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Biotransformation of Agricultural
Waste and By-Products
This page intentionally left blank

Biotransformation of Agricultural
Waste and By-Products
The Food, Feed, Fibre, Fuel (4F) Economy
Edited by
Palmiro Poltronieri
CNR-ISPA, Lecce, Italy
Oscar Fernando D’Urso
Bioesplora srl, San Michele Salentino, Brindisi, Italy
AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD

PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Elsevier
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The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK
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Copyright © 2016 Elsevier Inc. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
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Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our
understanding, changes in research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any
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To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability
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British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress
ISBN: 978-0-12-803622-8
For information on all Elsevier publications

visit our website at https://www.elsevier.com/
Publisher: John Fedor

Acquisition Editor: Kostas Marinakis
Editorial Project Manager: Sarah Jane Watson
Production Project Manager: Paul Prasad Chandramohan
Designer: Greg Harris
Typeset by TNQ Books and Journals
Contents
List of Figures�� xiii
List of Tables��xvii
List of Abbreviations��xix
About the Editors�� xxiii
About the Contributors�� xxv
Introduction��xxxv
Chapter 1: Crop and Plant Biomass as Valuable Material for BBB. Alternatives
for Valorization of Green Wastes��1
T. Dietrich, M.C. Villaran Velasco, P.J. Echeverría, B. Pop and A. Rusu
Abbreviations��1
1.1 Introduction��1
1.2 Plant Biomass as Feedstock for Industrial Applications: General Considerations��3
1.2.1 Temporal Availability and Compositions of Biomass�� 5
1.2.2 Logistic Requirements of Biomass�� 6
1.3 Plant Biomass: A Sustainable Feedstock for Industrial Applications��8
1.3.1 Current Applications�� 8
1.3.2 Resource for Value-Added Compounds�� 9
1.3.3 Feedstock for the Fermentation Industry�� 14
1.4 Conclusion��15
Acknowledgment��16
References��16
Chapter 2: Fruit Processing in Central America and Mexico��21
C. Velázquez, A.M. Pérez Carvajal and L.A. Prado Barragán
2.2 Fruit Production in the Region��22
2.2.1 Costa Rica�� 22
2.2.2 Other Central American Countries�� 25
2.2.3 Mexico�� 27
2.3 Fruit Industrialization in the Region��27
2.3.1 Costa Rica�� 27
2.3.2 Central America�� 28
v
Contents
2.4 Physicochemical Composition and Biological Activity of Fruits of the Region��30

2.4.1 Physicochemical Composition of Fruits of Costa Rica�� 30
2.4.2 Physicochemical Composition of Fruits Produced in Mexico�� 35
2.5 Biological Activities of Fruits From Costa Rica and the Central
America Region��39
2.5.1 Banana�� 39
2.5.2 Mango�� 39
2.5.3 Pineapple�� 40
2.5.4 Watermelon�� 41
2.6 Agro–Industrial Fruit Wastes and Its Potential as Biomass��41
2.7 Conclusions��42
References��43
Chapter 3: Plants By-Products and Fibers’ Industrial Exploitation��49
S. Narasimhan, B.S. Srikanth and P. Poltronieri
3.2 India Food Industry Wastes: Mango Case Study��50
3.2.1 Processed Mango�� 50
3.2.2 Barriers�� 50
3.2.3 Opportunities�� 53
3.3 Market Making in the Bioeconomy of Plants’ By-Products in Europe
and in the World��53
3.3.1 Feedstock Biomasses�� 55
3.3.2 Cellulose�� 55
3.3.3 EU Textile Industries and Vegetal Fibers�� 57
3.3.4 Innovative Companies Involved in Recycling of Plant Residues, Transforming
Them in Textiles, Biobased Biochemicals, and Fiber Applications�� 57
3.3.5 EU Business Analysis of Case Studies�� 60
3.3.6 Cargill Starches�� 63
3.3.7 Tomatoes Plant Waste Recycling Industrial Group�� 63
3.3.8 Novamont Case Study on Bioplastics�� 64
3.3.9 Wheatoleo�� 65
References��66
Chapter 4: Organic Recycling of Agricultural Waste Today: Composting
and Anaerobic Digestion��69
N. Mortier, F. Velghe and S. Verstichel
4.2 EU & American Policy on Organic Waste��69
4.2.1 Europe�� 70
4.2.2 South and Central America�� 73
4.2.3 North America�� 74
vi
Contents
4.3 Industrial Composting��78

4.3.1 Principle�� 78
4.3.2 Input�� 79
4.3.3 Pretreatment Steps�� 80
4.3.4 Composting Process�� 80
4.3.5 Composting Methods�� 83
4.3.6 Posttreatment Steps�� 88
4.3.7 SWOT Analysis�� 88
4.3.8 Composting Within the TRANSBIO Project�� 89
4.4 Anaerobic Digestion��90
4.4.1 Principle�� 90
4.4.2 Input�� 90
4.4.3 Pretreatment Steps�� 91
4.4.4 Anaerobic Digestion Process�� 92
4.4.5 Digestion Methods�� 95
4.4.6 Posttreatment Steps�� 100
4.4.7 Biogas Treatment�� 100
4.4.8 Biogas Valorization�� 105
4.4.9 SWOT Analysis�� 106
4.4.10 Anaerobic Digestion Within the TRANSBIO Project�� 116
References��122
Chapter 5: Biomass Pretreatment Strategies (Technologies, Environmental
Performance, Economic Considerations, Industrial Implementation)��125
C. Peral
5.2 Physical and Chemical Characteristic of Lignocellulosic Biomass��126
5.2.2 Hemicellulose�� 128
5.2.3 Lignin�� 130
5.3 Pretreatments��131
5.4 Physical Pretreatments��131
5.4.1 Mechanical Comminution�� 131
5.4.2 Extrusion Pretreatment�� 133
5.4.3 Liquid Hot Water Pretreatment�� 133
5.4.4 Pyrolysis and Torrefaction�� 134
5.4.5 Freeze Pretreatment�� 135
5.4.6 Irradiation�� 135
5.4.7 Pulsed Electric Field Pretreatment�� 136
5.4.8 Ultrasonic Pretreatment�� 136
5.5 Chemical Pretreatments��137
5.5.1 Acid Hydrolysis�� 137
5.5.2 Alkaline Hydrolysis�� 138
vii
Contents
5.5.3 Organosolv Pretreatment�� 139

5.5.4 Peroxide Pretreatment�� 140
5.5.5 Ozonolysis�� 140
5.5.6 Peroxyformic Acid�� 141
5.6 Physicochemical Pretreatments��141
5.6.1 Steam Explosion�� 141
5.6.2 Ammonia Fiber Explosion and Ammonia Pretreatment�� 142
5.6.3 Carbon Dioxide Explosion Pretreatment�� 143
5.6.4 Wet Oxidation Pretreatment�� 144
5.6.5 Ionic Liquid Pretreatment�� 145
5.7 Biological Pretreatment��145
5.7.1 Pretreatment With Fungal Species�� 146
5.7.2 Enzymatic Pretreatment�� 147
5.8 Combined Methods��147
5.8.1 Alkaline Pretreatment and Gamma Irradiation�� 147
5.8.2 Alkaline and Mechanical Pretreatment�� 147
5.8.3 Alkaline Pretreatment and Wet Oxidative Pretreatment�� 148
5.8.4 Combinatorial Biological Pretreatment�� 148
5.9 Industrial Implementation of Lignocellulosic Biomass Pretreatments��148
References��155
Chapter 6: Tobacco Seed Oil for Biofuels��161
P. Poltronieri
6.1.1 Feedstock Biomasses�� 163
6.1.2 Tobacco Mutagenesis and Genetic Studies�� 164
6.1.3 Selection of Tobacco Varieties for Biotic Stress, Pathogen, and
Nematode Resistance�� 166
6.1.4 Bioeconomy of Biofuels�� 167
6.1.5 Biokerosene Deployment and Sustainability Issues�� 173
6.1.6 Bioeconomics of Tobacco Seeds Oil for Aircraft Fuel: Energy
Tobacco Variety Solaris�� 178
6.1.7 Sustainability of Production and Exploitation of Solaris Tobacco;
Protein Cake as Feed�� 181
References��185
Chapter 7: Fermentative Production Methods��189
L.A. Prado Barragán, J.J.B. Figueroa, L.V. Rodríguez Durán, C.N. Aguilar González and C. Hennigs
7.2 Submerged Fermentation��189
7.2.1 Biotechnological Potential�� 190
7.2.2 Types of Bioreactors�� 191
7.2.3 Process Control�� 196
7.2.4 Microorganisms�� 200
viii
Contents
7.2.5 Substrates�� 200

7.2.6 Biomolecules Production�� 201
7.3 Solid-State Fermentation��202
7.3.1 Biotechnological Potential�� 202
7.3.2 Types of Bioreactors�� 202
7.3.3 Process Control�� 206
7.3.4 Microorganisms�� 210
7.3.5 Substrates�� 211
7.3.6 Biomolecules Produced by Solid-State Fermentation�� 213
7.4 Solid-State Fermentation Versus Submerged Fermentation:
General Considerations��213
7.5 Perspectives and Conclusions��215
References��215
Chapter 8: Composition of Plant Biomass Biotech Engineering of Cell
Wall to Optimize Biofuel Production��219
F. Francocci and I.B. Reca
8.2 The Plant Cell Wall: An Overview��219
8.3 Organization and Biosynthesis of Wall Components��220
8.3.2 Hemicellulose�� 221
8.3.3 Pectins�� 222
8.3.4 Lignin�� 222
8.4 Enzymes and Their Role in Cell Wall Modification (and Deconstruction)��223
8.4.1 Cell Wall Modification/Deconstruction: Learning From the Plant�� 223
8.4.2 Cell Wall Deconstruction/Modification: Learning From the Microbes�� 225
8.5 Manipulating Polysaccharides and Lignin Content or Composition
to Optimize Biofuel Production��226
8.5.1 Plants Cell Wall as Source of Energy�� 226
8.5.2 Cell Wall Recalcitrance, the Major Obstacle to Bioconversion�� 227
8.5.3 Modification of the Cell Wall Structure to Improve the
Saccharification Process�� 228
8.5.4 Lignin Modification to Improve Cellulose Accessibility�� 229
8.5.5 Hemicellulose and Pectin Modification to Overcome Cell
Wall Recalcitrance�� 229
8.6 Conclusions and Outlook��230
References��231
Chapter 9: Production of Dicarboxylic Acid Platform Chemicals Using Yeasts:
Focus on Succinic Acid��237
C. Pais, R. Franco-Duarte, P. Sampaio, J. Wildner, A. Carolas, D. Figueira and B.S. Ferreira
9.1 Markets and Applications��237
9.1.1 Drivers for Biobased Chemicals�� 237
9.1.2 Current Uses and Production of Dicarboxylic Acids�� 239
ix
Contents
9.2 Selection and Improvement of Yeast Strains��243

9.2.1 Yeasts as Platform Organisms for Dicarboxylic
Acid Production�� 244
9.2.2 Microorganisms Producing Succinic Acid: Natural Producers Versus
Metabolic Engineered Strains�� 245
9.2.3 Succinate Formation Pathways�� 245
9.2.4 Metabolic Engineering Strategies for Biotechnological
Production of Succinic Acid�� 248
9.3 Production of Biobased Dicarboxylic Acids��251
9.3.1 From Strain Isolation to Initial Physiological Studies�� 251
9.3.2 Linking Strain Physiology to Bioreaction Conditions�� 252
9.3.3 Requirements for Scale-Up Toward Commercial Operation�� 254
9.4 Downstream Processing of Biobased Dicarboxylic Acids��255
9.4.1 General Steps in Downstream Processing�� 255
9.4.2 Factors Influencing Downstream Processing�� 257
9.4.3 State of the Art DSP for Dicarboxylic Acids and New Trends�� 258
9.4.4 Designing an Effective Downstream Process�� 263
9.5 Challenges for Sustainable Microbial Production of Dicarboxylic
Acids Platform Chemicals��264
References��265
Chapter 10: Production, Stabilization, and Uses of Enzymes From Fruit
and Vegetable Byproducts��271
L.A. Prado Barragán, J.J. Buenrostro-Figueroa, C.N. Aguilar González and I. Marañon
10.2 Markets for Microbial Enzymes��272
10.3 Enzymes in Technical Applications��272
10.4 Encapsulation of Enzymes and Use in the Industry��273
10.4.1 Immobilization�� 274
10.4.2 Encapsulation�� 276
10.5 Uses of Immobilized Enzymes��282
10.5.1 Laundry and Dish Applications�� 283
10.6 Conclusion and Perspectives��284
References��284
Chapter 11: Sustainability Considerations on the Valorization
of Organic Waste��287
L. Six, F. Velghe, S. Verstichel and S. De Meester
11.2 Valorization Pathways for Organic Waste and the Waste Hierarchy��288
11.3 Sustainability��290
11.3.1 The Concept of Sustainability�� 290
11.3.2 Sustainability Assessment�� 291
x
Contents
11.4 Case Studies: Environmental Sustainability of Organic Waste

