions of firing the brick samples in the laboratory. Thus the data and
experiences, received in this work focus mainly on the development of recipes of mixing
different ingredients and experiences and in handling during the generation of plates and
bricks. This is the first important part of the planned work.
The next step to follow is testing the developed recipes under industrial firing conditions
as they are applied in industrial brick factories. They are characterised by special methods of
mixing, using pressure to form the bricks, pre-drying of the blanks and firing them with an
extremely slow heat increase and decrease to retain the structure and stability of the bricks
and obtain a higher classification than it is possible under laboratory conditions. This
development step is currently under work together with a brick factory nearby Meißen
(Saxony). The results will be shown if it is possible to attain significantly higher pressure
loads and stability of generated plates and bricks to build model houses in Vietnam.
Another aim will be the generation of bricks under the specific production conditions in
Vietnam with the aim to build and check model houses. The results will show the possibility
to start together with Vietnam authorities a planned farmhouse-building programme.
ACKNOWLEDGMENT
The project was financially supported by the Arbeitsgemeinschaft Industrieller
Forschungsgemeinschaften e.V. (AiF) - KU0016707GZ8.
Chapter 39
CRITERIA SYSTEM OF SUSTAINABLE AGRICULTURE

(CSSA - KSNL)
Gerhard Breitschuh1*, Hand Eckert2,

Ines Matthes3†, and Jürfgen Strümpfel3,
In cooperation with: Günter Bachmann3, Landwirt Martin Herold3,
Thorsten Breitschuh4‡ and Ulrich Gernand4
1
Thuringian Ministry for Agriculture and Environment, Erfurt
2
Am Rosenhang 6, Jena
3
Thuringian State Institute for Agriculture, Jena
4
VAVB e.V., Jena
ABBREVIATIONS AND UNITS OF MEASURE

ABAG general soil erosion equation
AF arable area
AK labour force
AZ quality of the soil
BF balanced land (LF minus untilled fallow)
BK-Region soil-climate region
BN rating
CC cross compliance (= other engagements)
GE cereal unit (about 100 kg wheat)
GJ gigajoule
GK soil classification (P, K, Humus-C)
GL grassland
GV livestock unit (about 500 kg living mass)
*
Thuringian Ministry for Agriculture and Environment, Beethovenstr. 3, Erfurt.
†
Thuringian State Institute for Agriculture, Naumburger Str. 98, Jena.
‡
4 VAVB e.V., Naumburger Str. 98, Jena.
372 Gerhard Breitschuh, Hand Eckert, Ines Matthes et al.
ha hectare
KSNL=CSSA criteria system for sustainable agriculture
KUL criteria of environmentally compatible agriculture
KSL criteria of socially compatible agriculture
KWL criteria of economically compatible agriculture
LF farmland
ÖLF ecologically and culturally significant areas without a productive
use
THG greenhouse gases (CO2, CH4, N2O)
TMLNU Thuringian Ministry of Agriculture, Nature Conservation and
Environment
TLL Thuringian State Institute for Agriculture
USL Environmental protection system for agricultural farms
VAFB Registered Association for Agricultural Research and Education in
Thuringia
VDLUFA Association of German Agricultural Institutes for Analysis and
Research
CRITERIA SYSTEM FOR SUSTAINABLE AGRICULTURE TO ANALYSE

AND ASSESS THE SUSTAINABILITY OF FARMS
The criteria system for sustainable agriculture (KSNL/CSSA) allows farmers to have the
sustainability of their farm analysed and assessed. The analysis provides an insight into the
operational weakness profile and action-orientedness to ensure sustainable development.
Figure 1. Summery of the results of all CSSA-criterias (example farm).

Criteria System of Sustainable Agriculture (CSSA - KSNL) 373
ÖLF-share: amount of ecological werthfull areas without a productive use
1. BRIEF INFORMATION ON CSSA

What is the Meaning of Agricultural Sustainability?
A development is called sustainable if it satisfies the needs of a growing number of

people in the long run and considers at the same time the limitations of natural resources and
a reduction of environmental pollution (committee of inquiry 1997). As a consequence,
sustainability marks a development, which is economically efficient and verifiably
compatible with the environment and the society.
How Does KSNL Work?
With a total of 34 evaluation criteria (11 for the economical, 14 for the ecological and 9
for the agro-social sector) KSNL reports about the sustainability situation of farms. The
evaluation criteria demonstrate in measures and numbers to what extent the farming situation
can be considered sustainable, indicating risks and goal conflicts opposing to sustainable
development.
How Did KSNL Come into Being?
Starting point was the system "Criteria for environmentally compatible agriculture"
(KUL), introduced initially in 1994 by the TLL in Jena. This system was developed
continually further in the following years in cooperation with numerous experts. It was put to
the test on more than 400 farms nationwide. On this basis, taking economic and agro-social
criteria into account, and because it is an extensive sustainability evaluation system the
―Criteria system for sustainable agriculture‖ (KSNL) was put up for discussion in the year
2000 (Breitschuh and Eckert 2000) and has been put to the test ever since then.
What Has the Evaluation Procedure KSNL to Offer?
With its unification of economic, social and ecological topics the Commonwealth of
States has declared sustainability a general political aim (Agenda 21, 1992). If this statement
shall not remain a mere declaration of intent and an empty cliché, instruments to describe a
situation must be established which can at the same time solve the existing communication
problems about the question ―what is sustainability?‖ KSNL serves exactly that purpose: it
draws up a farm‘s detailed strength/weaknesses profile and offers the farmer action-
orientedness towards sustainable development.
What are Farmers Getting out of KSNL?
 a convincing performance review of operational measures,

 the identification of avoidable shortcomings and their causes,
 objective-oriented optimisation of procedures and actions aiming at sustainability,
 cost reduction by pointing out efficiency reserves, and
 an better position to defend themselves against general reproaches.
To what Extent is the Evaluation Procedure KSNL Scientifically

Acknowledged?
KSNL consists of the three criteria systems KUL (criteria for environmentally compatible
agriculture), KWL (criteria for economically compatible agriculture) and KSL (criteria for
socially compatible agriculture). KUL is already scientifically accepted. The procedure was
run through an intensive expert discussion along with several system comparisons (VDLUFA
standpoint 1998), and is supervised by an expert committee. A similar situation applies to
KWL, which is based for the major part on statutory, audited accounting records and uses
modes of calculation that are part of operational analysis. As far as KSL is concerned, the
establishment of an expert committee is underway. KSNL went through an extensive system
comparison in 2007/08, carried through by experts of the KTBL working group ―Evaluation
systems for farms―.
Who Runs KSNL?
Farmers who want their farm to undergo a sustainability analysis must refer to the VAFB,
Naumburger Str. 98, D – 07743 Jena, Germany, phone: 0049-(0)3641/683-116.
The VAFB will supply them with a tender to contract. The VAFB is a registered society,
collaborating closely with the TLL in Jena, and take up mainly projects to implement
scientific insights financed by third-party funds.
What are the Costs for a KSNL Farm Analysis?
The temporary scale of fees stipulates a basic fee of 1220 € (small-scale farming below
100 ha without animal husbandry), extra charges apply for areas over 100 ha or animal
husbandry.
What about Data Security Precautions?
Each farm is generally analysed anonymously by using a code no., which, as a rule, is
assigned by an on-site consultant or the VAFB in Jena. Neither the consultant nor the VAFB
in Jena are authorised to pass the name of the farm on to a third party without the consent of
the farm manager.
How Does the Farm Receive the Analysis and Evaluation Results?
As a result of the analysis the participating farm receives
a) a documentation in the form of charts, worksheets and tables, explaining

comprehensively how the respective criteria value and its evaluation were generated,
b) a graphic illustration of the result, demonstrating at a glance the farm‘s strengths and
weaknesses, and
c) an analysis and interpretation report that identifies the causes for the disclosed
shortcomings and proposes effective countermeasures.
Does the Participating Farm Receive a Certificate?
The issuing of simple certificates for participants is not envisaged, instead, however, the
awarding of accomplishment certificates if the test requirements laid down for this certificate
have been met. The latter will be developed together with the German Safety standards
authority in Thuringia, attesting sustainable farming practices to the farm. The VDLUFA
awards since 1998 the certificate ―Agriculturally sustainable farm― in regard to the
assessment module KUL. Up to now, this certificate has been received by over 40 farms in 9
German States. Bearers of a certificate establish reference farms, demonstrating
environmental compatibility in their region.
2. SECTORS, TEST CRITERIA AND TOLERANCE RANGES

2.1. Brief Description of the Sectors
With KSNL (criteria system of sustainable agriculture) an evaluation procedure has been
introduced which allows farms to run through an extensive sustainability analysis in order to
recognize weak points and derive optional actions from that. Centrepieces of this procedure
are 34 criteria, comprising the sectors economical, environmental and social compatibility.
Defined impacts and/or states are expressed in measures and numbers and assessed by
predetermined tolerance ranges. (Figure 2)
KSNL comprises the three criteria systems KUL, KWL and KSL.
Figure 2. Criteria systems of the KSNL: KUL = Criteria of environmentally compatible agriculture.
KWL = Criteria of economically compatible agriculture. KSL = Criteria of socially compatible
agriculture.
2.1.1. Sector Environmental Compatibility (Criteria of Environmentally Compatible

Agriculture)
Aims:
 protection of the ecosystem = reducing the contamination of ecological subjects of

protection (soil, water, air, biodiversity) to an acceptable level
 keeping up productivity = ensuring the necessary increase of profitability and

avoiding developments limiting productivity
 preservation of ecological functions = ensuring the functions that have a positive
effect on the biosphere (i.e. species and landscape diversity) (cp. Figure3)
Table 1. Test criteria and optima; aimed at tolerance thresholds and location factors
Category Test criterion Dimension Optimum Tolerance Location factor

scoring value 1 threshold
rating 6
Surface N kg N/ha 0 ... 20 -50; +30...50 Leachate

balance
NH3 –emission kg NH3-N/ha = 25 50
Nutrient P-balance kg P/ha 0 -15 or +15 P-content

balance classification
Soil-pH- A ... E C D
classification
Humus balance kg humus-C/ha 0 ... 100 -75 ... 300 GK humus
Erosion t soil/ha Erosion of C = Quality of the Erosion/field

disposition 0.031 soil/8
Soil
protection
Risk of PT/PB2 1.0 1.25
compaction
Plant Plant protection %3 = 70 100 + 4