Valorization Pathways��296
11.4.1 Reuse as Feed�� 296
11.4.2 Recycling by Conversion to Higher Value Chemicals�� 297
11.4.3 Recycling by Composting�� 298
11.4.4 Energy Recovery and Recycling by Anaerobic
Digestion With Composting�� 299
11.4.5 Energy Recovery by Conversion to Biodiesel�� 302
11.4.6 Energy Recovery by Incineration�� 303
References��305
Chapter 12: GMO Acceptance in the World and Issues for the Overcoming
of Restrictions: Cisgenesis, RNA Transfer, Rootstock to Shoot
Delivery, Novel Methods of Transformation��309
P. Poltronieri
12.1.1 Genetically Modified Plants and Their Regulation Process in Different
Countries�� 309
12.1.2 Novel Plant Products�� 317
12.1.3 Gene Modification Using TALEN and Zinc-finger Nucleases,
CRISPR/Cas9 Nuclease System�� 322
12.1.4 Cisgenesis and Intragenesis�� 324
12.1.5 Genetically Modified Rootstock Grafting�� 325
12.1.6 Other Plant Improvement Techniques�� 327
12.1.7 Accelerated Transformation of Woody Plants and Early Flowering�� 328
12.2 Non-GMO Methods for Plants Improvement: Mutagenesis��329
12.2.1 Insertional Mutagenesis�� 330
12.2.2 Chemical Mutagenesis�� 331
12.2.3 Mutagenesis by Physical Agents�� 333
12.3 Regulation Harmonization��333
References��338
Index��343
xi

List of Figures
Figure 1.1 World production and world price indices (value 2000=100)
(Database FAO/OECD Agricultural Outlook 2014–2023). 2
Figure 1.2 Asparagus waste. 3
Figure 1.3 Garlic residues. 3
Figure 1.4 Green bean by-products. 4
Figure 1.5 By-product calendar of subproducts available in the Ebro valley (Spain)
(Personal communication with TRASA S.L.). 5
Figure 1.6 Transport costs of waste and by-products versus transport distance
(AWARENET).7
Figure 2.1 Central American major crops produced in the period 2009–2013.
Sugar cane data reported against secondary axis. 26
Figure 2.2 Flow diagram of the preparation of bananas to be used as carbon
source in lactic acid fermentation process. 42
Figure 4.1 Organic matter transformation into humus, biomass, and minerals. 78
Figure 4.2 Example of composting process (AP Business Technology Consultancy). 81
Figure 4.3 Typical temperature and acidity profile during composting. 82
Figure 4.4 Open air windrow system. 85
Figure 4.5 A fully automated turning machine. 86
Figure 4.6 Schematic overview of turning of table composting piles. 86
Figure 4.7 Drum composting reactor (DTEnvironmental). 87
Figure 4.8 The four steps in the anaerobic digestion process. 92
Figure 4.9 Evolution of biogas production on byproducts before bioproduct
extraction.118
Figure 4.10 Evolution of the biogas production of sweet corn before and after
bioproduct extraction. 119
Figure 4.11 Evolution of the biogas production of potato waste before and after
bioproduct extraction. 120
Figure 5.1 Structure of cellulose molecule. 127
Figure 5.2 Hydrogen bonds between cellulose polymer chains. 128
Figure 5.3 Structure of hemicellulose. 129
Figure 5.4 p-coumaryl-, coniferyl-, and sinapyl alcohol, building blocks of lignin. 130
Figure 6.1 Feedstock biomass options for production of ethanol, fuels,
and valuable chemicals. 164
Figure 6.2 Prospects of transformation of municipal solid wastes in Europe
into ethanol and fuels. 171
xiii
List of Figures
Figure 6.3 Scheme of production of biofuels and chemicals starting from

municipal solid wastes. 172
Figure 6.4 Diagram of CO2 emissions between 2005 and 2050. The graphic shows
the evolution of GHG emission under two contrasting economic
measures and policies: no action, and carbon neutral growth: by 2050
a net aviation carbon emission trajectory will reach a −50% reduction. 174
Figure 6.5 Comparison of Solaris variety (left) inflorescence size with that of
control tobacco (right). The differences in size of inflorescences and
seed content establish Solaris as a variety producing a high quantity
of tobacco seed oil. 182
Figure 6.6 Solaris plants from genetics to market (G2M) flow. From research to
agronomy studies, to process management, added value services, to
market.183
Figure 6.7 Tobacco seed oil from Solaris plants. Supply chain overview and
flow chart of processes to obtain biofuel. 184
Figure 7.1 Principle of a batch stirred tank reactor. 191
Figure 7.2 Principle of a continuous stirred tank reactor. 192
Figure 7.3 Principle of a fed-batch stirred tank reactor. 193
Figure 7.4 Principle of a bubble column reactor. 194
Figure 7.5 Principle of an airlift bioreactor. 195
Figure 7.6 Principle of a fluidized bed bioreactor. 196
Figure 7.7 Principle of a trickle bed bioreactor. 197
Figure 7.8 Microbial growth dynamics. 198
Figure 7.9 Column reactor. 204
Figure 7.10 Tray reactor. 205
Figure 7.11 Rotary drum reactor. 206
Figure 7.12 Continuous screw reactor. 207
Figure 9.1 Main succinate production metabolic pathways in Saccharomyces
cerevisiae: (A) oxidative branch of TCA cycle; (B) reductive branch of
TCA cycle; (C) glyoxylate pathway. Not all enzymatic steps are shown. 246
Figure 9.2 Schematic principle and processing steps during (A) reactive extraction
and (B) back extraction system with displacer as back extraction
compound.260
Figure 9.3 Schematic principle of electrodialysis. 262
Figure 10.1 Main methods of enzyme immobilization: (A) electrostatic adsorption,
(B) covalent binding, (C) cross-linking, (D) entrapment, and
(E) encapsulation. 275
Figure 10.2 Schematic diagram of a spray drying encapsulation process. 278
Figure 10.3 Schematic diagram of a bottom spray fluid bed coating process
(Wurster process) to obtain enzymes with a double layer coating,
changing the shell formulation atomized at the appropriated time. 279
Figure 10.4 Schema of a process combining spray drying and a fluid bed. 280
Figure 10.5 Schematic diagram of a top spray fluid bed agglomerating process. 280
Figure 10.6 Classification of methods to make and solidify drops. 281
Figure 11.1 The waste hierarchy. 289
Figure 11.2 Life cycle phases of a product (dotted line = recycling). 291
Figure 11.3 Life cycle assessment phases and their interactions. 292
xiv
List of Figures
Figure 11.4 Relationship between life cycle inventory, midpoint indicators,

endpoint indicators, and single endpoint score in ReCiPe 2008
(from left to right). 295
Figure 11.5 Climate change impact of food waste valorization routes: dry feed
production, wet feed production, composting, and landfilling. 297
Figure 11.6 Environmental impact of different composting technologies. 299
Figure 11.7 Landfilling everything or separating for 65% composting and 35%
landfilling.299
Figure 11.8 Climate change impact of composting, anaerobic digestion, and
incineration technologies (over 100 years). 300
Figure 11.9 Endpoint impact of composting, anaerobic digestion, and incineration
technologies (Eco-indicator 95+). 301
Figure 11.10 Environmental impact of municipal waste treatment with increasing
diversion of food waste from incineration to anaerobic digestion with
composting and small-scale composting. 301
Figure 11.11 Life cycle climate change impact of fuels derived from food waste,
agricultural products, and fossil resources (per MJ energy). 303
Figure 11.12 Climate change impact ranges of electricity generation by renewable
and nonrenewable sources. 303
Figure 11.13 Climate change impact ranges of heat production by renewable
and nonrenewable sources. 304
xv

List of Tables
Table 1.1 Vegetable Waste Amount in 2012. 4
Table 1.2 Valuable Compounds From Fruit and Vegetable Biomass. 12
Table 1.3 Currently Used Carbohydrate Carbon Sources in Industrial Fermentation. 15
Table 2.1 Main Seven Exported Fruits Form Costa Rica for the Year 2014, Expressed
in Thousands of US Dollars and in Tons. 23
Table 2.2 Agricultural Production in Tons for Central American Countries
in the Year 2013. 26
Table 2.3 Physicochemical Composition of Cultivated and Exported Fruits
From Costa Rica and Central America. 31
Table 2.4 Vitamin C Content (mg/100 g) of the Main Cultivated Fruits in Costa Rica
and Central America. 32
Table 2.5 Phenolic Compounds Content (mg GAE/100 g) of the Main Cultivated Fruits. 34
Table 2.6 Antioxidant Capacity Assessed by ORAC Method (TE Micromol/g) of the
Traditional Tropical Fruits. 35
Table 2.7 Composition of Green Coffee of Varieties Arabica and Robusta, Produced
in Mexico. 36
Table 2.8 Physicochemical Characterization of Pomegranate Seeds and Husk
in g/100 g of Fruit Produced in Mexico. 38
Table 3.1 Potential of Mango Waste Components. 51
Table 4.1 Recycling Targets for Biodegradable Waste as Prescribed by the Landfill
Directive.71
Table 4.2 Main Characteristics of Some Biodegradable Materials. 79
Table 4.3 Duration of Some Industrial Composting Processes (Large-scale Compost;
Deyerling & Fuchs; Productie Van Compost; Offaly). 82
Table 4.4 Levels of Rottegrad Based on the Potential Heating Ability of Compost. 83
Table 4.5 Overview Baumuster Composting Categories. 84
Table 4.6 Theoretical Amounts of Methane in Biogas. 93
Table 4.7 Analysis Results of Biogas Potential Tests on Byproducts Before Bioproduct
Extraction.117
Table 4.8 Analysis Results of Sweet Corn Products. 118
Table 4.9 Analysis Results of Potato Products. 119
Table 5.1 Effect of Various Pretreatments Methods on Composition of Lignocellulosic
Biomass.132
Table 5.2 List of Main Biorefinery Facilities, Status and Pretreatment Strategies. 150
Table 6.1 Content in Fatty Acids of Some Tobacco Varieties Selected. 180
xvii
List of Tables
Table 6.2 Characteristics of the Tobacco Oil Obtained by Pressing the Seed and
Filtering.181
Table 7.1 Main Characteristics of SSF Bioreactors. 203
Table 7.2 Differences Between Solid-State (SSF) and Submerged Liquid Fermentation
(SmF).214
Table 8.1 Worldwide Production of Bioethanol. 226
Table 9.1 Demonstration-Scale Facilities for the Production of Biobased Succinic Acid. 240
Table 9.2 Main Metabolic Engineering Strategies to Improve Succinate Production
in Different Yeast Species. 249
Table 9.3 General Steps in Downstream Processing, Highlighting Common
Principles and Unit Operations as well as the Respective Desired
Outcome of Each Step. 256
Table 11.1 Impact Assessment Methods Used in the International Life Cycle
Data System Midpoint Method. 293
Table 12.1 List of Genetically Modified Crops Cultivated Around the World. 311
Table 12.2 Genetically Modified Crops Approved in European Union. 312
xviii
List of Abbreviations
A4A Airlines for America

AFEX Ammonia fiber explosion
AG Agronomic trait
AHAS Acetohydroxy acid synthetase
ALH Acetolactate synthetase
AM Association mapping
AMIMCl 1-allyl-3-methylimidazonium chloride
APHIS Animal and Plant Health Inspection Service
ASTM American Society for Testing and Materials
ATP Adenine trinucleotide phosphate
BDO 1,4-butanediol
BMIMCl 1-butyl-3-methylimidazonium chloride
Bt Bacillus thuringensis
BTG Biomass Technology Group
C4H Cinnamate 4-hydroxylase
CAGR Compound annual growth rate
CCoAOMT CoA O-methyltransferase
CCR Cinnamoyl CoA reductase
CDE Carbon dioxide explosion
CesA Cellulose synthase A
CESA Cellulose synthase catalytic subunit
cgRNA Chimeric guide RNAs
CHP Combined heat and power
CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
DM Dry Matter
DME Dimethyl ether
DP Degree of polymerization
DSBs DNA double-strand breaks
DSP Downstream processing
EBFPI European Biofuels FlightPath Initiative
EBTP European Biofuel Technology Platform
ECHA European Chemical Agency
EDTA Ethylene diamine tetracetic acid
EFSA European Food Safety Agency
E-LCA Environmental life cycle assessment
ELISA Enzyme-linked immunosorbent assays
EMS Ethyl Methane Sulphonate
ENI Ente Nazionale Idrocarburi
xix
EPA Environmental Protection Agency

EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
ETI Effector triggered immunity
FAA Federal Aviation Administration
FDA Food and Drug Administration
FLT FLOWERING LOCUS T
FR Fungus resistance
FSK Fully Synthetic Kerosene
FT Fischer Tropsch
FVW Fruit and vegetable waste
GHG Greenhouse-Gas-Emissions
GMO Genetically modified organisms
HDR Homology-directed repair
HEFA Hydrotreated Esters and Fatty Acids
HFTL Heavy Fraction FT Liquids
HMF 5-hydroxymethylfurfural
HPLC High performance liquid chromatography
HR Homologous recombination
HR Hypersensitive response
HRJ/HEFA Hydroprocessed oils and fats
HT Herbicide tolerance
HT-IR Herbicide tolerance and insect resistance
IEA International Energy Agency
IL Ionic liquids
ILUC Indirect land use change
IR Insect resistance
KGD α-ketoglutarate dehydrogenase
LCA Life Cycle Analysis
LCC Life cycle costing
LFTL Light Fraction FT Liquids
LM Linkage mapping
LTR Long terminal repeat
MASBI Midwest Aviation Sustainable Biofuels Initiative
MCC Microcrystalline cellulose
MFC Microfibrillar cellulose
MFTL Medium Fraction FT Liquids
MiR MicroRNA
MLG Mixed linkage glucans
MS Male sterility
MSW Municipal solid waste
NADH Nicotinamide adenine dinucleotide hydrate
NBGT Nuclease-Based Gene Targeting
nDart1 Nonautonomous DNA-based active rice transposon1
NGS Next-Generation Sequencing
NHEJ Non-homologous end joining
NPP Novel Plant Products
ODM Oligonucleotide-directed mutagenesis
OMT O-methyl transferase
OR Origins of replication
PAL Phenylalanine ammonia lyase
PAMP Pathogen Associated Molecular Pattern
xx
PBS Polybutylene succinate