BK-region
protection intensity
Share ÖLF5 % >9 6 Quality of

farmland
Landscape
and species Crop species Index > 2.2 1.25 Field size
diversity diversity
Median field size ha = 10 40 Quality of

farmland
Energy balance GJ/ha = 80 – 50 – GV/ha, AZ

of farm 75*GV/ha 75*GV/ha
Energy
balance
Energy balance GJ/ha 80 + AZ-20 50 AZ
of crop
GHG special GHG kg CO2-Äq/GJ 136 BN 1 * 1.5 GV/ha, AZ

emission emission MP
1)
according to ABAG with a C-factor of 0.03 (seed grassland).
2)
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)).
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.
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 Tolerance threshold

Rating 1 rating 6
Availability of jobs %7 = 100 70

8
Age distribution % of AK variable variable8
Occupation
Percentage of women % > 43 or < 57 23 or 74
Qualification % variable9 variable9
Holidays Working days = 30 20

Conditions of
Working conditions Points = 12 6
employment
Gross wage level %10 = 95 70
Social activities Points = 11 6

Participation
Percentage of owner % = 66 51
7)
% of the required staff.
8)
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.
Figure 4. Graph of a KSL analysis result (sector social compatibility).

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 Agric-

ulture
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).
Table 3. Test criteria, targeted optima and tolerance thresholds
Category Test criteria Dimension Optimum Tolerance threshold

rating 1 rating 6
Profitability ratio (ordinary) % > 10 0
Return on total assets % >5 0

Profitability
Return on net assets % > 10 0
Rel. factor remuneration % > 130 90
Debt service competence % 31 150

Liquidity
Cash flow III EUR/ha LF 500 50
Equity ratio % > 95 60
Stability Changes in equity EUR/ha LF > 160 0
Net investments EUR/ha LF > 150 0
Income per AK TEUR/AK > 50 25

Added value
Revenue EUR/ha LF > 1200 700
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.

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 Difference between supplied and sold N
Method of evaluation Farm gate balance (kg N/ha) minus NH3-losses (kg NH3-N/ha)
Dimension kg N/ha balance area (LF minus untilled fallow)
Evaluation Rating 1 = 0 … 20 kg N/ha; Rating 6 = 30 … 50 kg N/ha
Protection goal Water, air (N2O), biodiversity, soil fertility
System level Total enterprise
Intent to steer Avoidance of N-surplus, preservation soil fertility
Receipts for bought-in and sold products (fertilizer, market, feed
Required data
products etc.)
Table 4. KSNL criteria profile illustrated by surface N balance
Data sources Accountancy, multiple application

Survey costs Low to medium
Location factor Leachate quantity (mm)
Plausibility check Integrated in analysis programme
Reproducibility High
Controllability Given (accounting records)
Justiciability Given for external, objective analysis
Communicability Balance and evaluation are easy to impart
Practicability Tested extensively (> 400 farms, 675 analyses)
Scientific consensus 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
 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, justiciable-
which 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
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.
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
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
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.
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 agro-
social 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 value-
creation 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.
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.
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.
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‖.

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 guideline-
oriented 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 decision-
making 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.

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
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): 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 manage-
ment (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
agricultural market, 380, 389

2 Agricultural residue, 68, 69, 70
agricultural sector, 389, 390, 393
21st century, 312, 314
agriculture, xiv, 21, 50, 86, 89, 98, 100, 104, 123,
128, 264, 265, 267, 271, 280, 304, 311, 312, 313,
A 314, 315, 357, 372, 373, 374, 375, 376, 380, 382,
384, 385, 389, 394
absorption, 11, 16, 17, 19, 53, 116, 196, 198, 284, Agrobacterium, 226
337 air pollutants, 158
abstraction, 327 albumin, 212, 221
access, 243, 244, 265, 268, 299, 321, 328 alcohol production, 112
accessions, 238, 241, 243, 244, 245, 249 algae, 347, 348, 349, 350, 354
accounting, 263, 374, 382, 384 alimentary canal, 100
acetic acid, 193, 203, 276 alkaloids, 204
acetone, 148, 153 allergic reaction, 57
acetylation, 20 alternative energy, 229, 291
acid, 41, 42, 43, 44, 47, 51, 52, 54, 55, 61, 93, 94, alternative fuels, 167, 168, 169
99, 113, 131, 132, 135, 136, 138, 139, 149, 192, alters, 193, 202
193, 194, 201, 202, 203, 205, 207, 210, 211, 212, aluminium, 51
214, 236, 238, 239, 241, 242, 261, 272, 274, 276, ambient air, 350
280, 299, 302, 303, 304, 305, 306, 307, 308, 309, amino, 210, 211, 275, 280
334, 347, 348, 351, 352, 353, 354, 360 amino acid, 210, 211, 275
acidic, 205, 236, 274 amino acids, 275
acidity, 194 ammonia, 81, 82, 83, 84, 85, 86, 275
acne, 334, 335 ammonium, 81, 82, 85, 86, 251, 252, 254, 255, 259,
acrylic acid, 304 260, 261
active compound, 11, 59, 60 amputation, 358
additives, xii, 76, 220, 226, 317, 365, 366 amylase, 132, 140
adhesion, 12 anaerobic digesters, 63, 64
adhesive properties, 316 anaerobic digestion, 271, 274, 277, 302
adhesives, 211, 316 analgesic, 334
adjustment, 212, 241, 384, 387 anatomy, 314
adsorption, 165, 205, 284 animal husbandry, 375, 383
advancement, 390 annealing, 224
adverse effects, 383 antibiotic, 342, 344
agar, 224, 333 antioxidant, 61, 64, 65, 148, 150, 151, 155, 334, 337
age, 149, 153, 330, 379 antipyretic, 334
aggregation, 61 APC, 132, 136, 137, 138
apoptosis, 65
396 Index
aptitude, 359 biodiversity, 2, 243, 376, 378, 382, 387

aquaculture, 210, 255 bioenergy, xiv, 68, 79, 89, 238, 263, 264, 265, 266,
aqueous solutions, 213 267, 268, 326, 348
Arabidopsis thaliana, 331 Biofilm, 251, 252
arginine, 280 biofuel, 59, 99, 101, 118, 284, 348
arsenic, 262 biogas, 1, 2, 3, 4, 6, 8, 22, 81, 84, 86, 87, 90, 94, 95,
Art of the Renewable Resources, viii 98, 128, 131, 140, 142, 266, 267, 271, 273, 274,
Asia, 12, 59, 78, 95, 334, 363 275, 277, 283, 284, 285, 286, 287, 288, 289, 290,
Asian countries, 363, 364 291
aspartate, 280 Biogas crop, 1
aspartic acid, 280 biological consequences, 148
assessment, 42, 43, 79, 187, 268, 288, 290, 303, 312, biological control, 148, 149
313, 355, 375, 384, 389, 392 biological processes, 326
assets, 264, 381, 387 biologically active compounds, 11
astringent, 60 biomarkers, 329
atherosclerosis, 61 biomaterials, xiii, 21, 23, 303, 357, 358, 359, 360,
atmosphere, 86, 87, 123, 158, 160, 221, 230, 364 361
atoms, 232, 354 biopolymer, 110
Austria, 79, 142, 149, 238, 286, 311 biopolymers, 19, 279, 315, 358
authorities, 365, 370 biosphere, 377
authority, 375 biostimulator, 41, 42, 43, 45
Automobile, 358 biosynthesis, 148, 326, 329
awareness, 1, 55, 320, 321 biotechnology, xiii, xv, 105, 299, 302, 306, 309, 316,
321, 350
birds, 63, 64, 245
B black liquor, 147, 149, 151, 153
bleaching, 13, 158, 194, 197, 206
Bacillus subtilis, 132, 337, 344 blends, 107
bacteria, xiii, 61, 96, 106, 224, 225, 226, 299, 306, blood, 60, 61, 65, 147, 148, 149, 150, 151, 152, 153,
308, 337 154, 155, 156
bacteriostatic, 106 blood plasma, 147, 148, 149, 150, 151, 152, 153,
balance sheet, 109, 112 154, 156
barriers, 101, 319, 323 bonding, 16, 26
base, 70, 94, 116, 198, 202, 226, 267, 268, 283, 300, bonds, 26, 163, 236, 280, 337, 354
316, 317, 336, 337, 347, 348, 350, 357, 363, 364 branching, 2, 43, 45, 46
basic needs, 379 breast cancer, 334
basic raw materials, 318 breathing, 42
bedding, 113 breeding, xii, xiii, 8, 39, 60, 61, 100, 105, 181, 241,
beet molasses, 113 242, 243, 244, 249, 312, 318, 319, 325, 329
Belgium, 103 Britain, 98
bending, 16, 17 brittleness, 18
beneficial effect, 41, 46, 124 building blocks, 305, 306
benefits, 12, 229, 230 butyl ether, 101
bioavailability, 212 buyers, 102
bio-based products, 299, 301, 308, 312, 314, 317 by-products, 11, 12, 105, 210, 220, 289, 320, 363
biochemistry, 157
Biocompound carrier, 251
bioconversion, 304, 305, 306 C
biodegradability, 193, 202, 293
Biodegradable, ix, 251, 255 cadmium, 257, 258, 259, 261, 262
biodegradable materials, 293 Cairo, 104
biodegradation, xiii, 261 calcium, 61, 86, 193, 202
biodiesel, xv, 180, 210, 232, 236, 239, 258, 283, 315, calcium carbonate, 86
347, 348, 349, 353, 354, 355 calibration, 149
Index 397
caloric intake, 12 circulation, 42, 230