PEF Pulsed-electric-field pre-treatment
PEG Poly ethylene glycol
PGPR Plant Growth Promoting
PH Plant height
PHB Polyhydroxybutirate
PLA Poly lactic acid
PLL Poly-l-Lysine
PMEIs Pectin methylesterase inhibitors
PPO Polyphenol oxidase
PQ Product quality
PVP Poly vinyl pyrrolidone
QTL Quantitative trait loci
RB Reverse breeding
RED Renewable Energy Directives
REDD Reducing Emissions from Deforestation and Forest Degradation
RG-II Rhamnogalacturonan II
RNAi RNA interference
RSB Roundtable and Sustainable Materials
SA Succinic acid
SAA South african airlines
SAR Systemic acquired resistance
SFE Supercritical fluid extraction
sgRNA Single-guide RNA
SIP Synthesized iso-paraffine
SiRNAs Small interfering RNAs
SKA Synthetic paraffinic kerosene with aromatics
SkyNRG Sky Energy
S-LCA Social life cycle assessment
SmF Submerged fermantation
SOD Superoxide dismutase
SPK Synthetic paraffinic kerosene
SPT Seed Production Technology
SSF Solid state fermentation
SWOT Strength, weaknesses, opportunities, threats
TALENs Transcription activator-like effector nucleases
TCA Tricarboxylic acid
T-DNA Transfer DNA
THF Tetrahydrofuran
TILLING Targeting Induced Local Lesions in Genomes
TSO Tobacco seed oil
UA United Airlines
USP Ultrasonic pre-treatment
VBS Vector Backbone Sequences
VR Virus resistance
WEP Wet oxidation pre-treatment
WWTP Waste water treatment plant
XTH Xyloglucan transglycosylase/hydrolase
XyG Xyloglucans
ZFNs Zinc finger nucleases
xxi

About the Editors
Palmiro Poltronieri is researcher at the Agri-Food Department of the National Research
Council, Italy. He holds a PhD in Molecular and Cellular Biology and Pathology, obtained
from Verona University in 1995. In 1996–1997, he was Japanese Society for Promotion of
Science postdoctoral fellow at University of Tsukuba, Japan. Since 1999, as a researcher at
the Institute of Sciences of Food Productions, he studied plant protease inhibitors and their
applications, and plant signaling in response to abiotic stresses in legumes. He is cofounder
of Biotecgen Company, involved in several European projects, and has supported as supervisor
the biotech company, Bioesplora. He is associate editor to BMC Research Notes and editor-
in-chief of the journal Challenges, MDPI, Basel, Switzerland.
Dr. Oscar Fernando D’Urso obtained a BSc in Biological Science in 2005 from the Univer-
sity of Salento and a PhD in Physiology in 2010. For 6 years, he has been the head of the
laboratory at Biotecgen in Lecce, Italy. He founded a small company, Bioesplora, San Michele
Salentino, Italy, in 2011, working in the field of novel diagnostic kits. Bioesplora counts on two
divisions: biotechnology and engineering. Bioesplora’s core business relies on molecular
biology services, and in the development and commercialization of microelectronics and
electronics devices. Bioesplora has a strong expertise in the development of microarray and
real-time-based protocol in clinical and environmental fields.
xxiii

About the Contributors
Cristóbal Noé Aguilar González is Dean of the School of Chemistry at Universidad
Autónoma de Coahuila, México. He is Chemist (1992) by the Universidad Autónoma de
Coahuila (Mexico); his MSc Program in Food Science and Biotechnology (1995) was held
in the Universidad Autónoma de Chihuahua, México. His PhD program in Fermentation
Biotechnology (2000) was developed in the Universidad Autónoma Metropolitana, Mexico.
He was working on a postdoc in Biotechnology and Molecular Microbiology (2001) in the
IRD, Marseille, France. Dr. Aguilar has published more than 130 papers published in indexed
journals, more than 35 articles in Mexican journals; 16 book chapters, 8 Mexican books,
4 editions of international books, 34 proceedings, and more than 250 contributions in scientific
meetings. Dr. Aguilar is a member, Level III of SNI (Mexican National System of Researchers)
from 1998, receiving several prizes and awards, among which the most important are:
National Prize of Research 2010 of the Mexican Academy of Sciences, the prize “Carlos
Casas Campillo” (2008) of the Mexican Society of Biotechnology and Bioengineering,
National Agro-Bio Prize (2005), and the Mexican Prize in Food Science and Technology.
Consejo Nacional de Ciencia y Tecnologia (CONACYT)-Coca Cola México, 2003. Prof.
Aguilar has developed more than 21 research projects, including 4 international exchange
projects Evaluation-orientation de la COopération Scientifique Nord (ECOS-Nord), alpha
network European Project; Marie Curie European project for mobility of researchers. He has
been advisor of 13 PhD theses, 19 MSc theses, and 40 BSc theses. He is now a regular
member of the Mexican Academy of Science (2014).
Lilia Arely Prado Barragán is full-time Professor at the Universidad Autónoma

Metropolitana-Iztapalapa (UAM-I). She graduated in Food Engineering at the Universidad
Autónoma Metropolitana–I (UAM-I) (Mexico) and performed technical studies on flavor
release, Master’s degree and PhD in Biochemistry and Food Science at the University of
Nottingham (England), performed a research program (2003) at the University of Victoria
(British Columbia, Victoria Canada) and in the Biotechnology and Molecular Microbiology
Laboratory in the Institute for the Research and Development, IRD, Marseille, France (2004).
She published more than 45 papers in indexed journals and 8 book chapters, and edited 2
national books, 15 proceedings, and more than 90 contributions in scientific meetings; she is
co-author of 3 patents on the production of biomolecules by solid state fermentation processes.
xxv
She is an expert in fermentation processes for the valorization of bioactive molecules from
agroindustrial by-products. She has experience in isolation of native strains with biotechnological
potential and has developed solid state fermentation processes for the production, isolation,
and characterization of biomolecules with enzymatic, antioxidant, antimicrobial, and
hypoglycemic activities. Dr. Prado has been awarded the “Sergio Sanchez-Esquivel” prize by
the Mexican Society of Biotechnology and Bioengineering (2009), has been recognized for
the desirable profile for full-time teachers by the Ministry of Education Programa Mejoramiento
del Profesorado (PROMEP), and belongs to the National System of Researchers, Level II
(SNI). She is member of the Commission on Graduate Biotechnology, a member of the
Mexican Society of Biotechnology and Bioengineering; she belongs to the Research Group of
Bioprocess, Fermentations and Natural Products; and she is the Coordinator of the Editorial
Committee of Biological Science Faculty (UAM-I) for the development of textbooks and
teaching materials.
José Juan Buenostro Figueroa is a food engineer. He obtained Master’s and doctoral (2008)
degrees in Food Science and Technology by the Universidad Autónoma de Coahuila and
accomplished postdoctoral research at the Biotechnology Department of the Universidad
Autonoma Metropolitana, Iztapalapa. He is coauthor of 2 bioprocess patents, 16 indexed
papers, and 28 contributions in scientific events, a member of Mexican Society of Biotechnology
and Bioengineering, and belongs to the Research Group of Bioprocess, Fermentations and
Natural Products.
Ana Carolas is lab leader and fermentation scientist at Biotrend. She holds an MSc in Biological
Engineering (University of Lisbon, Portugal) and has performed research at NIZO Food
Research BV, Ede, The Netherlands. Ana oversees all fermentation trials at Biotrend and has an
impressive experience of having personally run hundreds of fermentation trials in controlled
bioreactors with diversified biological systems, including yeasts, bacteria, and filamentous fungi.
Steven De Meester holds a Master’s in Bioscience Engineering with a specialization in
Environmental Technology at Ghent University. After his Master’s, he worked with a PhD
fellowship at Ghent University on the use and development of the Life Cycle Analysis (LCA)
methodology in the biorefinery sector, and has now a PhD degree. He is author or coauthor of
several scientific articles on environmental sustainability assessment. Within Organic Waste
Systems (OWS), he worked as Sustainability Assessment Services project manager and is
currently working at Environmental Organic Chemistry and Technology (EnVOC) group at
Ghent University, Belgium.
Thomas Dietrich is an engineer in biotechnology, and project manager and researcher at
Tecnalia Research & Innovation (Spain) in the area of bioprocesses and food ingredients. He
participated in several European-funded research projects and coordinated the biomass
valorization projects HELICAS and TRANSBIO. After receiving his engineering degree from
Technical University of Berlin (Germany) in 2000, he started his career as project manager in
xxvi
ttz Bremerhaven (Germany). In November 2004, he took over the responsibility for the
bioprocess engineering group at the food department of ttz. In the end of 2011, he moved to
the applied research center LEIA CDT (Spain), mainly working in projects related to food
ingredients and preservation, as well as utilization of microalgae for different approaches.
Additionally, he coordinated the European project AIRTV, aiming at environmental technology
verification of air emission abatement technologies, and was involved in EU projects:
FERMATEC, continuous bioreactor for bioethanol; CRUSTAMEL, preventing browning
(melanosis) of shrimps; FISHNOSE, electronic nose to detect quality of smoked fish,
SO2SAY, prevention of polyphenol oxidase-induced browning in fruits and vegetables; and
BreadGuard. Since 2011, he is part of Tecnalia, working in biomass valorization as well as
new sensor systems for food applications.
Pedro J. Echeverría is an agronomist engineer; his degree was obtained at U.P.N.A University
in Navarra, Spain. He is Occupational Safety and Health Senior Technician, and Master’s in
Environmental Management Engineering. He has more than 15 years of experience in the
agro-alimentary sector, basically devoted to the technical, environmental, and the R&D areas.
Between 1999 and 2007, he worked in different projects related to enterprise management,
always within the scope of CONSEBRO (enterprise association of agro-alimentary industries
that has a unit of services of enterprise management), and since 2008, he is Technical Director
of the company TRASA, Tratamiento de Subproductos Agroalimentarios, San Adriàn, Navarra,
Spain. The aim of the company is the treatment and valorization of subproducts from the
agro-food sector. During these last years, he participated in several R&D projects in
collaboration with other companies, technological centers, and universities related to the
remainders/agro-alimentary by-products and their possibilities of valorization (BISOSTAD,
CARBIO, VALBIO, VALSOST+).
Bruno Sommer Ferreira is co-founder and CEO of the BIOTREND company, located in
Cantanhede, Portugal, specializing in bioprocess development, optimization, and scale-up,
and he is co-founder of Silicolife, a leading computational biology company. He was visiting
scientist at the Institute of Biomaterials and Biomedical Engineering, University of Toronto,
Canada, and researcher at the Center for Biological and Chemical Engineering, Technical
University of Lisbon, Portugal. Bruno holds a PhD in Biotechnology, University of Lisbon,
and an MSc in Chemical Engineering, Instituto Superior Técnico, Portugal. Bruno has been
Vice-President of the Portuguese Bioindustries Association. He is a member of the expert
panel of ERA-Net Industrial Biotechnology and has contributed to advisory groups for the
Portuguese Government, the European Commission, and the OECD on the topic of the
bioeconomy. He advises public and private initiatives aiming to bridge the gap between
biotechnology research and commercial applications.
Diogo Figueira is downstream processing scientist at Biotrend, specializing in the development

of process operations for the recovery of biomolecules from fermentation products, both at
xxvii
laboratory and at pilot scale. He holds an MSc in Biotechnology (University of Aveiro,

Portugal) and has past research experience in the brewery industry.
Ricardo Franco-Duarte is a researcher from Center of Molecular and Environmental
Biology, in University of Minho, Portugal. He holds a Master’s in Molecular Genetics (2008),
and a PhD in Biology (2014). His research interests are focused mainly in yeast diversity and
genomics, molecular biotechnology, computational science, bioinformatics, metabolomics,
and phenomics. He participated in several national and international projects, such as
INNOYEAST, WineMetrics, PhenoMet, and Transbio, to name a few.
Fedra Francocci holds a PhD and MD in Industrial and Environmental Biotechnologies,
obtained at Sapienza University of Rome in 2006 and in 2010. She performed a PhD in
Botanical Sciences and Plant Biology, a collaborative thesis under “La Sapienza” University
in Rome and the Universidad Autónoma de Madrid, in which she studied the role of the cell
wall in plant development and defense against microbial pathogens. Her research interests are
pectin and cell wall recalcitrance to enzymatic hydrolysis during saccharification, and processes
to facilitate higher fermentative biomasses for biofuel production. She collaborated with the
Royal Holloway, University of London, to improve sugar release from plant biomass by
pretreating the plant material with a white rot fungus. She has several high impact publications
and is inventor the international patent EP2204451A1, “Use of plants with reduced levels of
de-esterified homogalacturonan in the cell wall or portions thereof for improving the
saccharification of plant biomasses.”
Caroline Hennigs, Engineer in Biotechnology, has been working for Naturstoff-Technik
GmbH in Osterholz-Scharmbeck, Germany, since 2015, where she is responsible for the
business development. After receiving her engineering degree from Hochschule für Angewandte
Wissenschaften Hamburg (Germany) in 1998, she started her career in the “cosmed” depart-
ment of Beiersdorf AG in Hamburg, Germany. In 2001, she became project manager in ttz
Bremerhaven (Germany). In 2005, she took over the responsibility for the natural resources
group at the food department of ttz. She has more 15 years experience in applied research in
bioprocesses and food ingredients using renewable resources. She has elaborated, coordi-
nated, and participated in more than 15 European and national research projects, as well as
direct R&D contracts within the European food industry.
Izaskun Marañon is researcher at Foundation TECNALIA Research & Innovation, Spain.

She has 15 years of experience in developing R&D projects in macromolecular chemistry,
working with synthetic and natural polymers. Her last 12 years of experience related to food
ingredients development and micro- and nanoencapsulation technologies for different
applications: food ingredients, nutraceutics, enzymes, yeasts, and bacteria. Since 2002, she is
active as member of the Bioencapsulation Research Group. She has five international
contributions, and she is an inventor in two international patents.
xxviii
Nike Mortier holds a Master’s of Science in Bioscience Engineering with specialization in

Environmental Technology at Ghent University. She is project manager for compostability
and ecotoxicity testing and has been working at OWS for 7 years.
Srinivasan Narasimhan, recipient of the Tamil Nadu Scientist award, obtained his PhD
in Chemistry from the University of Madras. Subsequently, he became associated with
Prof. Herbert C. Brown, Nobel Laureate in Chemistry (1979) in Purdue University, Lafayette,
Indiana, USA. He returned to India and served as Senior Scientist in IDL-Nitro Nobel Basic
Research Institute, Bangalore, and as Associate Director, Spice Science Foundation. He is
currently the Founder/Director of Asthagiri Herbal Research Foundation, an institution that
achieved accreditation from Department of Scientific and Industrial Research (DSIR),
Ministry of Science and Technology; Ayurveda, Yoga, Unani, Siddha and Homoeopathy
(AYUSH), Ministry of Health and Family Welfare; and University of Madras. He works
in interdisciplinary areas involving natural products, biotechnology, entomology, microbiology,
and analytical chemistry. His research has resulted in the development of several crop
protection formulations, health care products, and novel lead molecules. His innovative
technology on the application of pheromone technology to control sugarcane borers earned
him the Tamil Nadu Scientist Award and citation given by the Tamil Nadu State Council for
Science and Technology. He has supervised researchers, leading to PhD degrees from the
University of Madras, Sri Ramachandra Medical College and University, Bharathiar University,
and Bharath University. He has so far guided 36 PhDs, and 14 more are currently pursuing
research in chemistry and biotechnology. He has published more than 125 papers in
national and international journals, applied for 15 patents, and conducted 3 international
conferences. He has also established collaborations with Indian and foreign institutions for
conducting research. A few of them are: Vel’s University (Department of Biotechnology);
Greenwich University, UK; Nag Research Labs, Fremont, USA. He has generated a
number of projects funded by Department of Science and Technology (DST), Department of
Biotechnology (BDT), AYUSH, Indian Institute of Chemical Technology (CSIR), and a few
private sponsors. He has been serving as a member in the advisory committees of several
institutions. He also served as the task force member of the Department of Biotechnology,
Ministry of Science and Technology, India.
Célia Pais holds a PhD in Biology and a postgraduation in Plant Pathology by Imperial
College, London. She is an Associate Professor at the University of Minho, Braga, Portugal,
where she has been Vice-President of the School of Sciences, Head of the Biology Department,
and Coordinator of the doctoral program in Molecular and Environmental Biology. She
supervised numerous PhD and masters theses, and she has been involved in over 20 research
projects and networks, European and national, as principal investigator or participant. Presently,
she develops her research at the CBMA, University of Minho, focusing mainly on yeast
biotechnology, molecular biology, and taxonomy.
xxix
Carlota Peral is a researcher at TECNALIA Research & Innovation (Spain) in the area of
bioprocesses. She has 20 years of experience in developing R&D projects in the chemistry
industry, the last 5 years of experience being related with food ingredients development,
biomolecules extraction from agricultural by-products, and encapsulation technologies for
different applications. She has participated in several European-funded research projects.
Since 2011, she is part of Tecnalia, working in biomass valorization, mainly in the development
of pretreatment and downstreaming operations for the recovery of biomolecules from
fermentation products.
Ana Mercedes Pérez Carvajal holds a PhD in Food Science from the University of
Montpellier, France, obtained in 1992. She is researcher at the National Center of Food
Science and Technology (CITA) of the University of Costa Rica (UCR) since 2001, and she
has the coordination of the research area. Also, she is currently the Director of the PhD
Program in Sciences from the UCR. Her research interests are mainly the development of
functional foods, antioxidant activity assessment, analysis of bioactive compounds, and
membrane technologies applications in foods. She has published as coauthor more than 30
papers in high-impact factor journals, and she is co-inventor in three patents.
Palmiro Poltronieri is researcher at the Agri-Food Department of the National Research
Council, Italy. He holds a PhD in Molecular and Cellular Biology and Pathology, obtained
from Verona University in 1995. In 1996–1997, he was Japanese Society for Promotion of
Science postdoctoral fellow at Tsukuba University, Japan. Since 1999, as a researcher at the
Institute of Sciences of Food Productions, he studied plant protease inhibitors and their
applications, and the plant signaling in response to abiotic stresses in legumes. He is
cofounder of Biotecgen Company, is involved in several European projects, and has supported
as supervisor the biotech company, Bioesplora. He is associate editor to BMC Research
Notes, and editor-in-chief of the journal Challenges, MDPI, Basel, Switzerland.
Bianca Pop, environmental science engineer, is Senior Project Manager at Tritecc, Romania.
She is founder of the company, which, apart from being a member in various project consortia
since FP7, is also supporting other Romanian Small Medium Enterprises (SMEs) to have
access to funding for their innovative ideas. Ms. Pop is a former scholarship holder of the
German Environmental Foundation (DBU) and now a member of the scholarship awarding
committee. During the 1-year scholarship, she specialized in Germany through various
research projects.
Ida Barbara Reca is postdoctoral fellow at CNR-ISPA Institute in Lecce, Agri-Food
Department of the National Research Council, Italy. She graduated in Biology and
biotechnology from “La Sapienza” University in Rome in 2004. She holds a PhD in Plant
biotechnology obtained from “La Tuscia” University, Viterbo, and a PhD in Nutrition,
molecular and cellular aspects, from the “Paul Cezanne” University, Aix-Marseille. She
worked from 2009 to 2013 at the Great Lake Bioenergy Research Center, Plant Research
Laboratory, Michigan State University, on plan cell wall and hemicellulose biosynthesis.
xxx
Presently, she studies pathogen recognition and immunity conferred Leucine Rich Repeat
(LRR) proteins in plants.
Luis Víctor Rodríguez Durán is a biochemical engineer by the Universidad Autónoma de

Tamaulipas (Mexico) and holds a Master’s degree in Food Science and Technology by the
Universidad Autónoma de Coahuila (Mexico). He is author or coauthor of 12 scientific papers
in international peer-reviewed journals, 4 books chapters, and 17 contributions to congresses,
co-author of a Mexican patent on the extraction of biomolecules by solid state fermentation¸
and has participated in several projects funded by the National Council of Science and
Technology (Mexico). His research interests include the production, purification, character-
ization, and application of enzymes involved on the phenolic compounds release. His research
is focused on the application of solid state fermentation for the production of fungal enzymes
such as tannases, feruloyl esterases, and chlorogenate hydrolases.
Alexandru Rusu (PhD) is the Leader of Strategic Research Department in Biozoon GmbH,
Germany. He has extensive background in different areas of biotechnology and food research.
From the combined experience in academia and industry, he also gained a strong professional
know-how in managing national and international collaborative Research and Technical
Development (RTD) projects. Thus, he possesses a successful track record, both as a participant
and as coordinator, in managing numerous European and national RTD projects.
Paula Sampaio is a biochemist and holds a PhD in Molecular Microbiology by the University
of Minho, Braga, Portugal. She is an assistant professor at the same university and a researcher
at the Center of Molecular and Environmental Biology (CBMA), University of Minho. During
the last decade, she has focused her research on yeast molecular biology and taxonomy, as
well as on the yeast–host interactions. She supervised numerous PhD and masters theses, and
has been involved in several European and national research projects and networks.
Lasse Six holds a Master’s of Science in Bioscience Engineering with a specialization

in Environmental Technology at Ghent University. He has been working as a SACS
Sustainability Assessment Service & Compensations project manager at OWS for 2 years.
Bikshandarkoil Sivaraj Srikanth is a Management Consultant/Facilitator with 17 years

experience in market and business research, strategy planning and execution (including
merger and acquisitions/joint ventures), feasibility studies, operational audits, performance
improvement, market development across consumer packaged goods (CPG), retail, agro-services,
industrials (automotive and light engineering goods), infrastructure, and consumer services.
He had the opportunity to work with a few ministries of the Government of India, state
government departments like tourism, agriculture, and other government/quasi-government
bodies like Confederation of Indian Industries (CII), Federation of India Chambers of
commerce (FICCI), National Disaster Management Authority (NDMA), National Institute for
Smart Government (NISG), etc., in numerous capacities involving policy papers, capacity
building, and communication programs. His association with the educational ecosystem has
xxxi
been through management/business magazines publications (prepared articles/case studies),

British Council/English programs in India, preparing and running small programs in marketing
management, modern retailing, and strategic management with some notable B-schools in
India. In the agriculture domain, he had the opportunity to work with teams in some of the
leading agriculture firms, working on strategy and execution programs for “horticulture,
organic seeds, fruits and vegetables, farm implements, and inputs, etc.,” over the years. This
involved interface with R&D, strategy, and operations teams.
Carmela Velázquez is a full professor at UCR and currently the General Director of
CITA of this university since 2005. She holds a Master’s Degree in Food Science from
Iowa State University, United States, from 1998 and an Engineering Degree in Food
Technology from the UCR. Her main research has been focused in the use of agro-industrial
wastes as biomass for industrial fermentations in lines like lactic acid, ethanol, and
recently, poly-2-hydroxybutyrate (PHB). She has worked with the scaling-up process
from lab to pilot plant facilities with a special interest in the full use of the biomass for
different industrial applications. She also has developed a large amount of new products
with the local industry through extension programs developed by CITA and UCR. Se has
also specialized in different models of cooperation between academia and industry, and
has started a new spin-off between CITA and the Chamber of Food Industry in Costa Rica
to develop a platform of cooperation for the production of value added products with
scientific background. She has participated in several academic activities and represented
academia in several industrial and government organizations.
Filip Velghe is a project engineer at OWS and is responsible for anaerobic digestion lab tests
and follow-up of full-scale AD plants, and has 9 years experience with anaerobic digestion.
He is author or coauthor of several scientific articles on anaerobic digestion. Before OWS,
he worked for 2 years as coordinator and technological advisor of Biogas-E, a nonprofit
organization promoting the use of anaerobic digestion in agriculture. He is Master of Science
in Bioscience Engineering with specialization in Environmental Technology at Ghent
University and Master of Science in Physical Land Resources with specialization in Soil
Science.
Steven Verstichel holds a Master’s in Bioscience Engineering with a specialization in
Environmental Technology at Ghent University. He has been working for 15 years at
OWS and is the head of the Biodegradability, Compostability and Ecotoxicity (BCE)
department. He is the author or coauthor of several scientific articles on biodegradation
and compostability. Within the company, he is responsible for the organization of European
projects.
María del Carmen Villaran Velasco holds a PhD in Chemistry from the University of
Basque Country (Spain). She is the Director of Bioprocesses Area in Fundacion TECNALIA
Research & Innovation, a Technological Center, Miñano, Alava, in the North of Spain. She
xxxii
has more than 25 years’ expertise in developing and management of R&D projects at national
and international levels, related to the development of drying and dehydration processes for
the food industry, and the development of processes for food ingredients development and
stabilization. Since 2002, she is a member of the Bioencapsulation Research Group. She is
inventor in two international patents. She has collaborated in more than 55 projects subsidized
by the public organism at the national and international level, and more than 50 contracts with
private companies. She has more than 30 contributions to congresses and 31 papers in
national and international journals. She has tutored 3 PhD fellows.
Jessica Wildner graduated in Industrial Biology at the University of Applied Sciences in
Bremen (Germany). She had the opportunity to work for different research organizations in
Europe in the fields of food science and microbiology. Since about the past 10 years, she has
performed applied research and project management at ttz Bremerhaven (Germany), an
independent provider of market-driven research and development services in the areas of
food, environment, and health. Her main research interests include food microbiology and
biotechnology, valorization of by-products, integrated fermentation, and biorefinery concepts.
xxxiii