calorie, 231 CIS, 284, 287
calorimetry, 293 cities, 158, 159
cancer, 61, 64, 65, 334 citizens, 363
cancer cells, 61 clarity, 341, 389, 390
candidates, 305, 348, 354 classes, 74, 226, 355
capillary, 197, 198 classification, 53, 57, 166, 182, 184, 212, 302, 335,
capsule, 230, 233 359, 361, 369, 370, 371, 377, 382
carbohydrate, 97, 204, 231, 274, 275, 280, 324 cleaning, xii, 90, 192, 194, 202, 285, 287, 288
carbohydrate metabolism, 280 clients, 145
carbohydrates, 230, 231, 237, 271, 281, 305, 308, climate, 1, 59, 89, 90, 119, 168, 232, 233, 241, 248,
314, 315, 319 284, 299, 302, 308, 314, 323, 325, 357, 371
carbon, 24, 25, 26, 68, 91, 95, 168, 174, 230, 231, climate change, 299, 302, 308, 323
232, 236, 237, 252, 271, 272, 273, 274, 275, 276, clone, 93, 95, 181, 182, 184, 187, 188, 223, 224,
277, 299, 301, 305, 306, 319, 326, 328, 347, 349, 225, 226, 336
354 cloning, 188, 224
carbon atoms, 354 clothing, 53, 55, 57, 319
carbon dioxide, 68, 91, 95, 230, 231, 237, 271, 272, clusters, 73, 264, 308
273, 274, 275, 276, 277, 347 C-N, 16
carbon monoxide, 168, 236, 237 CO2, 1, 28, 68, 77, 82, 84, 123, 141, 142, 143, 144,
carbon neutral, 349 145, 167, 168, 169, 180, 214, 221, 232, 274, 275,
carboxylic acid, 93, 242 280, 290, 347, 348, 350, 372, 377
carotenoids, 114, 204 coal, 24, 25, 70, 90, 126, 127, 288, 347
catalyst, 194, 203, 204, 236 coatings, 142, 211, 212
cattle, 209, 210, 278 color, 49, 51, 52, 53, 54, 194, 204, 206, 212
C-C, 26 combined effect, 156
cell line, 64, 337 combustion, 21, 22, 23, 24, 26, 27, 28, 67, 70, 72,
cell phones, 358 73, 74, 75, 76, 77, 79, 86, 127, 142, 167, 168,
cellulose, xiii, 12, 13, 61, 131, 132, 149, 241, 242, 169, 176, 179, 231, 236, 237, 288, 294, 296, 334,
247, 276, 301, 325 347
Census, xi, 60 combustion processes, 75
Central Europe, 106 commerce, 284
certificate, 375, 379, 385, 386, 389, 390, 391 commercial, 38, 115, 168, 184, 210, 219, 220, 234,
challenges, 19, 209, 299, 300, 302, 389, 393 280, 286, 306, 337
chemical, 12, 13, 32, 33, 42, 55, 69, 71, 75, 109, 113, commodity, 39, 314, 358
132, 133, 147, 148, 151, 154, 155, 158, 193, 194, commodity markets, 314
197, 204, 211, 221, 229, 230, 231, 234, 235, 237, Commonwealth of Independent States, 284
238, 242, 244, 247, 279, 280, 283, 285, 287, 291, communication, 190, 374
300, 301, 302, 303, 304, 305, 308, 317, 318, 337, compaction, 67, 124, 377
348, 360 compatibility, 193, 202, 375, 376, 378, 379, 380,
chemical characteristics, 69 381, 385, 386, 387, 390, 392
chemical industry, 113, 304, 305 compensation, 142, 264
chemical properties, 42, 71, 229, 230, 234, 235, 237, competition, 109, 263, 264, 265, 266, 287, 303, 389
238 competitive advantage, 142, 145
chemical reactions, 42 competitiveness, 302, 380
chemicals, 12, 33, 124, 203, 280, 300, 304, 316, 320 compilation, 265
China, 62, 90, 104, 168, 364 composites, xii, 11, 12, 14, 16, 17, 18, 19, 20, 293,
chlorine, 24 294, 295, 296, 297
chloroform, 193, 203, 351, 352 composition, 1, 2, 6, 7, 8, 12, 23, 69, 75, 91, 93, 150,
chlorophyll, 204 192, 201, 202, 210, 211, 220, 229, 230, 231, 235,
cholesterol, 61 236, 237, 238, 239, 241, 242, 244, 247, 273, 275,
chromatograms, 93 326, 329, 347, 349, 350, 351, 353, 354, 365
chromatography, 91, 215, 257, 259, 353 composting, 22, 24, 25, 26, 27, 28
398 Index
compounds, 42, 43, 44, 45, 59, 87, 93, 106, 166, 185, 232, 243, 257, 258, 287, 312, 313, 319, 320, 322,
210, 230, 231, 237, 258, 281, 306, 308, 360, 361 323, 326, 330, 377, 384
compression, 176, 252 crop production, 124
conditioning, 16, 143, 213, 285, 289 crop residue, 25, 68
conductance, 330 crop rotations, 1
conference, xi, xii, xiii, xiv, xv, 20, 78, 394 crops, xii, xiv, xv, 1, 2, 5, 21, 22, 23, 24, 25, 26, 29,
conflict, 319, 380, 387, 393 41, 42, 43, 62, 78, 79, 81, 89, 97, 98, 103, 106,
conflict resolution, 393 113, 116, 118, 119, 123, 124, 125, 126, 127, 128,
consciousness, 118, 318 129, 145, 181, 186, 229, 230, 238, 258, 262, 286,
consensus, 382, 387 288, 311, 312, 313, 314, 315, 317, 318, 319, 320,
consent, 375 321, 322, 323, 324, 325, 326, 329, 348
conservation, 90, 95, 234, 238 crude oil, 102, 193, 194, 195, 196, 229, 230, 232,
constituents, 335, 337 234, 235, 236, 347
construction, 31, 37, 53, 105, 265, 308 crystalline, 12, 13, 134
consumers, 142, 314 crystallinity, 360
consumption, 21, 101, 102, 107, 109, 110, 112, 117, cultivar, 3, 4, 5, 32, 34, 37, 59, 60, 247, 259, 262
124, 143, 144, 145, 168, 169, 175, 179, 230, 231, cultivars, 1, 2, 3, 4, 5, 6, 7, 8, 32, 33, 37, 38, 47, 99,
237, 264, 279, 284, 293, 304, 308, 318, 387 106, 110, 181, 248, 249, 321
contaminated soil, 262 cultivation, 1, 5, 8, 38, 39, 43, 47, 86, 89, 90, 92, 97,
contaminated soils, 262 99, 100, 101, 103, 104, 105, 107, 109, 111, 113,
contamination, 189, 223, 224, 258, 376, 377, 378 114, 115, 116, 119, 121, 123, 124, 125, 126, 127,
content analysis, 33 128, 223, 232, 237, 243, 259, 277, 280, 281, 312,
control condition, 257, 259, 261 313, 314, 318, 320, 325, 334, 336, 350, 351, 354,
control group, 60, 150, 152 383, 385, 387
controversial, 389 cultivation conditions, 89, 277
cooking, 217, 229, 230, 348, 351 culture, xii, 106, 223, 307, 353
cooling, 14, 60, 134, 143, 245 culture conditions, 353
cooperation, x, 41, 42, 132, 249, 303, 305, 358, 360, cycles, xii, 183, 184, 224, 230, 335
361, 371, 373 cycling, 182
corns, 41, 44, 47 cysteine, 257, 258, 259, 261
correlation, 37, 75, 77, 148, 150, 152, 195, 233, 277, Czech Republic, xii, 47, 79, 131
329
correlation coefficient, 152, 233
correlations, xiii, 148, 153, 154, 330 D
corrosion, 23, 24, 75, 77
cortex, 119, 120 data analysis, 331
cosmetic, 31, 106, 304, 335, 337, 341 data collection, 266, 383
cosmetics, 53, 113, 114, 115, 212, 317, 333, 334, database, 267, 302, 304, 390
335, 344 debts, 387
cost, 12, 13, 182, 193, 198, 202, 212, 259, 264, 305, decay, 161, 162, 318
312, 313, 316, 320, 321, 322, 374 decision makers, 229, 230
cost effectiveness, 305 decision-making process, 389, 392
cotton, 13, 51, 186, 189 decomposition, 294
cotyledon, 210 decontamination, 258, 261
Council of Europe, 95 decortication, 316
course content, 393 decoupling, 287
covalent bond, 12 deficiency, 2
covering, 31, 143, 385 deficit, 2, 9, 42, 43
creep, 359 deflate, 143
critical state, 385 deformation, 18, 234
crop, xiv, 1, 2, 6, 8, 25, 42, 43, 68, 89, 97, 99, 103, degradation, 61, 79, 157, 165, 256
107, 122, 123, 124, 125, 126, 127, 145, 181, 210, degraded area, 106
degumming, 204
denitrification, 87, 251, 254, 255, 256
Index 399
Denmark, 14 drug addiction, 243