Introduction
The bioeconomy and sustainable growth are top topics, both in Europe and around the world.
There is a need to exploit all the added value in the present productions and to unravel new
markets and technologies for the future. An important topic in plant biotechnology showing
good prospective and economic potential is the exploitation of plants with modified cell
walls for improved processes and industries to be applied also to biofuel production.
This book presents an evaluation on plant species better exploitable for a particular
transformation. Crops are already covering large parts of cultivable soils, so it is not
conceivable to try to extend the cultures beyond the limit of available soils. A further
increase in productivity is also difficult to obtain. Agriculture for food products covers
the largest part of crop productions, while feeds represent the second largest market. A smaller
part is devoted to production of maize for biofuel. Life cycle assessment (LCA) and CO2
footprint assessment evaluate the production processes as sustainable or not. Transport of
small distances (within 100 km), and through shipment transfer, are the most economic
means to deliver the waste and by-products. Nowadays, the food industry residues are
recycled by composting and by biomass incorporation in the soil to sustain farmyard
manure and fertilizers in order to increase the organic matter in the soil. Composting is
presently the most used method for waste treatment, the largest applicative transformation in
the management of differentiated wastes. However, there is a need to digest enzymatically
complex fiber-rich polysaccharides to obtain simple sugars suitable for biofermentations.
Further advances in technology and plants design are supporting the exploitation and
valorization of vegetable and fruit by-products through fermentation (feed-batch liquid
fermentation, solid state fermentation) in biobased biochemicals and industrial enzymes
production. The need to substitute the decreased reserves of petroleum with chemicals
from fermentative processes and the request for an increase in crop yields pose new
challenges to scientists in Europe and in the world. New production systems on an
industrial scale exploit tobacco plants (seeds) for biofuel, while the residual proteins are
processed as feeds for livestock. A chapter is dedicated to the biotechnology attempts to
improve the efficiency of second-generation bioethanol production from lignocellulosic
plant material. Fours chapters describe the activities done within the framework of the
TransBio project (www.transbio.eu).
xxxv
Introduction
Chapter 1 describes how a suitable biomass feedstock for conversion to energy or refining to
biobased products is the first step of a biobased supply chain. It is a prerequisite before
establishing any conversion facility to review the four Ws in the overall approach: What type
of biomass, in What quantities it is available, at What time of the year, and What properties it
has. Proper logistic solutions must be implemented to establish economic feasible and
environmental sustainable value-added chains using crop and plant biomass as feedstock.
Afterward, valuable compounds can be extracted and/or biomass can be used as feedstock for
fermentation processes.
In Chapter 2, the fruit production and processing in Central America and Mexico are discussed,
considering the volume of production, the competitiveness that these crops have in the region
and world, and some sociocultural differences between the countries that help to explain the
different aspects of the agroindustry. Central American countries are world leaders in agricul-
tural production in tropical fruits like banana and pineapple, and Mexico dedicates impor-
tance to agriculture also and is a world leader in products like avocado, coffee, and citrus
fruits. The composition of these major tropical fruits are analyzed and compared, considering
not only nutrients, but also biological activities related to major molecules of special interest.
Chapter 3 discusses food processing industries and awareness that waste and by-products offer
new market opportunities. Research addresses the issue of mango wastes and by-products in
India, as one of the most growing Brazil, Russia, India and China (BRIC) countries, and in
Europe by taking a comprehensive approach at looking for sustainable processes to turn food
processing by-products and nonfood plant fibers into valuable industrial products (fabrics and
textiles, vegetal leather, elements for the automotive and building industry, cases for electronics
products or household appliances, composites, bioresins for plastic materials, and lightweight
structural composites based on biobased polymers, reinforced with cellulose pulp fibers). There
are many additional applications for plant wastes, including the use of wood fibers to produce
lower carbon, energy efficient construction materials with antiseismic properties, or the use of
biobased and biodegradable agricultural mulch films for improving agricultural productivity. The
bioeconomy must take into account the balance of industrial use of biomass resources for multiple
uses including food, feed, chemicals, fibers, and fuels. To achieve this, the concept of smart and
sustainable use of biomass should be applied. In this chapter, several types of plant by-products
are discussed, and case studies of companies exploiting new production lines for textiles, light-
weighted insulating components, and biobased biochemicals are presented. A Strengths, Weak-
nesses, Opportunities, Threats (SWOT) analysis of the most significant products is presented.
In Chapter 4, organic waste is discussed, along with the treatment options developed and
installed to deal with this waste. This chapter focuses on two commonly used technologies:
composting and anaerobic digestion. Landfilling and incineration are not discussed in this
chapter, as they are less sustainable than composting and anaerobic digestion, and are
therefore being faded out in waste management solutions.
xxxvi
Introduction
In Chapter 5, agricultural wastes are discussed as the most abundant and renewable sources in
the world. These wastes, commonly called lignocellulosic biomass, are composed mainly of
cellulose, hemicellulose, and lignin. Both the cellulose and hemicellulose fractions are
polymers of sugars and thereby a potential source for fermentation processes. Lignin can be
used for many purposes, including production of chemicals, heat, and energy. Lignocellulosic
biomass is recalcitrant to hydrolysis with cellulolytic enzymes. Therefore, a pretreatment step
is necessary for the production of fermentable sugars. Various pretreatments methods, such as
physical, chemical, physicochemical, biological, and a combination of them are available.
Selection of the best pretreatment depends on such factors as foreseen utilization of the main
biomass components, efficiency in separation, environmental concerns, compatibility with
subsequent fermentation, energy requirements, and costs that must be taken into account for
commercial implementation.
In Chapter 6, tobacco is described as a model plant used to study the effect of genetic inter-
ventions on biomass production, lignification, and plant growth. In addition, tobacco has been
studied for its ability to produce seed oil in established and new varieties devoid of nicotine.
One of these health-friendly varieties, Solaris, has been patented for the high amount of
seeds and seed oil of good quality for transformation into biofuels. Feedstock biomasses and
plant species are briefly introduced. The bioeconomics of biofuels, biokerosene, syngas, and
different types of biofuels for aircrafts are presented. The chapter gives an overview on the
economic measures and debate on biofuels, and second-generation alternative fuels in
Europe and around the world. Sustainability issues in this field are presented. Finally,
Solaris pilot plants in South Africa are discussed from a sustainable economy point of view.
Chapter 7 summarizes the main aspects of the fermentative methods and its application to
the biomolecules’ production. Traditionally, most enzyme production was implemented by
submerged fermentation (SmF); however, in the from 1990 up to 2015, an increasing trend
toward the use of the solid state fermentation (SSF) started to figure in the enzymes and
other microbial metabolites production. Title comparisons between SSF and SmF processes
must be done under the same fermentation and analytical parameters, and care should be taken
in comparing the same enzymatic units. There exist advantages and disadvantages in both
processes; however, the best process will be dictated by means of availability in infrastruc-
ture, substrates, and strain, and also to be considered is the downstream process. Then, it
shows how it is not a matter of a “better that” statement; it is rather what can be achieved
with the available chain of recourses.
In Chapter 8, plant cell walls are described as the most abundant renewable source of energy
of the earth. For thousands of years, their use mainly concerned animal feed and heat
power, as well as large employ in the paper industry. This abundant resource is today consid-
ered a promising source of energy that can be converted into biofuels for transport and
products for electricity and heat production, as well as biogas. Pathways in the biosynthesis
of fibers, sugars, and metabolites are presented with the focus on the life cycle of plant species.
xxxvii
Introduction
Cellular structures and organization of cell walls are analyzed to show polysaccharides
favoring fermentative processes. Cellulose, the main component of the plant cell wall, is
embedded in a matrix of other polysaccharides, and together they form the skeleton of the
plant tissue. This structure is naturally recalcitrant to the deconstruction carried out by
microbes and enzymes; therefore, breakthrough technologies are needed to overcome barriers
to developing cost-effective processes for the conversion of biomass into fuels and chemicals.
For all these reasons, the interest to deeply understand cell wall structure and composition of
plants is becoming even more essential. The progress made in understanding plant cell wall
biosynthesis and remodeling and the biotechnological approaches to reducing biomass
recalcitrance are here described.
In Chapter 9, the biotechnological production of biobased dicarboxylic acids is introduced as
a hot topic in industrial biotechnology, with many investments involved in the development,
piloting, and validation at demonstration scale of diverse processes using renewable raw
materials. This chapter will review the main markets and applications of some of the most
important dicarboxylic acids, and will briefly present their current chemical and biotechno-
logical production processes. Then, the chapter will mainly focus on the particular case of
succinic acid, and the microbial platforms that have been proposed will be reviewed, with
emphasis on yeast strains. Next, the basic requirements for setting up and scaling the
bioprocess and the required purification strategy to obtain an economically feasible process
yielding a product meeting the required specifications will be presented. Then, the specific
challenges of using very low-cost raw materials, such as agroindustrial residues, will be
highlighted.
Chapter 10 describes the problem of industrial exploitation of fermentation products,
introducing the markets for microbial enzymes. Enzyme production and stabilization
methods, with a focus on the techniques used, such as liposome encapsulation, are presented.
Industrial applications are described, such as proteases and lipases for laundry and dish
powder applications, and use of immobilized enzymes in food industry, hygiene products,
cosmetics, and medical and pharmaceutical applications.
Chapter 11 discusses the development and selection of sustainable waste management
solutions, as global waste production rates are expected to continue rising. The chapter
focuses on environmental sustainability considerations for organic waste management,
comparing the principles of the waste hierarchy to results obtained in comparative case
studies following the LCA method, and reviewing the competitiveness of existing organic
waste valorization systems. It can be concluded that the waste hierarchy is not sufficient for
decision-making in the field of organic waste valorization, and that LCA studies are needed in
order to avoid misconceptions. Furthermore, the referenced studies show that certain bio-
waste-based products are capable of competing with conventional processes, but these results
cannot be generalized and should be evaluated on a case-by-case basis, as seemingly small
differences can drastically change environmental impacts.
xxxviii
Introduction
Chapter 12 presents an overview on transgenesis, cisgenesis, and alternative and innovative

methods of genetic modification to obtain novel plant products, as well as nongenetically
modified methods based on mutagenesis. Genetically modified organism studies and genetic
improvements using genome editing are discussed, as well as novel techniques to achieve new
varieties and organisms for novel foods and feed applications. Finally, the various assessment
regulations around the world are discussed, as well as the need for harmonization among
different states. Novel plant products, in many cases, could go through a simplified authoriza-
tion process due to the assimilation to traditional breeding and mutagenesis techniques.
Acknowledgment
The research carried on in the project “TransBio: Bio-Transformation of by-products from
fruits and vegetables processing industry into valuable Bioproducts” has received funding
from the European Union’s Seventh Framework Program (FP7/2007–2013) under grant
agreement n° 289603.
xxxix

CHAPTE R 1
Crop and Plant Biomass as Valuable

Material for BBB. Alternatives for
Valorization of Green Wastes
T. Dietrich, M.C. Villaran Velasco
Tecnalia, Miñano (Alava), Spain
P.J. Echeverría
Tratamiento Subproductos Agroalimentarios S.L., San Adrián (Navarra), Spain
B. Pop
TRITECC SRL, Cluj Napoca, Romania
A. Rusu
Biozoon GmbH, Bremerhaven, Germany
Abbreviations
DM Dry matter
EU27 European Union (27 member States) Belgium (BE), Denmark (DK), France (FR), Germany (DE),
Greece (EL), Ireland (IE), Italy (IT), Luxembourg (LU), Netherlands (NL), Portugal (PT), Spain (ES), United
Kingdom (UK), Austria (AT), Finland (FI), Sweden (SE), Cyprus (CY), Czech Republic (CZ), Estonia (EE),
Hungary (HU), Latvia (LV), Lithuania (LT), Malta (MT), Poland (PL), Slovakia (SK), Slovenia (SI), Bulgaria
(BG) and Romania (RO)
EU15 European Union (15 member states) Belgium (BE), Denmark (DK), France (FR), Germany (DE), Greece
(EL), Ireland (IE), Italy (IT), Luxembourg (LU), Netherlands (NL), Portugal (PT), Spain (ES), United
Kingdom (UK), Austria (AT), Finland (FI) and Sweden (SE)
FVW Fruit and vegetable waste
SFE Supercritical fluid extraction
1.1 Introduction
Until about 15 years ago, bulk agricultural raw materials such as wheat, rice, and corn have
been continuously low in price because of increasing agricultural yields. This tendency has
drastically changed with competition between biomass for food versus biomaterials, chemi-
cals, or biofuels becoming a societal issue (Vandamme, 2009). Despite increasing world
production and yields, world prices are rising. Fig. 1.1 shows the world production increase
of wheat, coarse grain, and rice versus price changes.
Biotransformation of Agricultural Waste and By-Products. http://dx.doi.org/10.1016/B978-0-12-803622-8.00001-X
Copyright © 2016 Elsevier Inc. All rights reserved.
1
2 Chapter 1
Figure 1.1
World production and world price indices (value 2000 = 100) (Database FAO/OECD Agricultural
Outlook 2014–2023).
Currently, around 12% of the global land surface is used for crop cultivation (Foley et al.,
2005; Ramankutty et al., 2008). In order to stay in the planetary boundaries, Rockström et al.
(2009) propose that no more than 15% of the global ice-free land surface should be converted
to cropland. Especially in EU27, more than 40% of available lands were utilized as agricul-
tural area (Eurostat, 2015). Therefore, further increase of agricultural area is not an option, as
transformation of forests, grasslands, wetlands, and other vegetation types to agricultural land
is the driving force behind the serious reductions in biodiversity and impacts in water flows as
well as biogeochemical cycling of carbon, nitrogen, phosphorous, and other important
elements. Nitrogen and phosphorus are both essential elements for plant growth, so fertilizer
production and application is the main concern (Rockström et al., 2009). Besides others,
anthropogenic perturbation levels of land-system change and biochemical flows exceed
already proposed planetary boundaries (Steffen et al., 2015). Therefore, new alternatives must
be identified to ensure sufficient food supply as well as sustainable production of biomateri-
als, biochemicals, or biofuels. New approaches must consider the use of all available biomass
resources.
Crop and Plant Biomass as Valuable Material for BBB 3
1.2 Plant Biomass as Feedstock for Industrial Applications:

General Considerations
Significant amounts of agricultural residues and by-products are generated from agricultural
crop production and food processing such as straw, corncobs, corn stover, or cutoffs from
fruit and vegetable processing (Figs. 1.2–1.4).
These residues and by-products are rich in lignocellulosic materials or other value-added
compounds, which can be valorized via physical, chemical, and biotechnological treatments.
Most of the crop residues are available in the countries with a large agricultural sector and
high agricultural production as well as food processing industry. Scarlet et al. (2010) calcu-
lated that the total amount of crop residues produced in EU27 is estimated at 258 million dry
Figure 1.2
Asparagus waste.
Figure 1.3
Garlic residues.
4 Chapter 1
Figure 1.4
Green bean by-products.
ton per year (sunflower, wheat, rye, rapeseed, rice, maize, oats, barley). Of these residues,
approximately 109 million dry ton per year could be collected and used as feedstock for other
applications, if environmental and harvesting constraints are taken into account. Furthermore,
fruit and vegetable waste should be considered as valuable feedstock, as FVW are produced
in large quantities and are available throughout the world. According to FAO (2011), around
45 percent of fruit and vegetable biomass is wasted during agriculture, postharvest, process-
ing, distribution, and consumption. The organized sector of fruit and vegetable processing,
packaging, distribution, and consumption in India, the Philippines, China, and the United
States of America generate for example a total of approximately 55 million tons of FVW.
A large proportion of these wastes are dumped in landfills or rivers, causing environmental
hazards (FAO, 2013). Table 1.1 is showing total vegetable waste amount in EU28 and key
producing countries for agricultural production and manufacture of food products.
Nevertheless, general statistics are not sufficient in analyzing the potential of crop and plant
biomass resources as feedstock for valuable applications, as the use requires accurate data on
their availability by crop and biomass type, but also on their local and annual variability. The
Table 1.1: Vegetable Waste Amount in 2012 (Eurostat, 2015).
Vegetable Waste from Manufacture

Vegetable Waste from Agriculture, of Food Products, Beverages &
Forestry, and Fishing (t/year) Tobacco Products (t/year)
EU28 6,600,000 15,670,000
Italy 49,950 2,903,042
Poland 388,635 1,430,485
France 2693 1,241,609
Spain 653,604 723,994
Germany 77,765 565,365
Romania 3,192,623 346,825
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"I am so grieved, Norman. You have incurred a risk for my sake, and I fear we shall have
trouble," I said. Tears came to my eyes, for I felt it would be too dreadful to have our
happiness clouded over by this freak of my dear but rash brother.
"No crying, Berty. You attach too much importance to such a trifling matter. I tell you I will
speak to Frith."
There was no help for it. I could only hope for the best, and the sight of Stephen's
beaming face, as he leaped from the train, made me for the moment forget all but present
happiness.
"You two will like to walk to the Court," said Norman, after the first hand-shakings and
congratulations were over. "Parks will see to the luggage, and I want a word with the
station-master."
Stephen and I gladly obeyed, and left the station together. Norman turned to Frith, and in
his frank way, said—
"I owe you an apology for having meddled with the mail-bag. I was wrong, and you were
right; shake hands and receive my pledge never to repeat the offence. Here is the letter I
took. Examine the date—the contents, if you like-and satisfy yourself that I took only my
own property."
Norman's apology, his friendly manner, and bright, happy face, drove away all bitter
feeling from Frith's mind. He would have liked to grasp the hand so frankly offered, but
held back, knowing what he had written to head-quarters about the raid on the letter-bag.
"Surely you bear no malice, Frith," said Norman. "I own I was wrong, and I think all the
more of you for fearlessly doing your duty and rebuking me."
"It is not that, sir. I had a duty to perform—"
"Yes, yes; and you did it. No need for another word."
"But you are not, perhaps, aware that I felt compelled to report what had passed. My
letter must have reached head-quarters by this time, and there will be an inquiry."
"Do you think the knowledge that you have reported me makes me less anxious to shake
hands with you, Frith? Not a bit of it."
Again Norman extended his hand, and Frith grasped it, feeling the while woefully
concerned at the trouble which was hanging over the kindhearted but impetuous young
squire.
"There, now; that is done with, and I do not mind telling you why I was so eager to get
hold of the letter, to which I so improperly helped myself this morning. You are a young
man, and have just won a good and pretty wife, so you will be pleased to know that we
shall soon have a double wedding at the Court. At Overford we are very clannish—
something like one great family, and we talk over probable social changes very freely,
without troubling ourselves about little differences in means and position. You are not
Overford born, but you can claim affinity through your wife, and I am sure will sympathize
with us in our happy prospects."
Poor Frith looked rueful enough as he stammered out his congratulations, then added, "I
have something to confess and to regret. I was very angry when I wrote that report."
"And did not try to soften matters. I am hasty myself, and can quite understand how you
felt when I defied you. What shall I get, Frith—a stern reprimand, or have to pay heavily in
coin of the realm?"
"Neither, sir, I am afraid. I hope the actual penalty incurred may not be enforced."
"Well, good night; I must await my punishment with what patience I may. I hope it may be
a sentence to matrimony in a fortnight instead of a month."
And Norman started homeward feeling as light-hearted and happy as possible.
Stephen and I were eagerly waiting to tell him our news, for my beloved had distanced all
competitors, and every one foretold a brilliant professional future for him. After these more
interesting details had been gone through, I asked if Norman had made all smooth with
Frith.
"To be sure I have; and now your affair is over, Stephen, I shall not be tempted to break
my word by again meddling with Her Majesty's mail-bags."
"I do not understand the allusion," said Stephen. "Berty has been worrying herself about
something: I can hardly tell what. Let me have your version."
Norman coloured and looked a little ashamed, but told the story without sparing himself.
I spoke indignantly as he ended—"It was too bad of Frith to write straight off about such a
ridiculous affair. He might have waited for Norman to explain."
Stephen looked, as I thought, needlessly grave.
"Frith was undoubtedly right," he said. "There could be no explanation, dear. I should have
done as he did."
I took alarm, for Stephen's face was always eloquent, and I was sure he anticipated
coming trouble; but Norman interposed before I could ask any further questions.
"No gloomy retrospections—no evil forebodings. Frith and I are good friends, and future
misunderstandings impossible. Now for a pleasanter subject. We have fixed the week for
the weddings—what about the day?"
Stephen promptly suggested Monday, but was snubbed and silenced by me, though I knew
he and I must sail for the East very soon after our marriage. Finally, Thursday was agreed
upon, subject to Lucy's sanction.
We three lingered until a rather late hour, but after I had retired, Norman asked Stephen
why he looked so grave on hearing his story about the affair with the mail-bag.
"You are learned in the law. Tell me what my freak will cost, Stephen?"
"I am afraid it is not a matter of money," replied Stephen; "but, you know, I have had no
actual experience of exactly such a case."
His unwillingness to answer directly made Norman uneasy, and, laying his hand on that of
his friend, he asked—
"Is it a matter of imprisonment? Surely I shall not have to pay so dearly for what was only
a piece of foolish bravado! But let me know the worst that can happen."
Stephen told him in two words—"Seven years."
Poor Norman! He was utterly unprepared for such a response, and felt certain that Steve
was wrong. At the worst, he had only anticipated a heavy fine or a sharp reprimand, and
he was stunned by the words. The colour forsook his face, and he dropped back on the
seat from which he had risen, utterly overcome, gasping out, "You must be mistaken."
"I wish I were," said Stephen; "but I do happen to know the exact law in such a case, for I
was concerned in one a few months back. There was, however, this difference between it
and yours. The man who opened the mail-bag did it to obtain a letter not intended for him,
and the contents of which enabled him to carry out successfully a plan for a gigantic
robbery. But had the plot failed, the abstraction of the letter alone rendered him liable to
seven years' imprisonment."
"There must be mitigations," said Norman.
"Unfortunately, there are none. The law has long been in existence, and has remained
unaltered to the present day. The penalty for such an offence is seven years or nothing."
Norman did not speak in reply, but made a rush to the library, and began to rummage
amongst a collection of law books, for the Savells had been on the Commission for
generations past. After some time, he found what he wanted, and it confirmed the opinion
expressed by Stephen. He laid down the book in dumb despair, and seemed to age visibly
before Steve's pitying eyes, and scarcely to heed the more hopeful words he strove to
utter.
Stephen, indeed, did not feel hopeful, so, naturally, his words had not a very genuine ring
with them. He knew, as Norman might have done, how important it is to prevent the mails
from being tampered with. Every subject of the Queen has an interest in their safe
keeping, and every loyal subject should be their protector. So, with these thoughts in his
mind, no wonder his words carried little consolation to Norman's.
He, poor fellow, spent an almost sleepless night, and came down in the morning looking
haggard and unrefreshed. He implored Stephen not to tell either me or Lucy what was
hanging over him, and awaited as best he might the result of his rashness.
It was hard to preserve a calm face and go into all the details in connection with wedding
festivities without betraying his dread that his plans might never be carried out. But he
went through his task with the courage of a martyr, and, after a long wearying day, lay
down to dream that he was a prisoner in a felon's dress, and hopelessly severed from Lucy
and the home of which she was to have been the mistress.
CHAPTER V.
THE Savell Arms, as the one inn at Overford was named, stood near the station and within
sight of the Court, and furnished fitting accommodation for those who could be contented
with exquisite cleanliness and simple country dainties, served by a village maiden instead
of a town-bred waiter.
Two days after the unfortunate affair with the mail-bag, a stranger arrived at Overford,
and sought accommodation at The Arms, guided thither by the station-master, who
doubted whether the little inn would furnish a fitting shelter for so great a personage as
one of Her Majesty's Post Office inspectors. But the new-comer was charmed with its
appearance, so different from the huge wilderness in which he was often obliged to
sojourn, and said the quiet would be delicious.
He had come down, first and foremost, about this mail-bag business, but he had other
places to inspect, and would make Overford the centre, as trains were fairly convenient for
visiting them.
After due rest and refreshment, the inspector held conference with Frith.
"This is an unpleasant affair," he said, "but you have acted most creditably, especially
considering the position of the offender. Your King's Court squire seems to regard himself
as above all law, but he will have to learn a new lesson."
Frith looked as miserable as though the compliment just paid him had been a threat of
dismissal, and replied, "The matter looks worse on paper than it was. Mr. Savell would not
have taken such liberties but for Mr. Joynson, who was here before me."
And Frith, with the permission of his superior, told the whole story, softening matters as far
as possible, and making excuses for Norman.
Mr. Fisher, the inspector, listened attentively, but, instead of making an immediate reply, he
read over Frith's written statement. For a moment, the thought came into his mind that
Frith had been bribed during the interval, to make the case seem better than it was, in
order to save the offender. He looked keenly into Frith's face, in which he noted an
expression of truthfulness and honesty, which contradicted the suspicion; but he
remarked, "I find a great difference between the tone of your report and your verbal
account."
The young man's face flushed, but he met the keen glance with honest eyes and words—
"I was angry when I wrote, and I am afraid I was thinking more about Mr. Savell having
set me at defiance than of his offence against the law. I am not angry now, and though I
can only tell the truth, I can do so without passion, and would gladly undo the effect of my
first harsh words."
"What has caused this change of feeling?"
"After I had sent off the letter, I began to think of Mr. Savell's many excellences. He is a
capital landlord, a true friend to the poor, a kind brother, and he ought to be married in a
month to one of the sweetest girls in the world. He has been naturally accustomed to think
a great deal of himself, as the largest landowner of the neighbourhood, and old Joynson
had let him do as he liked with the mail-bags, and never said him nay. There are many
things to be said in excuse for Mr. Savell."
"Were not all these things equally true when you wrote your report of the affair?"
"Yes, sir, but I was in a passion. A man does not like to be defied when doing his duty. I
should have reported my own brother, or the Prince of Wales, I hope, if he had done what
Mr. Savell did. All I wish is that I had written in a better and kinder spirit, instead of
making the worst of things. That same evening, Mr. Savell came and apologized in the
frankest way, and brought the letter to show me. He has no idea, I believe, of what he has
laid himself open to, but he was sorry for having insulted me when I was doing my duty.
Besides, he owned he had set a bad example, when his very position should have made
him anxious to set a good one. Must you take any proceedings, sir?" asked Frith, after a
brief pause, during which Mr. Fisher was evidently thinking things over.
"I must indeed, Frith. The mail-bags are trusted to the honour of the people, as it were,
tied only with a string and secured by a seal which a child's hand could loose and break.
They are flung down on the platforms as if they were of little importance, yet think what
sacred deposits they enclose. Everybody is interested in their safety, and the law, while
trusting so much, imposes a heavy penalty on any violation of the trust. An instance of the
kind is rare indeed. Now, if we were to gloss over this offence of Mr. Savell, what would the
world say?"
"I suppose they would say that a rich man was allowed to do what a poor man was not;
that a labourer who meddled with the mails would be marched straight to gaol, whilst the
squire went scot free," replied Frith, ruefully. "But, oh dear! What will become of Miss
Pemberton and Miss Savell? It will break their hearts!" And the young man groaned
audibly.
A brief question or two, and Frith became loquacious enough, and Mr. Fisher heard all
about the intended marriages, the charms of the brides-elect, and the learning of Stephen
Hastings, who was to fill some high position abroad, he had been told.
The young man used all his eloquence, but his story came to an end at length; and in spite
of it, Mr. Fisher found himself compelled to apply for a summons against Norman Savell, to
appear and answer before the magistrates for the offence he had committed.
The application caused no small commotion in the mind of the gentleman who received it.
There was no magistrate within a wide radius who was not personally acquainted with my
brother, and the lawyer to whom Mr. Fisher wished to entrust the conduct of the case flatly
refused it. He was Norman's own agent and legal adviser.
The magistrate could not refuse to grant a summons, but owned that he did it with
extreme reluctance.
"Let me see," he said, "this is Saturday. We meet on Monday, and I dare pledge myself for
Mr. Savell's presence to answer any charge against him. And, Mr. Fisher, you are alone in
this neighbourhood. Will you, by way of passing the time, dine with me to-morrow? My
carriage shall fetch you from the inn, in time for church, and take you back in the evening,
unless you would prefer taking a bed here to be ready for Monday morning."
Mr. Fisher thanked the speaker, but declined the invitation.
"No doubt you are right, but," here the worthy magistrate lowered his voice, "you must let
me say a word for my friend Savell. A worthy young man. Impetuous, if you like, but
incapable of anything dishonourable. I am as sure as though I had seen it, that the letter
he took was his own."
"I have no doubt of it," was the reply. "I can feel no prejudice against Mr. Savell, of whom I
hear so much that is excellent; but I have an official duty to perform, though I do it with
regret."
"Of course. You can have no wish to be hard, and no doubt you will do what you can,
consistently with duty, to soften matters. We magistrates can only administer the laws, we
cannot alter them but there are such things as recommendations on behalf of an unwitting
offender. Sometimes, a case need not be pressed. The justice of the law can be tempered
with mercy, and you, Mr. Fisher, can recommend leniency in dealing with this case. Good
day, and I shall hope to meet you under more agreeable circumstances."
Mr. Fisher felt his hand grasped by the speaker, who subsequently congratulated himself
that he had put in an effectual plea for Norman. Indeed: he told me afterwards of his
efforts on my brother's behalf, and assured me that he had been enabled to render him a
signal service without in the least compromising his own dignity or that of the bench.
CHAPTER VI.
MR. FISHER attended Overlord Church on the following morning, and had no difficulty in
recognising Norman and myself. The likeness between us betrayed the relationship, and he
guessed that the third occupant of the pew must be Stephen. In his face, too, Mr. Fisher
saw a reflection of an old friend's. He knew afterwards that Sir Vernon Hastings, Stephen's
eldest brother, and he were old friends and had been schoolfellows.
Norman's face had an anxious expression that morning which neither Lucy nor I could
account for.
Stephen was in the secret, for he had informed my brother as the two men were strolling
in the park together that a warrant for his arrest was certain to be issued. They had
agreed to say nothing to Lucy or myself until the secret could be kept no longer.
"No use to give them needless anxiety. If the worst comes they will know soon enough,
without meeting trouble on the way," said Norman and Stephen assented.
But who can keep a secret in a village where events are few and eyes and ears always
open? Norman and Stephen might keep their counsel; but, in spite of their reticence, a
rumour got abroad that the young squire was going to be tried for his life for breaking
open the mail-bag.
As I was preparing for dinner on Sunday afternoon, I was struck by the combined
dolefulness and mystery observable in my attendant's manner. An inquiry on my part
brought a burst of tears from Ellen.
"Are you ill, or have you been quarrelling With Tom?" I asked; for the girl, a pretty, modest
creature, was engaged to a farmer's son in the neighbourhood.
"Oh dear no, Miss Berty. We never quarrel, and I am quite well."
"Then tell me what is amiss. You have only to, speak, if I can do anything to remove the
trouble."
"It is nothing about myself, miss. It is the young squire."
"My brother!" I exclaimed. "What do you mean? He was well an hour ago. What has
happened since then?"
Glancing towards the mirror, I caught a glimpse, of my face, from which every trace of
colour had fled, and Ellen, frightened at the effect of her words, said—
"You must not be alarmed, miss. Mr. Savell is all right in himself, only it is the trouble
about the mail-bag that is on all our minds. They do say in the kitchen that it is a hanging
matter for anybody, if he was a duke, to break the seal of one. Anyway, we all know that
the squire is to be brought before the magistrates, and tried for it to-morrow morning."
"Nonsense, Ellen! People are not hanged for trifles nowadays. As to trying my brother, you
may be sure he knows nothing about it, or I should have heard also. There was a little
dispute with Frith at the station, but Mr. Savell made all right, and there the matter ended.
You are a good, tender-hearted girl; but there is nothing to grieve about, or I should be
crying too," said I, smiling at my little maid.
"But, ma'am—Miss Berty, I know for a fact that Frith wrote to the Queen, or somebody
very great, and a gentleman was sent from London to have the young master tried. And
Parks saw a policeman from town show a paper to the squire, and then the two walked
away together."
"You are wrong, Ellen. My brother would have told me, were there any ground for such a
report."
"Indeed I am not mistaken," said Ellen. "I wish I were. The London gentleman was at
church this morning; you must have seen him yourself, Miss Berty."
"There was a stranger, a fine, tall man, and he sat—"
"In Tom's father's pew," interposed Ellen, eagerly, and forgetting her good manners. "He is
staying at The Arms, and Frith has been backwards and forwards to see him there. Frith is
cut up enough that he will have to give evidence against the squire, though he was vexed
with him at first. But, dear, dear! There goes the five minutes' bell, and you are not ready
for dinner."
My attendant had almost suspended operations during the conversation, for though her
tongue went fast enough her hands were idle, and I was a too deeply interested listener to
notice the lapse of time.
I hastened to make up for lost minutes, and went downstairs with a troubled face, and
little appetite for the coming meal.
A glance at Norman's face confirmed my fears as to the truth of what I had heard. He was
evidently ill at ease, and though Stephen tried hard to keep up a cheerful conversation, he
failed ignominiously. He could not feel indifferent as to the result of Norman's escapade, or
ignore my brother's evident depression.
When the servants had left the room, Norman made an excuse for his dulness. "I have a
stupid headache," he began; but I interrupted him.
"Heartache, you mean, Norman. Why did you try to hide it, dear, instead of letting Lucy
and me share your troubles, whether small or great? It is useless to try and keep secrets
in a place like this. The whole village is in a state of excitement about your coming 'trial.'
And though it is not quite a 'hanging matter,' as reported, it is serious enough to cloud my
dear brother's brow and make him anxious."
My allusion to the "extreme penalty of the law" did good service by giving a ludicrous
aspect to the affair, and both Norman and Steve burst into a hearty laugh.
"No, Berty, they surely do not think your brother will pay for his fault with his life! That is
quite too ridiculous," said Stephen, wiping mirthful moisture from his eyes. Another
prolonged laugh followed, and I was glad that I had repeated Ellen's absurd tale, since it
had chased the cloud from Norman's brow for the moment. When he could speak, he told
me all.
"I only wished to save you and Lucy from over-anxiety," he said. "You know, dear, I have
perfect confidence in you both."
"Not enough to understand that there is truer kindness in letting us share the trouble of
those we love than in hiding it, Norman," I replied, as I stood with my arm round his neck,
and his bonnie curly head resting on my shoulder.
Then we three resolutely strove to put aside our gloomy forebodings, and to talk as if
doubts and fears were strangers to our minds. If we had peeped just then into the
housekeeper's room, we should have seen the good dame and my waiting-damsel mingling
their tears as they sipped their tea together.
The elder woman was bemoaning Mr. Norman's rashness, which had brought disaster and
shame on King's Court.
"And only ten days off Christmas! What a contrast it will be this year to old happy
Christmases which I have spent here, seeing I came a girl in the time of Mr. Norman's
grandfather! Mourning instead of feasting and making everybody happy. To think I should
live to see it!"
The sound of that burst of laughter from the dining-room reached the tea-drinkers, but did
not prove infectious. The housekeeper shook her head and moaned audibly, whilst Ellen
remarked that it quite made her shudder to hear it. It was like a person laughing with a
rope round his neck!
The justices' room at Greystone was crowded on the Monday morning, and there was a full
attendance of magistrates. Everybody wanted "to hear Squire Savell tried," but, owing to
want of space, only a few were gratified.
The case was stated. The lawyer for the Crown expatiated on the gravity of the offence.
Frith's evidence was taken, and confirmed by one or two other very reluctant witnesses.
There could be no doubt that the squire had committed the offence with which he was
charged.
But Frith had to submit to cross-examination, and managed to say that he believed Mr.
Savell had no idea of the risk he was incurring, as he had been permitted to open the bags
by his own predecessor in office, Mr. Joynson.
"Was there ever a complaint that letters were lost at Overford?"
"Never in my time, or, I believe, before," said Frith.
"Did you remonstrate with Mr. Savell? And if so, how did he act?"
Frith looked distressed, but replied, "I did speak to him, but he laughed, and before I could
interpose, he again opened the bag and took out a letter. I was angry, and spoke sharply,
and, being annoyed, I wrote strongly too. I should like to say that I have no doubt my
predecessor's remissness encouraged Mr. Savell in the belief that there could be no harm
in his opening the bag and taking out his own letters."
I doubt if there was one individual present who wished that Norman might suffer for his
fault, beyond a fine or reprimand, and many approving looks were turned on Frith, for it
was known that the young squire had set his authority at naught.
Norman refused to employ his lawyer, saying he could tell a plain tale without help. When
called on for his defence, he frankly admitted his fault, and repeated his apology to Frith in
public.
"I can say nothing in excuse for my offence," he added, "beyond what the principal witness
has already suggested—namely, that I had been so long permitted to transgress that I no
longer realized that I was transgressing the law. I was in the wrong, however, and must
abide the consequences."
"Do you really wish to press this charge?" asked the presiding magistrate of the inspector.
"If so, we have no alternative but to commit, and we have ascertained how severe is the
penalty the law imposes."
"I have no present alternative," replied Mr. Fisher. "I am here in my official capacity, and
must report to my chief. If you remand Mr. Savell until, say, Thursday, I will report
progress, make a recommendation, and act according to further instructions."
The magistrate nodded intelligently. There was a brief consultation, at the close of which
Norman was remanded. There was a perfect rush of candidates eager "to bail the squire,"
and soon the audience dispersed, with the conviction that whatever else might result from
a second examination, the culprit would not suffer the extreme penalty of the law.
"I have seen that inspector with my eldest brother," said Steve, as he and Norman left the
court. "I will hunt him up and find out who he is."
"Not till after Thursday. People would say you were bringing outside influence to bear, were
you to claim acquaintance now. We will give no one a handle to lay hold on," said Norman;
and Stephen assented.
CHAPTER VII.
"WHAT are you doing here, you gipsies?"
Lucy Pemberton and I were hurrying to Greystone Station, hoping to get back to King's
Court without being seen by Norman and Stephen. They had ridden over early, not wishing
to go by train. We girls had chosen the latter mode of conveyance, and had reached
Greystone before them, though we started later. From a quiet corner, we had heard all that
had passed during the examination, and thought of arriving at home before the young
men, but were discovered on the road to the station.
"We had shopping to do, and thought we would come and complete it during your
absence," said I, boldly, whilst Lucy did not answer, but blushed quite guiltily.
"Where are your purchases?" demanded Stephen.