density values, 74 drugs, 115, 317
Department of Agriculture, 333 dry matter, 1, 2, 3, 4, 5, 6, 8, 85, 90, 94, 106, 124,
Department of Energy, 102, 308, 309 127, 280
deposition, 308 dry matter content, 1, 2, 3, 5, 8
deposits, 61, 236, 364 dry matter yield, 1, 4, 8, 85
depth, 35, 283 drying, 26, 33, 63, 71, 100, 203, 219, 234, 367, 370
derivatives, 93, 102 durability, 44, 47, 67, 69, 73, 77, 78
desolventizing, 209, 213, 214, 216, 217, 219, 220, dusts, 27
221 dyeing, 49, 51, 53, 55, 62, 64, 114, 116
destruction, 71, 95 dyes, 49, 50, 53, 55
detectable, 174, 175, 178, 224
detection, xiii, 224, 226
detergents, 52, 113, 212 E
detoxification, 212, 214, 221, 258
developed countries, 238 East Asia, 334
developing countries, 95 ecology, 12, 318, 378
deviation, 4, 14, 18, 377 economic crisis, 322
diesel engine, 167, 169, 173, 179, 180, 236, 353 economic development, 263
diesel engines, 167, 169, 236, 353 economic efficiency, 380
diesel fuel, 101, 169, 171, 175, 178, 179, 180, 236, economic evaluation, 79
237, 348 economic growth, 300, 319
diffusion, 86, 333, 336, 341, 342, 343, 344 economic performance, 264, 383
digested residue, xiv, 81, 82, 83, 84, 85, 86 economics, 124, 263, 284
digestibility, 4 economies of scale, 289
digestion, 114, 271, 274, 275, 277, 302, 351 education, v, 379
dioecious hemp, 241, 242, 243 Efficiency of Four Crop Species, viii, 123
discomfort, 334 effluent, 5
diseases, 3, 61, 114, 148, 334 effluents, 308
dispersion, 27, 108, 193, 194, 196, 202, 211 Egypt, 104
displacement, 196, 199 elaboration, 111
disposition, 377, 385, 387 election, 340
distillation, 37, 38, 199 electrical properties, 360
distilled water, 203, 213, 259, 350 electrical resistance, 20
distribution, 67, 68, 69, 70, 71, 74, 75, 283, 284, 285, electricity, 62, 86, 87, 142, 144, 229, 230, 267, 283,
379, 386, 390 284, 291, 347, 348
divergence, 189, 238 electrolysis, 142
diversification, 323 electron, 157, 165, 337
diversity, 181, 186, 187, 241, 249, 319, 377, 383, electrophoresis, 184, 224
387 elongation, 45
DNA, viii, xiii, 95, 147, 148, 149, 150, 151, 152, e-mail, 21, 59, 81, 103, 104, 263, 299
153, 154, 155, 156, 181, 182, 183, 184, 185, 186, embryogenesis, 223, 224, 227
188, 189, 190, 224, 225, 226, 254 emerging markets, 168
DNA damage, 148, 155 emission, 12, 24, 77, 83, 84, 123, 168, 169, 174, 236,
DNA polymerase, 182, 183, 184 284, 291, 359, 377, 378, 387
DNA sequencing, 184 employees, 264
domestic markets, 108 employment, 167, 302, 379
dosage, 22, 27, 387 emulsions, 210, 211
dosing, 11, 14, 15, 19 encapsulation, 212
double bonds, 236, 354 endothelial dysfunction, 61, 65
drinking water, 148, 149, 151, 153 end-users, 321
drought, 1, 2, 41, 42, 43, 44, 45, 47, 90, 321 energy consumption, 124, 143, 144, 145, 284
drug addict, 243 energy density, 67, 69, 74
400 Index
energy efficiency, 67, 69, 73, 123, 124, 127, 128, exploitation, 95, 258, 337
129, 141, 142, 144, 145 exports, 98, 263
energy input, 123, 124, 126, 128 exposure, 51, 149, 152, 163, 258
energy supply, 145, 264, 284 extraction, xv, 91, 101, 116, 185, 186, 189, 204, 209,
engineering, xiii, 89, 132, 249, 251, 258, 305, 306, 212, 213, 214, 215, 216, 219, 220, 229, 230, 234,
308, 358, 365 238, 258, 318, 322, 333, 336, 337, 341, 342, 343,
England, 155 347, 349, 350, 351, 352, 353, 354, 355
Enhanced Oil Recovery, 192, 194, 199, 207 extracts, 260, 261, 317, 333, 335, 337, 341, 342, 343,
entrepreneurs, 388 344, 351
environment, xiv, 12, 21, 29, 42, 52, 108, 229, 232, extrusion, 71, 75, 252
233, 236, 237, 316, 318, 357, 373, 389
environment factors, 232, 233
environmental aspects, 291, 308 F
environmental conditions, 242, 252, 318, 321, 329
environmental factors, 233 factories, 101, 104, 108, 319, 323, 348, 369
environmental impact, 290, 308, 312, 313, 319, 348 families, 334
environmental protection, 50, 102, 394 farm land, 91, 92, 93, 94
enzyme, 11, 12, 13, 14, 15, 16, 19, 60, 132, 133, 134, farmers, 118, 128, 312, 319, 321, 322, 323, 372, 383,
136, 137, 138, 140, 193, 202, 212, 280, 326, 329 387, 388, 392, 393
enzymes, 11, 13, 20, 131, 132, 134, 136, 137, 138, farmland, 357, 372, 377
140, 299, 320 farms, 100, 321, 323, 372, 373, 374, 375, 378, 380,
epidermis, 31 382, 383, 384, 385, 386, 388, 389, 390, 391, 392,
equilibrium, 161, 195 393
equipment, 62, 63, 116, 142, 144, 193, 203, 204, fat, 6, 7, 12, 13, 16, 17, 204, 238, 242, 302, 315, 348
218, 229, 234, 238 fatty acids, xv, 201, 202, 236, 238, 247, 276, 348,
equity, 380, 381, 387 353, 354
erosion, 90, 91, 95, 371, 385, 387 feedstock, xv, 68, 102, 201, 202, 214, 215, 283, 285,
erythrocytes, 149 287, 288, 291, 300, 303, 312, 313, 314, 315, 317,
ESI, 261 320, 322, 323, 348
ESR, 157, 158 fermentation, 89, 95, 98, 214, 220, 284, 285, 299,
ester, 16, 113, 169, 171, 173, 191, 193, 194, 195, 302, 303, 305, 306, 307, 308, 309, 355
197, 199, 202, 203, 204, 205, 206, 207, 236, 353, fertility, 5, 124, 382
354 fertilization, 32, 36, 86, 94, 232
ethanol, xiv, 3, 102, 110, 111, 117, 118, 149, 193, fertilizers, 33, 124, 126, 128
203, 336, 340, 342, 343, 352 fiber, 6, 13, 20, 61, 97, 103, 104, 105, 106, 107, 108,
ethers, 101 109, 186, 214, 242, 247, 249, 308
ethyl alcohol, 110 fiber content, 6, 247, 249
EU, 98, 99, 104, 106, 108, 109, 110, 112, 115, 183, fibers, 104, 106, 109, 121, 210, 211
185, 209, 212, 242, 284, 286, 287, 290, 302, 303, fibra flax, 41
304, 309, 312, 313, 321, 322, 323, 324, 357 fibre plant, 223, 314, 324
Europe, v, 9, 12, 29, 62, 95, 98, 102, 103, 104, 106, filament, 365
119, 210, 242, 280, 287, 300, 309, 312, 313, 319 fillers, 211, 293, 297, 360
European Commission, 98, 103, 104, 292, 311 films, 211, 212, 220
European Community, 22 filters, 77
European market, 118 filtration, 215, 218, 336
European Parliament, 309 financial, 8, 104, 111, 255, 266, 292, 323, 380, 392
European Regional Development Fund, 303 financial condition, 111
European Union, 284 financial support, 8, 255, 292, 323
evaporation, 336 fingerprints, 95, 181
evidence, 188, 360 fire retardancy, 297
exchange rate, 230 fire retardants, 297
expectorant, 334 first generation, 169
expertise, 315, 355 fish, 209
fission, 300
Index 401
flame, 168, 360, 361 gel, 210, 215, 224

flame retardants, 360, 361 gel formation, 215
flammability, xiii, 293, 294, 297 gene bank, xiii, 241
flavonoids, 60, 61, 64, 114 genes, 90, 190, 329
flax fiber, 106 genetic diversity, 181, 186, 187
flexibility, 62, 143, 233 genetic factors, 37
flooding, 90, 199 genetic marker, 190
flour, 74, 363 genetic resource, xiii, 233, 241, 243, 244, 245
flowers, 52, 60, 232, 242 genetically modified plants (GMP), 279
fluid, 195, 198, 218, 219, 281 genetics, 186, 312, 326
fluidized bed, 209, 218, 219, 220, 221 genome, 280
fluorescence, 149 genotype, 33, 229, 241, 242, 249
foams, 210, 211 genotyping, 183, 185, 187, 188
folic acid, 61 genus, 60, 118, 354
food, xii, xv, 12, 21, 31, 59, 61, 63, 89, 90, 97, 98, geometry, 12
99, 100, 101, 109, 111, 112, 113, 128, 148, 209, Georgia, v, 47
211, 212, 243, 257, 258, 271, 280, 299, 300, 304, Germany, v, xi, xii, xiii, xiv, xv, 1, 2, 4, 8, 9, 11, 13,
306, 311, 312, 313, 314, 315, 317, 323, 326, 348, 14, 20, 24, 67, 70, 77, 78, 79, 81, 95, 98, 104,
363, 389 108, 118, 141, 167, 182, 191, 201, 221, 223, 251,
food chain, 258 263, 265, 279, 284, 285, 286, 287, 304, 311, 325,
food industry, 21, 31, 59, 63, 271, 315 333, 347, 350, 369, 374
food production, 257, 258, 348 germination, 259
food products, 97, 98, 111 glass transition, 360
force, 70, 77, 99, 194, 195, 197, 198, 234, 300, 312, glass transition temperature, 360
371 global warming, 2, 68, 347
formation, xiv, 23, 28, 32, 33, 91, 92, 93, 94, 157, glucose, 60, 65, 132, 133, 134, 136, 137, 138, 139,
162, 165, 167, 168, 169, 191, 192, 193, 194, 195, 306
197, 202, 206, 207, 210, 215, 236, 237, 252, 259, glucosidases, 60
261, 271, 272, 273, 274, 275, 276, 277, 325, 326, glucoside, 64
327, 328, 329 glucosinolates, 212
formula, 4, 42, 43, 44, 193, 197, 202, 232 glutamate, 113
foundations, 393, 394 glutathione, 258, 262
fragments, 182, 184, 224 glycerol, 193, 194, 205, 236
France, xii, 69, 98, 104, 284, 311, 313, 314 goods and services, 263, 264
free energy, 194 governments, 323
freedom, 388, 392 Grain by-product, 11, 12
friction, 52, 176 granules, 13, 14
frost, 3, 90, 91, 281 graph, 233, 378
fruits, 59, 60, 61, 63, 116, 232, 238 grass, 12, 21, 89, 90, 94, 95, 140
fuel cell, xiv, 142 grasses, 89, 94, 118, 262
fuel consumption, 21, 124, 168, 169, 175, 179 grazing, 3
fuel prices, 68 Great Britain, 98
functionalization, 214, 221 Greece, 311, 314, 324
funding, 303, 304 green alga, 355
funds, 109, 374, 380, 383, 388 green land, 98
fungus, 96 greenhouse, xiv, 21, 68, 81, 82, 84, 86, 245, 246,
fusion, 300 263, 283, 290, 299, 347, 348, 372, 378
greenhouse gases, xiv, 68, 81, 82, 84, 372
greenhouses, 385
G grounding, 76
growth, 2, 3, 9, 41, 42, 43, 44, 45, 46, 47, 59, 60, 68,
gas, nutrient loop, 81 82, 83, 85, 91, 92, 96, 98, 134, 231, 232, 241,
gasification, 70, 86, 168, 284, 285, 302, 334 242, 252, 258, 261, 300, 314, 317, 319, 326, 329,
GC-content, 185
402 Index
330, 331, 334, 335, 336, 337, 338, 339, 344, 348, hydrophobic properties, 211, 212
350 hydroxide, 14, 135
growth rate, 60, 331, 348 hydroxyl, 148, 162, 163
growth temperature, 330 hydroxyl groups, 162, 163
guidelines, 124 hyperglycemia, 61
Guinea, xii, xv, 229, 233, 235, 238 hypothesis, 37
H I
habitat, 42, 43 ideal, 67, 95, 348

halogen, 360 identification, 65, 97, 181, 189, 232, 264, 325, 374
hardness, 90, 91, 191, 192, 234 illumination, 350
harvesting, xiii, 38, 39, 93, 99, 105, 116, 242, 245, image, 55, 71, 77, 172, 360, 389, 390
247, 280, 314, 316, 320, 333, 336, 337, 355 image analysis, 71, 77
health, 59, 61, 223, 300, 363, 364, 379 images, 145, 172, 179
health care, 59 immunity, 321
heat release, 293, 294, 295, 296 impact assessment, 303
heat transfer, 74 impact strength, 14, 17, 361
heating rate, 14 imports, 320, 357
heavy metals, 69, 108, 257, 258, 261 improvements, 197, 214, 221, 266, 268, 317
height, 3, 43, 60, 242, 248, 326, 337, 338, 339, 344 impurities, 194, 204, 206, 232, 307, 308, 348
hematopoietic system, 147 in vitro, xiii, 106, 155, 182, 185, 186, 223, 224
hemicellulose, 12, 16, 61, 301 in vivo, 155
hemisphere, 334 income, 302, 319, 323, 357, 379, 380
hemp, xiii, 13, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, independence, 357
49, 53, 54, 55, 97, 98, 103, 104, 105, 106, 107, India, 47, 61, 90, 102, 168, 239, 367
108, 127, 181, 186, 189, 241, 242, 243, 244, 245, individuals, 182, 184, 185, 186, 189
247, 248, 249, 293, 294, 295, 296, 314, 316, 318, Indonesia, xii, xv, 191, 192, 195, 201, 203
319, 320, 322 industrial processing, 110, 112
hemp fiber, 106, 242 industries, 12, 21, 31, 59, 68, 110, 113, 191, 192,
herbal medicine, 114, 119 198, 202, 293, 315, 320, 321, 322, 323, 334, 337
heritability, 318 industry, xv, 12, 13, 20, 21, 31, 41, 59, 62, 63, 64,
heterogeneity, 151 98, 100, 101, 103, 104, 105, 106, 108, 109, 110,
hexane, 193, 203, 213, 216, 218, 351, 352, 353, 354 112, 113, 115, 141, 144, 168, 202, 264, 271, 280,
high school, v 293, 300, 302, 303, 304, 305, 308, 311, 312, 313,
history, 95 314, 315, 316, 317, 318, 319, 320, 321, 323, 324,
hives, 294 357, 358, 359
homeostasis, 147 inequality, 319
homes, 144 inflammatory cells, 337, 344
homogeneity, 44, 47, 320 inflation, 289
horizontal integration, 329 inflorescence harvest, 35, 37, 38
human, 12, 57, 61, 64, 65, 106, 115, 116, 148, 155, infrastructure, 141, 283, 284
209, 210, 221, 281, 299, 317 ingredients, 337, 365, 369
human body, 106 inhibition, 42, 61, 65, 182, 258, 306, 342
human health, 61 inhibitor, 83, 84, 85, 305, 342
humidity, 14, 35, 244, 245 inorganic fillers, 360
humus, 63, 64, 82, 86, 336, 377 institutions, 249, 314
husbandry, 375, 383 insulation, 107, 316
hybridization, 60 insulin, 60
hydrocarbons, 168, 230, 236 integration, 13, 170, 267, 280, 286, 288, 323, 328,
hydrogels, 279 329, 330
hydrogen, 16, 141, 142, 193, 203, 337 interface, 12, 194, 211
hydrolysis, 131, 132, 133, 204, 272, 302, 306, 352 interference, 281
Index 403
intervention, 155 legislation, 110, 167, 323

investment, 264, 288, 289, 320, 322, 355, 392 lentigo, 61
investments, 289, 319, 321, 322, 381, 387 leukemia, 61
investors, 321, 322 level of education, 379
iodine, 193, 194, 203, 204, 205, 206, 207, 236 liberation, 70, 76
ionization, 308 life cycle, 290
ionizing radiation, 147, 148, 154 lifetime, 142
ions, 197, 258, 308 light, 2, 49, 51, 53, 55, 143, 169, 170, 172, 183, 184,
IR spectra, 14, 163 224, 231, 363, 364, 365, 367, 378
Iran, 364 lignin, xv, 12, 13, 16, 17, 53, 61, 69, 71, 79, 106,
irradiation, 148, 149, 151, 152, 153, 156 157, 158, 160, 161, 162, 163, 164, 165, 301, 319
irrigation, 336 linen, 49, 51, 52, 53, 54, 55, 57, 103, 104, 105, 107,
isolation, 144, 183, 185, 186, 189, 190, 245, 355, 108
366 lipid peroxidation, 65, 147, 148, 155, 156
isomers, 360 lipids, 16, 17, 148, 150, 155, 348, 349, 350, 355
isotope, 85 liquid fuels, 101, 110, 168
issues, 312, 313 liquid phase, 196, 205
Italy, 13, 284, 311, 313, 314, 316 liver, 61, 149, 150
liver cancer, 61
livestock, 101, 285, 371, 377, 383
J logistics, xii, xiv, 79, 86, 141, 142, 143, 144, 145,
312, 313, 323
Japan, 91, 92, 93, 94, 149, 364 Logistics Centers, viii, 141
jatropha curcas, 201, 202 Louisiana, 199
job creation, 302 low temperatures, 178
Jordan, 323 LPO intensity, 147, 148, 150, 151, 152, 154
LPO regulatory system, 148, 151
K LSD, 34, 35, 36, 46
LTD, 113
KBr, 14 lubricants, 299, 313, 315
kinetic model, 326, 327, 329 lung cancer, 64
kinetic parameters, 278
kinetics, xiv, 147, 157, 166, 271, 305
M
KINS program, 149
KOH, 193, 194, 203, 204, 205, 206, 207 machinery, 108, 109, 124, 126, 280, 316, 323
macromolecules, 306
L magnesium, 61
majority, 92, 264, 387
laboratory tests, 32, 44, 83 man, 2, 319, 383
labour force, 371 management, 145, 318, 319, 380, 385, 386, 387, 389,
lactic acid, 299, 303, 304, 305, 306, 307, 308, 309, 394
360 manufacturing, 12, 55, 62, 316, 358, 360, 361
lactobacillus, 309 manure, 81, 86, 277, 288
landscape, 235, 377, 383 market penetration, 303
leaching, 82, 86 market segment, 319, 321
lead, 1, 84, 86, 136, 143, 151, 168, 169, 175, 176, marketing, 289, 316, 322, 335
178, 214, 217, 221, 223, 236, 261, 262, 280, 284, Maryland, v
325, 326, 359, 393 mass, 45, 75, 78, 91, 111, 115, 132, 134, 136, 137,
lead-acid battery, 143 138, 139, 168, 172, 175, 217, 218, 230, 231, 247,
leadership, 313 248, 258, 272, 273, 274, 277, 284, 294, 296, 318,
learning, 35 326, 327, 328, 329, 348, 371
legality, 23 mass loss, 294, 296
mass spectrometry, 91
404 Index
material resources, 22 Minneapolis, 79

materials, xiii, 12, 13, 17, 19, 20, 21, 22, 55, 62, 63, missions, 1, 68, 81, 83, 141, 143, 167, 168, 169, 175,
64, 68, 69, 70, 71, 72, 74, 75, 102, 105, 108, 115, 179
117, 119, 121, 122, 168, 209, 219, 247, 285, 286, mitochondrial DNA, 224
288, 293, 294, 299, 300, 301, 304, 305, 306, 307, mixing, 14, 72, 168, 236, 365, 369, 370
314, 316, 324, 334, 357, 358, 359, 360, 361, 363, models, 272, 325, 326, 327, 328, 329
364, 365 modernization, 109
matrix, 12, 17, 251, 252, 255, 294 modifications, 17, 167, 326
matter, 1, 2, 3, 4, 5, 6, 8, 73, 85, 90, 94, 106, 124, moisture, 11, 14, 16, 42, 44, 68, 71, 72, 74, 77, 95,
127, 168, 169, 175, 179, 214, 230, 231, 236, 273, 211, 238, 244
280, 383 moisture content, 14, 68, 71, 74, 77, 238
measurement, 169, 170, 179, 195, 197, 206, 207, molasses, 102, 113
293, 344 molecular biology, 105
measurements, 25, 43, 44, 149, 150, 153, 247, 259, molecular mobility, 19
339 molecular weight, 198, 236, 258
meat, 209, 211 molecules, 13, 42, 194, 236, 280, 281, 306, 335
mechanical properties, 11, 20, 234, 238, 359, 360 monoecious hemp, 241, 242, 249
media, 306, 348, 350 monosodium glutamate, 113
medical, xiii, xv, 229 monounsaturated fatty acids, 353
medicine, 61, 63, 114, 316 morphogenesis, 42, 44, 47
melt, 11, 14, 19, 253 morphology, 11, 12, 314
melting, 13, 14, 23, 183, 185, 360 Moscow, xi, xii, xiii, xv, 147, 155, 156, 157
melting temperature, 13, 14, 23, 183 motivation, 9
membranes, 150, 306, 308 moulding, 11, 14, 294, 358, 360, 361
MES, viii, xv, 191, 192, 193, 194, 195, 196, 197, mucus, 224, 226
198, 199, 201, 202, 203, 205, 206 mulberry, xv, 59, 60, 61, 62, 63, 64, 65
Metabolic, x, 308, 325, 326 multiplication, 89
metabolic pathways, 325, 326, 327, 328, 329 Myanmar, 364
metabolism, 280, 282, 326, 327, 328, 330 mycorrhiza, 91
metabolites, xiii, 42, 329
metal ion, 258
metal ions, 258 N
metals, 69, 108, 212, 257, 258, 261, 262
meter, 98, 193, 203, 232 Na2SO4, 193, 203
methane yield, 1, 4, 8, 94 NaCl, 193, 203, 259, 261, 337
methanol, 193, 194, 203, 204, 236, 348, 351, 352 nanocomposites, 166
methodology, 365 narcotic, 248, 322
methyl ester sulfonate, 191, 197 National Research Council, 95
methylene blue, 193, 203 natural fillers, 293, 297
Mexico, 261 natural gas, 142, 143, 283, 284, 285, 287, 291, 347
mice, 61, 147, 148, 149, 150, 151, 152, 153, 154, natural resources, 271, 373
156, 281 natural science, 387
microclimate, 57 nature conservation, 390
micromycetes, 79 Nd, 64
micronutrients, 82 necrosis, 223
microorganisms, 93, 252, 281, 299, 306, 337, 342 negative experiences, 368
micropropagation, 223 net investment, 387
microscope, 193, 203 Netherlands, 166, 286, 311, 314, 317
migration, 64 neutral, 68, 77, 142, 213, 223, 280, 348, 349, 384
mildew, 190 neutral lipids, 348
mineralization, 82, 86 New Zealand, 53, 57, 122
miniature, 348 niche market, 320
Ministry of Education, 8, 255 Nigeria, 364
nitric oxide, 237, 337, 344
Index 405
nitrification, 83, 84, 85, 86, 87, 251, 252, 256 Parliament, 309
nitrite, 337 participants, xii, 375
nitrogen, xiv, xv, 23, 32, 35, 36, 38, 81, 82, 84, 85, particle size distribution, 68, 69, 70, 71, 74, 75
86, 87, 124, 126, 157, 158, 160, 163, 164, 165, pastures, 98
168, 169, 170, 178, 179, 308, 319, 326, 328, 330, pathogens, 65, 243, 336
336, 337, 338, 344, 385 pathways, 283, 284, 285, 287, 288, 325, 326, 327,
nitrogen compounds, 87 328, 329
nitrogen dioxide, xv, 157, 158, 160, 164, 165 PCR, ix, xiii, 181, 182, 183, 184, 185, 186, 187, 188,
nitrogen gas, 170, 178, 179 189, 190, 223, 224, 225, 226
nitrous oxide, 81, 82, 83, 84 peptides, 257, 258, 259, 260, 261
North America, 12, 334 peripheral blood, 148
nucleic acid, 42 peroxidation, 65, 147, 148, 155, 156
numerical analysis, 180 peroxynitrite, 65
nursing, 358 Peru, 52
nutrient, xii, 1, 2, 4, 6, 8, 81, 86, 223, 299, 306 pesticide, 39
nutrients, 2, 3, 6, 33, 82, 83, 86, 89, 280, 306, 339 pests, 3, 12, 61, 63, 64
nutrition, 12, 105, 209, 210, 221 petroleum, 101, 193, 198, 202, 203, 300, 316, 317
Petroleum, 198
pH, 13, 33, 44, 82, 133, 136, 149, 193, 194, 203,
O 205, 206, 207, 212, 213, 259, 305, 336, 377
pharmaceutical, 59, 63, 105, 113, 114, 115, 280, 304,
oil production, 96, 99, 198, 232, 348 314, 315, 316, 317, 334, 335
oilseed, xv, 97, 98, 99, 100, 101, 209, 210 pharmaceuticals, xii, 100, 212, 311, 312, 313, 334
oligomers, 258 pharmaceutics, 335
operating costs, 142, 308 PHB, 251, 253, 255
operating range, 219 phenol, 157, 165, 193, 203, 316
operations, 77, 145, 232, 306, 318 phenolic compounds, 185, 281
opportunities, 89, 105, 115, 312, 313, 321, 379 phenolphthalein, 193, 203
optical measuring devices, 167 phenoxyl radicals, 160
optical parameters, 172 Philadelphia, 121
optimization, 28, 168, 169, 229, 271, 305, 326, 361 Philippines, 95
oral presentations, xii, xiii, xiv, xv phloem, 62
organic matter, 231 phosphate, 13, 193, 202, 351, 352
organism, 148 phospholipids, 348
organs, 42, 328 phosphorous, 61
osmosis, 320 phosphorus, 124
overpopulation, 119 photometric analysis, 337
overproduction, 61, 99 photosynthesis, 42, 231, 232, 326, 329, 330, 348
overtime, 323 physical and mechanical properties, 234
ownership, 104 physical characteristics, 44, 69
oxidation, 61, 148, 168, 204, 276, 353 physical properties, 238
oxidation products, 148 physicochemical characteristics, 193
oxygen, 60, 158, 220, 237, 293 physicochemical properties, 204, 205, 206, 207
oxygen consumption, 293 physics, 166
Physiological, 53
P phytochelatins, 257, 258, 261, 262
phytoremediation, 90, 257, 258, 261
Pacific, 95, 122, 309 pigmentation, 64
paints, 100, 101 pitch, 206
Pakistan, 364 plant growth, 82, 83, 85, 241, 329, 331, 339
palm oil, 192, 349 plant protection, 33, 41, 106, 114, 119, 387
PAN, 39 plasmid, 224
parallel, 162, 170 plastics, 12, 211, 357, 358, 360, 363, 364
406 Index
platform, 281 210, 212, 213, 216, 220, 221, 229, 230, 238, 242,
plausibility, 268 302, 306, 307, 314, 316, 318, 319, 320, 321, 322,
Poland, v, viii, xi, xii, xiii, xiv, xv, 39, 41, 42, 49, 59, 350, 363, 364
60, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, producers, 98, 102, 118, 122, 132, 142, 266, 267,
107, 108, 109, 110, 112, 113, 114, 115, 116, 117, 315, 358
118, 119, 120, 121, 122, 123, 128, 129, 241, 242, production costs, 181, 264, 288, 289, 290, 291, 350,
243, 249, 257, 293, 311, 313, 314, 320, 321, 322, 355
323, 324 production function, 385
polarity, 12, 197 production quota, 112
policy, 128, 244, 320, 321, 322, 325, 380, 383, 387, production technology, 286
389, 390 professionalism, v
policy makers, 322 profit, 283, 318
politics, 264, 390 profitability, 109, 112, 127, 128, 301, 318, 377
pollutants, 158, 258 project, 5, 38, 77, 81, 82, 87, 89, 90, 95, 102, 116,
pollution, 63, 64, 90, 119, 158, 229, 257, 258, 318, 143, 169, 170, 174, 176, 178, 181, 218, 220, 223,
373, 377 281, 288, 303, 311, 312, 317, 326, 328, 329, 333,
poly(3-hydroxybutyrate), xiii 335, 337, 340, 364, 370, 384
polyhydroxybutyrate, 281 proliferation, 61
polymer, 19, 166, 199, 251, 254, 279, 280, 282, 293, proline, 42
304 promoter, 280
polymer nanocomposites, 166 propagation, 181, 223, 279
polymerase, 183, 184, 189 propane, 290
polymerase chain reaction, 189 prosperity, v
polymers, xii, 158, 166, 251, 252, 255, 293, 304, protection, 1, 33, 41, 50, 53, 55, 57, 90, 95, 97, 102,
308, 313 106, 114, 119, 122, 181, 243, 284, 334, 357, 372,
polymorphism, 189, 248 376, 377, 378, 379, 387, 394
polyoxyethylensorbitanmonooleate, 148 protein components, 262
polypeptide, 211 protein synthesis, 42
polypeptides, 258 proteins, xiii, 13, 42, 155, 209, 210, 211, 212, 213,
polyphenols, 185, 188 214, 215, 216, 220, 221, 257, 258, 259, 260, 261,
polypropylene, 11, 14, 18, 19, 20, 293, 294 271, 275, 276, 277, 302, 329
polyunsaturated fat, 353 public awareness, 320
polyunsaturated fatty acids, 353 public opinion, 279
population, 12, 189, 299, 336 publishing, v
potassium, 24, 51, 61, 113, 124, 126, 193, 203 pulp, 31, 39, 106, 113, 158, 320
potato, 111, 277, 280, 281 purification, 37, 185, 193, 194, 203, 285, 309, 355
potential benefits, 12 purity, 149, 210, 215, 243, 307
power generation, 68, 300 PVP, 182, 183, 185, 186
power plants, xiii, 69, 77, 143, 334 pyrolysis, 70, 79, 288, 302, 348
precipitation, 2, 308, 336
preeclampsia, 154
pregnancy, 148, 154 Q
preparation, xv, 14, 22, 24, 42, 132, 133, 136, 137,
138, 139, 224, 232, 287, 306, 317, 336, 337 quality control, 71, 315
preservation, 357, 377, 380, 382 quality standards, 70
President, v, 280 quercetin, 60, 61
President Clinton, 280 quotas, 110
pressure gradient, 71
pressure groups, 394 R
prevention, xiv, 61, 81, 84, 86, 358
primary products, 264, 333 radiation, 53, 55, 57, 90, 106, 143, 147, 148, 149,
processing, xv, 12, 13, 71, 98, 101, 102, 104, 105, 154, 155, 156, 231, 232
108, 109, 110, 111, 112, 113, 145, 182, 204, 209, Radiation, 154, 155, 156
Index 407
radiation damage, 155 resolution, 14, 71, 74, 170, 171, 182, 327, 393
radical reactions, 158, 160 resource utilization, 308
radicals, 157, 158, 160, 161, 162, 163, 164, 165 resources, xiii, xv, 12, 19, 22, 68, 86, 97, 102, 108,
rape, 1, 43, 46, 47, 62, 97, 98, 99, 100, 101, 102, 121, 123, 128, 131, 143, 229, 233, 238, 241, 243,
173, 214 244, 245, 264, 266, 267, 271, 279, 284, 299, 300,
rape seed, 173 302, 303, 305, 306, 311, 316, 317, 319, 333, 334,
rapeseed, protein, 209 357, 361, 373, 380
raw materials, xiii, 21, 68, 69, 70, 72, 74, 75, 102, respiration, 230, 231, 237
115, 117, 119, 168, 209, 286, 300, 301, 304, 305, response, 42, 147, 151, 259, 261, 347
307, 314, 316, 318, 324, 334, 357, 358, 365 restoration, 258
reactant, 192, 193, 194, 202 restrictions, 187, 287
reactants, 193 restructuring, 104
reaction mechanism, 155 rice husk, 12, 13, 14, 15, 16, 17, 18, 19
reaction temperature, 133, 203, 206 risk, 2, 24, 68, 75, 82, 322, 323, 363, 364, 383, 385,
reactions, 12, 42, 57, 75, 157, 158, 160, 165, 166, 392
182, 183, 184, 185, 273, 274, 384, 387 risk assessment, 392
reactive groups, 158 risks, 243, 322, 373, 378, 383
recombination, 161 RNA, xiii, 185, 189, 190
recommendations, 313, 314, 315, 317, 318, 323 rodents, 63, 64
recovery, xv, 191, 192, 193, 194, 198, 201, 202, 207, Romania, v, 311, 313, 314
229, 230, 234, 238 room temperature, 13, 14, 161
recovery process, 207 root, 2, 46, 65, 86, 90, 91, 93, 96, 226, 258
recycling, 285 root system, 90, 93
reforms, 104 roots, 44, 60, 61, 86, 90, 91, 92, 93, 94, 116, 189,
regenerate, 230, 231, 237 258, 262
regional economic impacts, 263 rotations, 2, 73, 326
regionalization, 318 roughness, 12
regions of the world, 241, 242 routes, 283, 285, 286, 287, 291, 312, 313, 314, 315,
regression, 148, 153, 154 326
regulations, 106, 209, 212 rowing, 106, 209, 257
reinforcement, 12, 17, 360 rubber, 236
remediation, 262 rules, 123, 303, 329
renewable energy, 68, 90, 118, 121, 128, 230, 264, rural areas, xii, 116, 119, 263, 264, 287, 302, 357,
348 380, 388, 394
renewable fuel, 102, 113 Russia, v, xi, xii, xiii, xv, 147, 149, 154, 157, 247
repair, 155 rye straw, 68
reparation, 224, 317
repellent, 39
repetitions, 266, 336 S
reproduction, 380, 383
safety, 102, 171, 238, 279, 280, 319
requirements, 1, 62, 99, 102, 106, 210, 211, 213,
217, 300, 305, 306, 314, 317, 320, 358, 360, 375, salinity, 191, 192, 194, 195, 199, 202, 206, 207
393 salinity levels, 202, 206, 207
research institutions, 249 salts, 299, 304, 306
researchers, 317 samplings, 358
reserves, 99, 374, 389 saponin, 113
residuals, 299 saturation, 198, 259, 260, 261
savings, 144, 168
residues, xii, xiv, 11, 12, 21, 22, 23, 24, 25, 26, 27,
67, 68, 70, 81, 82, 83, 84, 85, 86, 257, 259, 280, scaling, 289
285, 286, 288, 300, 302, 316 science, 20, 249, 320, 322, 387
resins, 308, 311, 312, 313, 314, 315, 316 scientific progress, 300
resistance, 11, 19, 20, 41, 42, 43, 47, 49, 61, 77, 89, scope, 5, 243, 290, 313, 314, 327, 358, 385, 387
90, 101, 190, 192, 194, 319, 358, 359, 360, 366 sea level, 2
seasonality, 231
408 Index
second generation, 168 species, xiv, 3, 49, 50, 52, 59, 60, 114, 115, 116,
secondary data, 266 118, 119, 121, 123, 124, 127, 147, 151, 153, 181,
secretion, 31 186, 187, 223, 224, 225, 226, 227, 241, 261, 329,
security, 284, 348, 384 334, 336, 337, 338, 339, 340, 341, 344, 351, 352,
seed, 3, 12, 31, 32, 33, 99, 100, 173, 203, 204, 210, 353, 354, 377, 387
214, 229, 230, 233, 234, 236, 238, 239, 241, 242, specifications, 171, 265, 266, 268, 388
244, 245, 247, 248, 253, 259, 261, 349, 377 spectroscopy, 158
seedlings, 257, 259, 260 spin, 157
self-image, 55 spleen, 150
semen, 119 spray formation, xiv, 167
sensing, 350 Spring, 43, 44, 45, 126
sensitivity, 147, 148, 154, 323 spring barley, 45, 123, 124, 125, 126, 127, 128
sequencing, 184, 188, 224 Sri Lanka, 364
shape, 67, 68, 70, 71, 72, 73, 74, 175, 195, 217, 390 stability, 67, 90, 182, 184, 185, 194, 238, 256, 322,
shoot, 45, 90, 92 366, 368, 369, 370
shoots, 60, 92, 116, 258 stabilization, 77, 90, 210, 215
shortage, 12, 42, 43, 167, 210, 320, 333 stable radicals, 157, 158, 161, 165
showing, 259, 328 stakeholders, 312, 314, 321
shrubs, 334 standard deviation, 4, 14, 18, 377
silica, 23, 364 standardization, 37
silk, 49, 51, 52, 53, 54, 55, 56 starch, 3, 6, 13, 97, 109, 110, 111, 112, 122, 193,
silkworm, 60, 61, 62 203, 231, 276, 277, 301, 302, 306, 325, 326
silver, 261 state, xiv, 6, 61, 74, 75, 76, 104, 128, 148, 165, 189,
simulation, 325, 329 243, 266, 307, 385
skilled personnel, 316 states, 74, 304, 318, 376, 388
skin, 55, 57, 61, 204, 334, 335 sterile, 184, 337
sludge, 253, 271 sterols, 348
social development, 229 stinging nettle, 181, 223
social environment, 389 stoichiometry, 326
social interests, 383 stomach, 334
social structure, 385 stomata, 42, 330
society, 115, 118, 119, 229, 314, 318, 373, 374, 394 storage, 69, 70, 73, 74, 77, 82, 144, 145, 204, 212,
sodium, 13, 24, 113, 135, 149, 193, 203, 224, 308 220, 231, 244, 245, 277, 280, 318, 325, 326, 327,
sodium hydroxide, 14, 135 353
software, 144, 145, 169, 172, 184, 224, 314, 329 storage and retrieval vehicles (SRVs), 144
soil erosion, 371 straw pellets, 68, 72, 73, 74, 75, 77
soil pollution, 90 stress, 1, 41, 42, 45, 47, 214, 258, 259, 261, 262,
soil type, 336 312, 389
solidification, 236 stretching, 16, 163
solubility, 44, 47, 212, 213, 215, 342 structure, 16, 42, 53, 54, 102, 104, 109, 110, 112,
solution, 44, 86, 134, 142, 145, 148, 149, 185, 191, 167, 169, 197, 211, 231, 244, 264, 280, 320, 370,
195, 203, 215, 257, 259, 260, 261, 297, 307, 308, 380
316, 337, 387, 392 substitutes, 316
solvents, 203, 304, 313 substitution, 279, 299, 306
somatic embryogenesis, 223, 224, 227 substrate, 1, 42, 63, 83, 86, 252, 254, 255, 271, 272,
Sorghum, vii, xiv, 1, 2, 3, 4, 5, 9 273, 274, 275, 276, 277, 278, 285, 306
South Dakota, 78 substrates, xiv, 65, 86, 220, 267, 271, 272, 273, 275,
sowing, 6, 8, 32, 33, 35, 36, 37, 38, 39, 99, 106, 108, 277, 283, 285, 286, 306
112, 124, 241, 243, 244, 245, 320 sucrose, 325
Spain, 90, 102 sugar beet, 44, 46, 47, 97, 98, 113, 117, 123, 124,
specialists, 322 125, 126, 127, 128, 313
specialization, 319 sugar mills, 113
specialty crop, 313 sulfate, 86, 193
Index 409
supervision, 390, 393 tensile strength, 17

supplementation, 148 tension, 44, 47, 171, 175, 191, 192, 193, 194, 195,
supply chain, 79, 266, 312, 313, 318, 320, 358 196, 197, 198, 202, 205, 206, 207
surface area, 194 test procedure, 389
surface chemistry, 12 testing, 3, 5, 27, 73, 76, 131, 142, 293, 336, 360,
surface properties, 13 361, 365, 369
surface tension, 44, 47, 175, 207 testosterone, 334
surfactant, xv, 44, 156, 191, 192, 193, 194, 195, 196, textiles, 13, 53, 55, 103, 107, 211
197, 198, 199, 201, 202, 205, 206, 207 Thailand, 78, 190, 364
surfactants, 41, 191, 199, 207 thermal decomposition, 294
surplus, 68, 98, 382 thermal energy, 143
survival, 92, 279 thermal properties, 11, 359, 360
survival rate, 92 thermal resistance, 359, 360
susceptibility, 8 thermal stability, 185
sustainability, 141, 142, 145, 168, 283, 284, 300, thermodynamics, 12
308, 312, 313, 355, 372, 373, 374, 375, 383, 384, thermograms, 26
385, 386, 388, 389, 390, 393 thermolysis, 161
sustainable development, xii, 102, 123, 372, 373, thermoplastics, 13, 212, 293
374, 383, 384, 385, 388, 389, 390, 393, 394 time frame, 289
sustainable energy, 145, 264, 283, 355 tissue, xiii, 106, 223, 226, 258, 323, 355
sweat, 49 toluene, 193, 203
Sweden, 78, 104, 286, 287 tones, 62, 63, 100, 110
Switzerland, xii, 286, 287 tonic, 64
symptoms, 258, 259 total energy, 231
synchronize, 171 total product, 102, 112, 117, 231
synthesis, 42, 166, 232, 258, 279, 280, 302, 360 toxic effect, 44
synthetic fuels, 168 toxicity, 147, 148, 151, 259, 261, 262, 281
toxicology, 281
TP53, 154
T trade, 55, 112, 284
trainees, 393
talc, 360 training, 116, 379, 390
talent, v traits, 181, 238, 312, 313, 317, 318, 329, 330
tanks, 142, 277 transesterification, 193, 203, 204, 236, 348, 351
tannins, 62, 114 transformation, 104, 230, 231, 237
target, 184, 215, 314, 315, 392 transformations, 82
tax incentive, 323 transition temperature, 360
taxes, 264 transmission, 53
taxonomy, 60 transparency, 389, 390
techniques, 71, 105, 189, 229, 230, 232, 237, 249, transport, 42, 77, 142, 219, 229, 230, 234, 236, 258,
320 264, 287, 291, 301, 323, 340
technological progress, 380 transportation, 67, 69, 70, 73, 74, 77, 82, 236, 283,
technologies, xii, 79, 86, 105, 121, 141, 142, 143, 284, 293, 323
144, 145, 230, 238, 264, 285, 293, 300, 303, 312, treatment, 14, 15, 17, 19, 61, 79, 81, 82, 86, 100,
317, 320, 322 114, 133, 134, 135, 136, 158, 206, 216, 217, 218,
technology, 12, 20, 55, 86, 89, 102, 105, 141, 143, 253, 255, 256, 261, 275, 286, 288, 289, 297, 302,
144, 145, 191, 192, 198, 209, 213, 218, 221, 284, 377
286, 290, 302, 317, 318, 320, 322, 325, 358, 359 turbulence, 174, 175, 178
technology transfer, 302 turnover, 111, 231
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, U
184, 192, 193, 194, 196, 206, 214, 224, 233, 245,
253, 278, 284, 321, 330, 336, 350, 360, 368 UK, 14, 104, 143, 166, 311
410 Index
Ukraine, 104 waste water, 90

Ultra-high pressure injection, 167 wastewater, 253, 255, 256
ultrasound, 320, 343, 351 water, 2, 4, 5, 6, 9, 12, 13, 14, 32, 37, 41, 42, 43, 44,
unification, 374 47, 51, 52, 69, 70, 72, 73, 75, 76, 77, 82, 86, 90,
uniform, 70, 72, 242, 381 91, 143, 144, 145, 148, 149, 151, 153, 191, 192,
United Nations, 78, 269, 394 193, 194, 195, 197, 198, 202, 203, 204, 206, 207,
universities, 132, 311 210, 213, 217, 218, 220, 230, 231, 232, 251, 252,
updating, 380 256, 259, 264, 284, 299, 300, 304, 308, 318, 340,
urban, 287 347, 348, 350, 351, 352, 365, 376, 378, 384, 390
urban areas, 287 weakness, 372
USA, 9, 19, 20, 47, 57, 78, 79, 90, 91, 121, 166, 221, weight changes, 26
280 weight gain, 16
USSR, 157 weight loss, 15, 26
UV, vii, 49, 53, 55, 57, 106, 149, 224, 359 wettability, 12, 192, 193, 194, 198, 202
UV light, 224 wetting, 12, 170
UV radiation, 53, 55, 57, 106 wheat straw, 68
UV-radiation, 149 white blood cells, 148, 155
winter rapeseed, 123, 124, 125, 127, 128
winter wheat, 45, 123, 124, 125, 127, 128
V Wisconsin, 227
wood, 12, 14, 17, 18, 21, 22, 23, 24, 27, 39, 50, 62,
vacuole, 258 63, 68, 69, 73, 78, 79, 95, 118, 158, 266, 267,
vacuum, 14 283, 286, 288, 297, 316, 334, 340, 366
validation, 267, 326, 327 World Bank, 90, 95
valuation, 359, 373, 381, 383, 387 World War I, 103
variables, 322, 386, 387 worldwide, 12, 31, 168, 209, 210, 229, 249, 279,
variations, 126, 197, 354 304, 305, 363
varieties, xiv, 70, 100, 121, 181, 189, 241, 242, 243,
244, 247, 249, 329
vegetable oil, 99, 101, 201, 232, 236, 237, 315 X
vegetation, 35, 86, 89, 93, 94, 245, 246, 247, 248,
335, 338, 340 X-irradiation, 148, 149
vehicles, 142, 144, 168, 229, 230
velocity, 168, 174, 180, 195, 198, 216, 219
Venus, ix, xii, 299, 303, 304, 309 Y
vessels, 61, 304, 306
vibration, 16, 350 yarn, 55, 107, 108
Vietnam, 363, 364, 365, 367, 370 yeast, 113, 275, 306, 337
viruses, xiii, 61 Yeasts, 309
viscoelastic properties, 11 yield, 1, 2, 3, 4, 5, 6, 7, 8, 31, 36, 37, 41, 44, 45, 46,
viscose, 53, 320 62, 63, 85, 94, 99, 110, 113, 116, 118, 126, 127,
viscosity, 171, 194, 198, 236 164, 165, 209, 212, 213, 214, 215, 221, 229, 233,
visualization, 197, 328, 329, 331 234, 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 Z

wages, 380
Washington, v, 95 zinc, 61
waste, 11, 12, 21, 22, 90, 108, 118, 144, 278, 285, ZnO, 288
288, 318, 319, 348
waste heat, 144

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MATERIALS SCIENCE AND TECHNOLOGIES

Nova Science Publishers, Inc.

NOTICE TO THE READER

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

Published by Nova Science Publishers, Inc. † New York

Professor Gennady Efremovich Zaikov

Chapter 1 Sorghum as Biogas Crop 1

Chapter 9 Prevention of Disposal of Greenhouse Gas from Digested Residues

Chapter 21 Rapeseed Proteins – Recent Results on Extraction and Application 209

Gennady Zaikov, Frank Pudel1 and Grzegorz Spychalski2

This volume is a proceeding of the 16th International Conference for Renewable

1. Plant Breeding for non-food Application

2. Renewable Resources for Material Applications

 Biobased composites (Natural fibres, Nano particles etc.)

3. Processing, Characterisation and Application of Secondary Plant Metabolites

 Biomass conversion technologies

4. Biomass and Efficient Energy Generation

 Energy crops and organic residues

5. Sustainable Development in Environment, Economy and Sociology

(Leibniz-Institut für Polymerforschung Dresden e.V., Germany; Universitätsklinikum Carl-

SORGHUM AS BIOGAS CROP

Anja Hartmann, Karen Zeise and Maendy Fritz

2. AIM AND BACKGROUND

3. MATERIAL AND METHODS

3.2. Experimental Design

Table 1. Number of cultivars/strains included in the specific testing group

Subspecies Type Number

3.3. Crop Measurements

4. RESULTS AND DISCUSSION

Dry matter content

4.2. Yield Performance of Sorghum and Maize under Different Growing

Dry matter yield

4.3. Nutrient Composition and Theoretical Methane Production and Yield

Table 2. Nutrient composition [% DM] of sorghum types. Results represent

Subspecies/Type Crude protein Crude fat Crude fibre Starch Sugar

ENZYME MODIFIED GRAIN BY-PRODUCT

Abdullah Al Mamun, Andrzej K. Bledzki and Jürgen Volk1

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

Fourier-Transform Infrared Spectroscopy (FT-IR)

RESULTS AND DISCUSSION

Figure 1. Enzyme activity on temperature.

Duration and Dosing Rate

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

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

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

The impact strength of a composite is influenced by many factors, including the

 The following fibre modification parameters were observed; optimum temperature

BIOMASS AS COMBUSTION FUEL – EXPERIENCES

Grete Bach1*, Stefanie Bachmann, Daniel Kolb,

Table 1. Growth of biomass potentials in the European Community 2010-2030 /3/

Use of biomass Potential Potential Potential

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

Table 2. Dosation- and Combustion Systems for Biomass-Fuels

Kind max. diameter suitable suitable

bulk material < 50 mm worm conveyer dosage from below

bulk material < 100 mm dosage in pieces grate combustion, BFB

bales < 50 mm pneumatic dosage worm Dust combustion

bales whole crane, Stoker dosage grate combustion, Cigar bumer

Pellets < 30 mm worm conveyer dosage from below

Pellets < 120 mm dosage in pieces grate combustion, station. Fluid

2. ANALYTICAL PROPERTIES OF DIFFERENT BIOMATERIALS