"Do ladies usually carry home their parcels?" asked Lucy, recovering her self-possession.
"Ours, no doubt, await us at the station."
"Confess, now. You spent five minutes in a shop, buying twopenny worth of Berlin wool,
and the rest of the time in the court-house. You could not stay quietly at home, dear,
loving hearts," said Norman, his face aglow with pleasure as he bent towards Lucy.
I answered, "If I am as anxious about my brother's well-being as he has always been to

bring me good tidings, surely I have no need to be ashamed."
"Right, dearest," whispered Stephen; and Norman asked, "And you, Lucy?"
"Do you think I could bear suspense better than Berty where you were concerned,
Norman?" was her answer; but she added, as if fearful of appearing less than quite candid,
"We really did some shopping here."
"The results will be found at the station, doubtless."
"Indeed they will, Norman; though we did not finish our buying to-day. So many things are
wanted for Christmas, you know."
"You will buy the rest on Thursday, will you not, Lucy?"
"Norman, how did you know?"
My brother's merry laugh disconcerted Lucy a little; but he became grave in a moment,
and said:
"My darling, do I know your tender nature so little as not to guess that if it were
impossible for you to remain at home to-day, it will be no less so on Thursday? Suppose,
now, the worst were to come, and I were actually sentenced to imprisonment, would you
wait for me, Lucy?"
Would she wait? Aye, a lifetime if need were; and though she did not say these words, her
look was enough for Norman.
At the station I pointed triumphantly to a couple of bales containing pieces of red flannel
for rheumatic old folk at Overford, striped shirting for working men, and sundry clothing
materials for bestowal on families where the children were out of proportion to the income.
Norman poked holes in the papers to verify my list, and I added triumphantly, "There are
blankets besides; but we shall not require your aid in seeing to our purchases, as they do
not go to King's Court under our personal charge. They will be delivered at the house."
We were complimented on our marketing, and a hope expressed that our shopping might
always be as expeditiously performed and to equally good purpose.
We bent in acknowledgment of the compliment, and then I was going to make some
allusion to what had passed during the examination before the justices; but Norman
stopped me.
"Berty, dear," he said, "we will say no more about the matter until Thursday. I have told
all, and no one credits me with anything worse than a rash act, of which I am now
ashamed. Let us be as happy as we can. Whatever comes, I shall have strength to bear
it."
He looked at Lucy, pressing more closely the little hand which rested confidingly in his, and
receiving a responsive pressure.
Look and clasp said more than words. They meant from him, "You are mine, whether in
prosperity or adversity." And hers replied, "Yours in life and till death."
CHAPTER VIII.
MR. FISHER was an object of great interest during the interval

between the magistrates' sittings. His landlady declared that
no pleasanter gentleman had ever stopped at The Arms. His
residence there brought almost too many customers, for
people were constantly dropping in to catechize the servants,
to the hindrance of work, but little to their own satisfaction.
On Wednesday, Frith was seen to carry a huge packet of

letters across to The Arms, and on the contents of one, the
biggest of all, it was supposed the squire's fate would depend.
Mr. Fisher's landlady was so eager to gather information, that

she waited on her guest herself, but could only report that he
had put the letters aside and gone on with his dinner, as
though they were of no consequence.
The Overford butcher, who was especially eager for news,

remarked, "The gentleman had such a steak for his dinner to-
day as few people get in a country place. And the mistress
here knows how a steak should be sent up. The gentleman
showed his sense by not letting it get cold. Letters will keep. I
thought that when I cut that steak, 'He shall have a good one.
A man is always kinder-disposed when his meat has been
tender and he has made a good dinner.'"
The butcher spoke like a village philosopher; but there may

have been an underlying reason for his anxiety for a happy
result to Norman. It might be that an opposite one would
affect the free-handed distribution of joints of beef for
Christmas dinners which he was accustomed to supply.
"I say that if the Post Office gentleman had been a king, he
could not have been better cared for or waited upon by a
handsomer landlady."
This compliment came from the schoolmaster, who was

suspected of writing poetry and of admiring the comely widow
who ruled The Arms.
"I suppose you never ventured to say a word for the squire?"
inquired a farmer.
"Oh dear, no. How could anything I might say help Mr. Savell?"
replied the landlady.
"Why not? One of my lads read out of a storybook how a

mouse helped a lion out of a net; and surely the missis here is
better than a mouse."
"You allude to a common fable, my friend," said the

schoolmaster, loftily.
"Fable or not, it teaches a good lesson," retorted the farmer;

"and it is the good things that get to be common, because
folks read them."
"You all know," interposed the landlady, "how gladly I should

speak, if I could help the squire. But if I dropped a word about
Mr. Norman being such a kind gentleman and landlord, Mr.
Fisher would give a little nod and turn to something else. So I
judged that he did not choose to be talked to about that
matter, and that I might do more harm than good if I went on.
Sometimes one may do more by holding one's tongue than by
speaking."
"You have learned a difficult lesson. It is a grand thing to be a

fair woman with discretion," replied the schoolmaster; and the
landlady, who was not wholly superior to compliments, felt
secretly gratified at receiving such from the best scholar in
Overford, setting aside the Court and the vicarage.
Thursday came at last, and brought a happy ending to all the

suspense and anxiety of the preceding days.
The solicitor who represented the Post Office authorities

stated that, being convinced that Mr. Savell had no felonious
intentions when he broke open the mail-bag, and seeing that
he had already publicly expressed his regret, they had given
him instructions, with the permission of the magistrates, to
withdraw the charge. Only Mr. Savell must pay all the
expenses.
The room was thronged as before, and the moment it was

understood that the squire would not be sent to prison, there
was a rush from within to let outsiders know the result. Then
came a burst of cheering for the magistrates, the squire, and
the inspector, led on by the butcher, who anticipated a more
liberal Christmas distribution than ever. He was not
disappointed.
Who can paint either the effects of joy or sorrow? Only can we
judge what others feel by looking into our own hearts, and by
the experience of our own past lives.
Before we left the court, Mr. Fisher came up to Stephen and

said, "Surely we have met before. Are you not a younger
brother of my old schoolfellow, Sir Vernon Hastings?"
Stephen clasped the offered hand, and told Mr. Fisher that he
recognised him at first sight, but would not claim
acquaintance with him, lest by doing so he should furnish food
for gossip.
"If what I hear is correct, the Overford folk will now have a
pleasanter topic to occupy them."
"Yes," replied Stephen, "we may now look forward to a double
wedding. We all owe you a debt of gratitude for the manner in
which you have used your influence on behalf of Norman."
"I am glad I could recommend the course that has been

adopted, because no one here could doubt Mr. Savell's
innocence of any evil design in what he did."
Stephen would fain have persuaded Mr. Fisher to come to

King's Court for Christmas, but this could not be. Duty called
him in another direction. He did come, though, for the 14th of
January, and met his friend Sir Vernon, and several other
members of the Hastings family, for "they were many."
It was a very happy gathering, with only one subject of regret

in connection with it. I, Bertha Savell, just transformed into
Mrs. Stephen Hastings, would have to say a long farewell to
my only brother and my old home. This thought would come,
and it made the only cloud amid so much brightness.
Sir Vernon alluded to it regretfully.
"Though Stephen has done so well, and gained a really

wonderful position to start with, I wish we could keep him and
his wife in England. I am new to the estates, and have but
little loose capital."
"Because you have spent what you had in helping on a troop

of younger brothers," said Stephen.
"Hush! Dear boy. Am I not in the father's place? I would not

have you without work to do, but if it could have been here
instead of in India!"
"Do not suppose that Bertha will be quite a dowerless bride,"

said Norman. "Uncle Bernard might declare that he would give
her no share in his wealth, but he knew in whose hands he
left her. Berty has always refused to talk of money, and Aunt
Bella has made no sign, but immediately after my uncle's
death, I set aside ten thousand pounds, and caused the
money to be invested in my sister's name. That is hers
absolutely."
I tried to interrupt Norman, but he insisted that this act of his

was the barest justice, and that he hoped Stephen would see
his way to give up the Indian appointment after all, for, with
our united means, we could make a fair start in England.
But Stephen had his share of pride, and insisted that he would
not permit my little dowry to be the staff on which he would
lean.
We had visited the quaint iron safe in the early morning, and I
had duly taken therefrom the jewels which made up Uncle
Bernard's wedding gift.
The memory of his goodness to Norman and me, and the

sight of his empty chair, caused the only tears I shed on that
happy day. I was admired in my finery, and Lucy was more
admired with no adornment save her own grace and beauty.
Tenants were feasted, costly gifts examined, kindly wishes

spoken, and prayers offered for a bright future, both for the
pair who were to remain at the Court and those whose home
was to be far-away from it.
There was one more ceremony to begone through.
Amidst all the stir we had nearly forgotten the sealed packet,
which was found to be addressed in Uncle Bernard's
handwriting, "To the husband of my niece, Bertha Savell, of
King's Court."
Stephen somewhat nervously broke the seals and displayed

the contents. They were a closely-written legal document, and
a letter addressed to Stephen Hastings, which explained the
whole, and ran as follows—
"MY DEAR STEPHEN,—Most likely, when you

read this, the hand that penned it will have lost
its cunning; but surely a man's last days will be
rendered the happier for having done something
to promote the prosperity of the young who are
to follow him."
"It has needed little penetration on my part to

discover your love for my niece, and your
worthiness of the regard which she gives you in
return. Therefore, I only need to glance a little
further into the future, and picture you united to
her, for better, for worse."
"I should like to help you; but I want you first

to help yourself, and should prefer your marrying
with the prospect of having your way to make,
except as regards the share of my means which I
am sure Norman will give to his sister."
"I know you are hoping to obtain a better

position to start with, by going to India, than you
could look for here, and I trust you will succeed,
because the effort and the success, if attained,
will be good for you. But stay in England, that the
two last of the Savells may not be parted."
"I know, too, that you will have the choice;

because, though I have left King's Court, clear of
all incumbrance, to Norman, I have bought a little
estate for you, dear Stephen—not for Berty.
There is a pretty house upon it, and it is within a
convenient distance of London,—where, I
presume, you will choose to practise."
"Secure of a modest income, you will be able to

turn your talents to account in the best manner."
"Messrs. Partington and Howe, my solicitors,

will furnish all particulars, in addition to what this
letter and accompanying document give you."
"And now, with a prayer for your lasting

happiness, I remain,"
"Sincerely and
affectionately yours,"
"
BERNARD SAVELL."
So Stephen and I stayed in England, and blessed the memory

of him who had been my more than father during his life, and
whose kindness had reached us even from beyond the grave.
We two are very happy still, and so are Norman and Lucy. I
think I may say with confidence that, though we wives are
now "into the forties," and much less slender than we were on
January 14, 1865, we and our husbands are, in each case,
more truly one than we were on the day we became so.
Our double wedding led to two more; for Mr. Fisher met his
fate in the person of Stephen's sister, Lilian, and the poetical
schoolmaster long since gave up his post to become landlord
of the Savell Arms, by marrying the landlady. And as
Christmas comes round, the old story of Norman's raid on Her
Majesty's mail-bag, and the trouble it caused, is still told, with
the trite remark, as a closing sentence, "All's well that ends
well."
As a very last word, I should like to add that, though names,
place and circumstances are necessarily altered, the main
facts of this little chronicle are absolutely true, and that some
one, whose real name I could easily give, was placed in
precisely the position above narrated through rashly meddling
with the mails.
ONLY A SERVANT.
CHAPTER I.
"DON'T fret so, Miss Joyce. It grieves me to see you, and

crying never yet cured heart ache. When things are at the
worst they mend; and if so, better days must be at hand for
you."
The speaker, Sarah Keene, was a homely-looking, rather hard-

featured woman of fifty, evidently a servant, for she was
busily engaged in ironing some dainty laces and muslin. But
while her features were rugged, they were expressive of good
sense, and full of affectionate sympathy.
The Miss Joyce to whom she spoke had just entered the
laundry and thrown herself on an old chair, where she was
weeping bitterly. She was a girl nearly twenty-one years of
age, above the middle height, slender and graceful, and with
one of those faces which attract, even when seen amongst
many with far greater pretensions to beauty.
Joyce's features were not faultless, or her complexion of
alabaster fairness, which last would be very unpleasant, were
it possible in a healthy girl. But her large, dark eyes were
richly fringed with long lashes, whilst her broad, clear brow
was framed by chestnut hair, which lay in soft, wavy masses
on her shapely head. The expression of her face, though sad,
was singularly sweet and winsome.
That Joyce Mirlees was a thorough lady could be told by a

glance, though her gown was of coarse, common black stuff,
and its scanty crape trimmings were of the poorest
description. It was unrelieved even by a simple linen collar.
Only a band of the crape edged the throat, and ornaments she
had none.
Yet the house in which the girl lived was the dwelling of
wealthy people. The great rooms teemed with costly furniture
and all the exquisite accessories which money could supply.
The grounds were extensive and tastefully laid out, the
stables were well-filled, and luxurious vehicles of many kinds
were at the command of the master and mistress of The
Chase, as the place was named.
In the drawing-room, three ladies were seated. They wore

mourning dresses, but these differed widely from the poor
garment which was thought good enough for Joyce Mirlees.
Everything that could make mourning rich, tasteful, and
handsome had been done to set off the portly person of Mrs.
Walter Evans and the slender figures of her two handsome
daughters. A few moments would, however, have shown to
any stranger that only the semblance of refinement existed in
Mrs. Evans. Wealth she had in abundance. She was the
daughter of a successful speculator and for her wealth alone
had Walter Evans sought her as his wife. He gained this, but
paid dearly for it.

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Biotransformation of Agricultural waste

and by-products : the food, feed, fibre,

The Political Economy of Agricultural and Food Policies

Food wastes and by-products : nutraceutical and health

Saving Food : Production, Supply Chain, Food Waste, and

Valorization of Agri-Food Wastes and By-Products:

Food Waste Management: Solving the Wicked Problem 1st

Handbook of Grape Processing By-Products: Sustainable

Sustainable food waste-to-energy systems Babbitt

Sustainability of the Food System: Sovereignty, Waste,

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD

For information on all Elsevier publications

Publisher: John Fedor

2.4 Physicochemical Composition and Biological Activity of Fruits of the Region�����30

4.3 Industrial Composting������������������������������������������������������������������������������������������78

5.5.3 Organosolv Pretreatment�������������������������������������������������������������������������������� 139

9.2 Selection and Improvement of Yeast Strains������������������������������������������������������243

11.4 Case Studies: Environmental Sustainability of Organic Waste

Figure 6.3 Scheme of production of biofuels and chemicals starting from

Figure 11.4 Relationship between life cycle inventory, midpoint indicators,

A4A Airlines for America

EPA Environmental Protection Agency

PBS Polybutylene succinate

Lilia Arely Prado Barragán is full-time Professor at the Universidad Autónoma

Diogo Figueira is downstream processing scientist at Biotrend, specializing in the development

laboratory and at pilot scale. He holds an MSc in Biotechnology (University of Aveiro,

Izaskun Marañon is researcher at Foundation TECNALIA Research & Innovation, Spain.

Nike Mortier holds a Master’s of Science in Bioscience Engineering with specialization in

Luis Víctor Rodríguez Durán is a biochemical engineer by the Universidad Autónoma de

Lasse Six holds a Master’s of Science in Bioscience Engineering with a specialization

Bikshandarkoil Sivaraj Srikanth is a Management Consultant/Facilitator with 17 years

been through management/business magazines publications (prepared articles/case studies),

Chapter 12 presents an overview on transgenesis, cisgenesis, and alternative and innovative

Crop and Plant Biomass as Valuable

1.2 Plant Biomass as Feedstock for Industrial Applications:

Table 1.1: Vegetable Waste Amount in 2012 (Eurostat, 2015).

Vegetable Waste from Manufacture

"It is not that, sir. I had a duty to perform—"

And Norman started homeward feeling as light-hearted and happy as possible.

Stephen looked, as I thought, needlessly grave.

"There must be mitigations," said Norman.

"What has caused this change of feeling?"

Mr. Fisher thanked the speaker, but declined the invitation.

"It is nothing about myself, miss. It is the young squire."

"There was a stranger, a fine, tall man, and he sat—"

"Was there ever a complaint that letters were lost at Overford?"

"Never in my time, or, I believe, before," said Frith.

"WHAT are you doing here, you gipsies?"

"Where are your purchases?" demanded Stephen.

I answered, "If I am as anxious about my brother's well-being as he has always been to

"The results will be found at the station, doubtless."

"Norman, how did you know?"

MR. FISHER was an object of great interest during the interval

On Wednesday, Frith was seen to carry a huge packet of

Mr. Fisher's landlady was so eager to gather information, that

The Overford butcher, who was especially eager for news,

The butcher spoke like a village philosopher; but there may

This compliment came from the schoolmaster, who was

"Why not? One of my lads read out of a storybook how a

"You allude to a common fable, my friend," said the

"Fable or not, it teaches a good lesson," retorted the farmer;

"You all know," interposed the landlady, "how gladly I should

"You have learned a difficult lesson. It is a grand thing to be a

2.4 Physicochemical Composition and Biological Activity of Fruits of the Region��30

4.3 Industrial Composting��78

5.5.3 Organosolv Pretreatment�� 139

9.2 Selection and Improvement of Yeast Strains��243

11.4 Case Studies: Environmental Sustainability of Organic Waste

Figure 6.3 Scheme of production of biofuels and chemicals starting from

Figure 11.4 Relationship between life cycle inventory, midpoint indicators,

1.2 Plant Biomass as Feedstock for Industrial Applications: