Analysis Dr. Hans Ferdinand

Modern Methods of Plant Analysis
Editors
H.F. Linskens, Nijmegen/Siena/Amherst
J.F. Jackson, Adelaide
Volume 20
Springer-Verlag Berlin Heidelberg GmbH

Volumes Already Published in this Series:
Volume 1: Cell Components
1985, ISBN 3-540-15822-7
Volume 2: Nuclear Magnetic Resonance
1986, ISBN 3-540-15910-X
Volume 3: Gas Chromatography/Mass Spectrometry
1986, ISBN 3-540-15911-8
Volume 4: Immunology in Plant Sciences
1986, ISBN 3-540-16842-7
Volume 5: High Performance Liquid Chromatography in Plant Sciences
1986, ISBN 3-540-17243-2
Volume 6: Wine Analysis
1988, ISBN 3-540-18819-3
Volume 7: Beer Analysis
1988, ISBN 3-540-18308-6
Volume 8: Analysis of Nonalcoholic Beverages
1988, ISBN 3-540-18820-7
Volume 9: Gases in Plant and Microbial Cells
1989, ISBN 3-540-18821-5
Volume 10: Plant Fibers
1989, ISBN 3-540-18822-3
Volume 11: Physical Methods in Plant Sciences
1990, ISBN 3-540-50332-3
Volume 12: Essential Oils and Waxes
1991, ISBN 3-540-51915-7
Volume 13: Plant Toxin Analysis
1992, ISBN 3-540-52328-6
Volume 14: Seed Analysis
1992, ISBN 3-540-52737-0
Volume 15: Alkaloids
1994, ISBN 3-540-52738-9
Volume 16: Vegetables and Vegetable Products
1994, ISBN 3-540-55843-8
Volume 17: Plant Cell Wall Analysis
1996, ISBN 3-540-59406-X
Volume 18: Fruit Analysis
1995, ISBN 3-540-59118-4
Volume 19: Plant Volatile Analysis
1997, ISBN 3-540-61589-X
Volume 20: Analysis of Plant Waste Materials
1999, ISBN 3-540-64669-8
Analysis of Plant
Waste Materials
Edited by
H.F. Linskens and J.F. Jackson
Contributors
P.J.S. Bain S. Baskaran N.S. Bolan M.S. Erich G. Fenton

P. First L.Y. Foo S.R. Gupta B. Hamilton M. Kennedy
D. List Y. Lu V. Malik R.H. Newman A. Robertson
F. Schur I.M. Sims J.e. Tarafdar S. Thiagarajan
With 44 Figures
Springer
Professor Dr. HANS FERDINAND LINSKENS
Goldberglein 7
D-91056 Erlangen, Germany
Professor Dr. JOHN F. JACKSON

Department of Viticulture, Encology and Horticulture
Waite Agricultural Research Institute
University of Adelaide
Glen Osmond, S.A. 5064
Australia
ISBN 978-3-642-08431-7 ISBN 978-3-662-03887-1 (eBook)

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Introduction
Modern Methods of Plant Analysis
When the handbook Modern Methods of Plant Analysis, was first introduced in
1954, the considerations were:
1. the dependence of scientific progress in biology on the improvement of existing

and the introduction of new methods;
2. the difficulty in finding many new analytical methods in specialized journals
which are normally not accessible to experimental plant biologists;
3. the fact that in the methods sections of papers the description of methods is
frequently so compact, or even sometimes so incomplete, that it is difficult to
reproduce experiments.
These considerations still stand today.
The series was highly successful, seven volumes appearing between 1956 and
1964. Since there is still today a demand for the old series, the publisher has
decided to resume publication of Modern Methods of Plant Analysis. It is hoped
that the New Series will be just as acceptable to those working in plant sciences and
related fields as the early volumes undoubtedly were. It is difficult to single out the
major reasons for the success of any publication, but we believe that the methods
published in the first series were up-to-date at the time and presented in a way that
made description, as applied to plant material, complete in itself with little need to
consult other publications.
Contribution authors have attempted to follow these guidelines in this New
Series of volumes.
Editorial
The earlier series of Modern Methods of Plant Analysis was initiated by Michel V.
Tracey, at that time in Rothamsted, later in Sydney, and by the late Karl Paech
(1910-1955), at that time at Tiibingen. The New Series will be edited by Paech's
successor H.F. Linskens (Nijmegen, the Netherlands) and John F. Jackson
(Adelaide, South Australia). As were the earlier editors, we are convinced "that
there is a real need for a collection of reliable up-to-date methods for plant analysis
VI Introduction
in large areas of applied biology ranging from agriculture and horticultural experi-
ment stations to pharmaceutical and technical institutes concerned with raw
material of plant origin".
The recent developments in the fields of plant biotechnology and genetic
engineering make it even more important for workers in the plant sciences to
become acquainted with the more sophisticated methods, which sometimes come
from biochemistry and biophysics, but which also have been developed in com-
mercial firms, pharmaceutical laboratories, non-university research institutes, and
medical establishments.
Concept of the Series
Many methods described in the biochemical, biophysical and medical literature

cannot be applied directly to plant material because of the special cell structure,
surrounded by a tough cell wall, and the general lack of knowledge of the specific
behaviour of plant raw material during extraction procedures. Therefore all
authors of this New Series have been chosen because of their special experience
with handling plant material, resulting in the adaptation of methods to problems
of plant metabolism.
Nevertheless, each particular material from a plant species may require some
modification of the described methods and usual techniques. The methods are
described critically, with hints as to their limitations. In general it will be possible
to adapt the methods described to the specific needs of the users of this series;
however, references have been made to the original papers and authors. During the
planning of this New Series the editors tried to ensure that the aims and general
layout of the contributions are within the general guidelines indicated above, but
in addition they tried not to interfere too much with the personal style of each
author.
There are several ways of classifying the methods used in modern plant analy-
sis. The first is according to the technological and instrumental progress made
over recent years. These aspects were taken into consideration in Volumes 1 to 5
and 11 of this series describing methods in a systematic way according to the basic
principles of the methods.
A second classification is according to the plant material that has to undergo
analysis. The specific application of the analytical methods is determined by the
special anatomical, physiological, and biochemical properties of the raw material
and the technology used in processing. This classification was used in Volumes 6
to 8, 10, 14, 16 and 18.
A third category is according to the classes of substances present in the plant
material and the subject of analytical methods. the latter was used for Volumes 9,
12, 13, IS, 17, and 19 of the series.
Introduction VII
Naturally, these three approaches to developments in analytical techniques for

plant materials cannot exclude some small overlap and repetition; but careful
selection of the authors of individual chapters, according to their expertise and
experience with the specific methodological technique, the group of substances to
be analyzed, or the plant material which is the subject of chemical and physical
analysis, guarantees that recent developments in analytical methodology are de-
scribed in an optimal way.
Volume Twenty - Analysis of Plant Waste Materials
In previous volumes we have dealt with many different areas under the general
plant analysis theme, many to do with analysis of plant products for today's
consumer society. A quick inspection of the volume list on the reverse of the first
page of this volume shows topics dealing with such products as wine, beer, non
alcoholic beverages, vegetables, vegetable products and fruit. It is fitting then that
we should deal now with analysis of plant wastes, some of these wastes being
byproducts from production for the consumer society, and some being natural
"wastes" such as leaf litter.
DOC or dissolved organic carbon and its measurement are the subject of the
first chapter in this book. Its an all important factor in dealing with plant waste
products, since DOC controls a large number of physical, chemical and biological
processes in soil and water bodies, e.g. the quality of water, the metabolism of
aquatic populations through oxygen depletion and many other processes. This
first chapter deals with the many ways of measuring DOC and the importance of
using a suitable method for the particular situation and source of material.
This theme of DOC in aqueous wastes is taken up and broadened in the second
chapter, which deals with the analysis of paper mill sludges and effluents not only
for DOC but also for nitrogenous compounds and trace elements, a very important
topic for the control of water quality downstream from paper producing sites.
Then follows a chapter dealing with the analysis of sewage from a brewery. The
preservation of the image of the brewery to the general public is at stake here. We
present a chapter which looks at the anaerobic treatment modification for sewage
disposal procedures involving an up flow anaerobic sludge blanket purification
plant incorporating post aeration which strips volatile (and smelly) sulphur com-
pounds from the effluent. The details of analytical methods required to control
such process are presented, the result being for the brewery a reduction in energy
consumption, a decrease in remaining sludge, the production ofbiogas (methane)
for heating, and improved odour control.
While beer and paper manufacture provide complex problems for waste and
its analysis, even the simple pressing of fruit to produce fruit juice can pose
problems and analysis is needed in dealing with the products and wastes. Thus
apple pomace, which is the press-cake resulting from the pressing of apples for
VIII Introduction
juice, can be used for various products and so its analysis is essential as many
factors can influence the composition of pomace. The stage of ripening of the
apple, the variety of apple used and juice extraction technique all have an effect,
and need to be controlled and monitored. All this is dealt with in a chapter on
analysis of apple pomace and its products.
Kiwifruit is known throughout the world for its attractive green fleshed
fruit, pioneered by New Zealand horticulturalists, and so a chapter is included
on the analysis of wastes generated by this industry - not only for rejected whole
fruit, but also for kiwifruit pomace left after kiwifruit juice manufacture. Novel
products can be generated from these wastes and so analysis of these are also
examined here.
Finally, we come to the topic of a "natural" plant waste, leaf litter and its
decomposition. The decomposition of leaf litter is an important soil biological
process regulating nutrient cycling and soil fertility, and it is no surprise that we
include two chapters on this topic. Thus we have a chapter on the analysis of tree
leaf decomposition in arid soils, and complete the volume with a final chapter
dealing with the measurement of leaf litter decomposition. This is a very compre-
hensive treatment of the subject, emphasising such methods as the litter bag or
litter basket methods for studying litter decomposition, and combination with
such analytical methods as gas chromatography, mass spectrometry and 13C-NMR.
Pulse labelling of 13C of plant material, so useful for in situ studies, is discussed,
while the chapter finishes with respirometric measurements, determination of
enzymatic activity and nitrogen mineralization rates.
Acknowledgments. The editors would like to express their gratitude to all con-
tributors for their efforts in keeping to production schedules, and to Dr. Dieter
Czeschlik and the staff of Springer-Verlag, especially Mr. Christiane Glier and Mr.
K.-H. Winter for their cooperation in preparing this and other volumes of the
series, Modern Methods of Plant Analysis.
Adelaide and Nijmegen/Siena, Northern Spring 1999. H.F. LINSKENS

J.F. JACKSON
Contents
Methods of Measurement of Dissolved Organic Carbon of Plant Origin

in Soils, Manures, Sludges and Stream Water
N.S. BOLAN, S. BASKARAN, and S. THIAGARAJAN .......................... .
Introduction ................................................. .
2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1 Soils, Sludges and Stream Water .............................. 6
2.2 Extraction of DOC .......................................... 6
2.3 Amendment of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.4 Effect of Drying ............................................ 7
2.5 Measurement of DOC ....................................... 7
2.5.1 Spectrophotometric Method ......................... ... 7
2.5.2 Wet Oxidation ........................................ 7
2.5.3 Dry Combustion ...................................... 8
2.6 Fractionation of DOC ....................................... 8
2.6.1 Molecular Weight Fractions ............................. 9
2.6.2 Sorption Chromatography .............................. 9
3 Results and Discussion ......................................... 10
3.1 Methods of Measurement of DOC ............................. 10
3.1.1 Spectrophotometric Method ............................ 10
3.1.2 Dry Combustion ...................................... 11
3.1.3 Wet Oxidation ........................................ 12
3.2 Molecular Weight Fractions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.3 Effect of Drying on DOC .................................... 14
3.4 Effect of pH on DOC ........................................ 14
4 Conclusions ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
References ...................................................... 16
Analysis of Papermill Waste Water Treatment Residuals

and Process Residues
M.S. ERICH and P. FIRST. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1 Introduction .................................................. 21
2 The Pulping Process and the Composition of Papermill Sludge ........ 22
3 Sampling and Chemical Characterization of Papermill Sludge ......... 23
x Contents
3.1 Sampling.................................................. 23
3.2 pH and Macronutrients ..................................... 24
3.3 Trace Elements ............................................ 25
4 Polychlorinated Dibenzo-p-dioxins and Dibenzofurans
in Papermill Sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.1 Chemistry and Toxicity of Polychlorinated Dibenzo-p-dioxins
and Dibenzofurans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.2 General Analytical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.3 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.4 Method Performance Tests .................................. 29
4.5 Sample Preparation and Analyte Extraction . . . . . . . . . . . . . . . . . . . . . 30
4.6 Cleanup of Sample Extracts .................................. 32
4.7 Identification and Quantification of Polychlorinated
Dibenzo-p-dioxins and Dibenzofurans by High Resolution
Gas Chromatography/High Resolution Mass Spectrometry. . . . . . . . . 34
4.8 Bioassays for Polychlorinated Dibenzo-p-dioxins
and Dibenzofurans Equivalents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
5 Conclusions and Future Perspectives .............................. 37
References ...................................................... 38
Analysis of Sewage from Anaerobic Purification of Effluent

from a Brewery
F. SCHUR........................................................ 41
1 Introduction .................................................. 41
2 Anaerobic Effluent Treatment ................................... 42
3 Methods of Analysis ............................................ 45
3.1 Sewage and Sewage Sludge. . . . . .. . . .. . . . . . . .. . ... .. . .. . .. .. .. 45
3.1.1 Sampling (EDI 1983) ................................... 45
3.1.2 Carbon (EDI 1983) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.1.3 Organic Nitrogen (EDI 1983) ............................ 48
3.1.4 Ammonia/Ammonium (EDI 1983) ....................... 49
3.1.5 Phosphorus (EDI 1983) ................................. 51
3.1.6 Sulphate (EDI 1983) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1.7 Sulphite (EDI 1983) .................................... 54
3.1.8 Sulphide (EDI 1983) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.2 Air and Biogas ............................................. 59
3.2.1 Hydrocarbons (VDI 1975) .............................. 59
3.2.2 Ammonia (VDI 1974) .................................. 61
3.2.3 Hydrogen Sulphide (VDI 1982) .......................... 64
3.2.4 Mercaptanes (Meier 1975) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4 Mass Balances (Schur et al. 1995, 1996) ............................ 67
5 Conclusions ................................................... 73
References ...................................................... 73
Contents XI
Apple Pomace and Products Derived from Apple Pomace:

Uses, Composition and Analysis
M. KENNEDY, D. LIST, Y. Lv, L.Y. Foo, R.H. NEWMAN, I.M. SIMS, P.J.S. BAIN,
B. HAMILTON, and G. FENTON ........................................ 75
1 Introduction .................................................. 75
2 What Use Is Apple Pomace? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3 The Goal of Apple Pomace Analysis ............................... 83
4 The Composition of Apple Pomace ............................... 84
5 Analytical Techniques .......................................... 102
5.1 Routine Methods of Analysis ................................. 102
5.2 Water Content/Dry Matter Content . . . . . . . . . . . . . . . . . . . . . . . . . . .. 102
5.3 Bulk Density. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 103
5.4 Crude Protein. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 104
5.5 Apple Pomace Buffering Capacity ............................. 105
5.6 Bioavailable Energy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 105
5.7 Polyphenol Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 106
5.8 Antioxidant Analysis ....................................... 107
5.9 NMR Analysis: A Novel Method of Characterising
Apple Pomace ............................................. 109
6 Discussion .................................................... 113
References ...................................................... 113
Kiwifruit Waste and Novel Products Made from Kiwifruit Waste:

Uses, Composition and Analysis
M. KENNEDY, D. LIST, Y. Lv, L.Y. Foo, A. ROBERTSON, R.H. NEWMAN,
and G. FENTON ................................................... 121
1 Introduction .................................................. 121

2 What Use Is Kiwifruit? .......................................... 121
3 The Composition of Kiwifruit .................................... 126
4 Analytical Techniques .......................................... 126
4.1 Dry Material. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 138
4.2 Enzyme Activity Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. l39
4.2.1 Protease ............................................. 140
4.2.2 Carbohydrate Modifying Enzymes ....................... 141
4.2.3 Oxido-Reductases ..................................... 141
4.3 Polyphenol Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 142
4.4 Antioxidant Analysis ........................................ 143
4.5 NMR Analysis ............................................. 144
5 Discussion .................................................... 147
References ...................................................... 147
XII Contents
Analysis of Tree Leaf Decomposition in Arid Soils

J.C. TARAFDAR .................................................... 153
1 Introduction ................................................. 153
2 Important Trees in Arid Soils .................................... 154
3 Methodological Approaches for the Decomposition Process. . . . . . . . . .. 154
3.1 The Perfusion Method ...................................... 154
3.1.1 Perfusion Apparatus of Lefroy et al. (1995)
and Its Components ................................... 155
3.1.2 Management of the Perfusion Apparatus .................. 158
3.1.3 CO 2 Measurement ..................................... 158
3.1.4 Nutrient Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 159
3.2 The Litterbag Technique .................................... 159
3.3 Tracer Technique. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 160
3.4 Measurement of Soil Respiration. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 161
4 Analysis of Leaf Material Before and After Decomposition ............ 161
4.1 Determination of Cell Wall Constituents in Leaf Samples ......... 161
4.1.1 Reagent and Apparatus ................................ 162
4.1.2 Procedures ........................................... 162
4.1.3 Filtration............................................. 163
4.1.4 Cleaning of Crucibles .................................. 163
4.2 Estimation of Crude Fibre (Acid Detergent Fibre ADF) ........... 164
4.2.1 Reagent and Apparatus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 164
4.2.2 Procedure ............................................ 164
4.3 Determination of Cellulose, Lignin and Insoluble Ash ............ 164
4.3.1 Reagents ............................................. 164
4.3.2 Procedure ............................................ 165
4.4 Determination of Cell Contents and Hemicellulose. . . . . . . . . . . . . .. 166
4.5 Direct Estimation of Cellulose, Hemicellulose and Lignin ......... 166
4.6 Fibre Degrading Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 166
4.6.1 Carboxymethyl Cellulose (Endo-l,4-~-Glucanase,
EC 3.2.1.4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 166
4.6.2 a-Amylase (1,4-a-D-Glucanohydrolase, EC 3.2.1.1) . . . . . . . . .. 168
4.6.3 Xylanase (1,4-~-Xylan Xylano Hydrolase; Endo-l,4-~-Xylanase,
EC 3.2.1.8) ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 169
4.6.4 ~-Glucosidase (~-D-Glucoside Glucohydrolase,
EC 3.2.1.21) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 169
4.6.5 a-Glucosidase (EC 3.2.1.21) ............................. 170
4.6.6 ~-Xylosidase (1,4-~-D Xylan Xylohydrolase: Exo-l,4-~-D
Xylosidase, EC 3.2.1.37) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 171
4.7 Protein-Degrading Enzymes ..................... ............ 171
4.7.1 Urease (Urea Amidohydrolase, EC 3.5.1.5) ................. 171
4.7.2 Proteases . . . . . . .. . . . .. .. . . .. . .. ... . . . . . .. . .. .. . .. . . . .. 172
4.7.3 Transaminases ........................................ 173
4.7.4 Glutamate Dehydrogenase (GDH)(L-Glutamate: NADP +
Oxidoreductase EC 1.4.1.4) .............................. 174
Contents XIII
4.8 Determination of Gross Energy (GE) .......................... 175

4.8.1 Principle ............................................. 175
4.8.2 Things Required ...................................... 176
4.8.3 Chemicals and Reagents ................................ 176
4.8.4 Procedure ............................................ 176
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 178
References ...................................................... 178
Measurement of Leaf Litter Decomposition

S.R. GUPTA and V. MALIK .......................................... 181
Introduction .................................................. 181
2 Plant Litter Sampling and Preparation ............................ 182
3 Characterization of Resource Quality of Litter ...................... 182
3.1 Physical Properties of Leaves ................................. 182
3.2 Chemical Composition of Litter .............................. 184
3.2.1 Soluble Carbohydrates and Amino Acids .................. 184
3.2.2 Analysis of Cellulose, Hemicellulose and Lignin ............ 185
3.2.3 Polyphenols .......................................... 186
3.2.4 Plant Nutrient Analysis ................................. 187
4 Lignocellulose Transformation ................................... 187
5 Methods for in Situ Litter Decomposition Rates . . . . . . . . . . . . . . . . . . . .. 188
5.1 The Litterbag Technique. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 188
5.2 Litter Basket Technique ..................................... 190
5.3 13C Nuclear Magnetic Resonance (NMR) . . . . . . . . . . . . . . . . . . . . . . .. 191
5.4 Tracer Techniques ......................................... 193
5.4.1 Tagging Technique .................................... 193
5.4.2 14C Technique ......................................... 193
5.4.3 l3C Stable Isotope Technique ............................ 193
6 Analysis of Decomposition Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 195
7 Laboratory Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 196
7.1 Respirometric Techniques ................................... 196
7.2 14C -C0 2 Evolution Rates .................................... 197
7.3 Measurement of Enzymatic Activity ........................... 198
7.4 Nitrogen Mineralization from Decomposing Litter ............... 198
8 Conclusions................................................... 201
References ...................................................... 201
Subject Index ................................................... 209

List of Contributors
BAIN, J.S., Natural Products Proce si 19, Industrial Research Ltd. P.o. Box 31-310,
Lower Hutt, New Zealand
BASKARAN, S., Research Fellow, RC for Sustainable Cotton Production, Department

of Agrl. Chemistry and Soil Science, The University of Sydney, Sydney, NSW
2006, Australia
BOLAN, N.S., Massey University, Palmerston North, New Zealand

ERICH, M. SUSAN, Department of Applied Ecology, 5722 Deering Hall, University of
Maine, Orono, Maine 04469-5722, USA
FENTON, G., Natural Products Processing, Industrial Research Ltd. P.O. Box 31-310,
FIRST, P., Abt Associates Inc., 55 Wheeler Street, Cambridge, MA 02138, USA
Foo, L.Y., Natural Products Processing, Industrial Research Ltd. P.O. Box 31-310,
GUPTA, S.R., Department of Botany, Kurukshetra University, Kurukshetra 136119,

Haryana India
HAMILTON, B., Natural Products Processing, Industrial Research Ltd. P.O. Box 31-
310, Lower Hutt, New Zealand
KENNEDY, MAX, Natural Products Processing, Industrial Research Ltd. P.O. Box 31-
LIST, D., Frucor Processors (NZ) Ltd., PO Box 45, Hastings, New Zealand
Lu, Y., Natural Products Processing, Industrial Research Ltd. P.O. Box 31-310,
MALIK, V., Department of Botany, Kurukshetra University, Kurukshetra 136119,

Haryana India
NEWMAN, R.H., Natural Products Processing, Industrial Research Ltd. P.O. Box 31-
ROBERTSON, A., Natural Products Processing, Industrial Research Ltd. P.O. Box 31-
XVI List of Contributors
SCHUR, FRITZ, Doemens e.V., D-82166 Grafelfing/Munich, Germany and Dr. Fritz
Schur & Partner KG, CH-4312 Magden, Switzerland
SIMS, LM., Natural Products Processing, Industrial Research Ltd. P.O. Box 31-310,
TARAFDAR, J.C., Central Arid Zone Research Institute, Jodhpur 342 003, Rajasthan,
India
THIAGARA)AN, S., University of Idaho, Mosco, USA
Methods of Measurement of Dissolved Organic
Carbon of Plant Origin in Soils, Manures,
Sludges and Stream Water 1
N.S. BOLAN, S. BASKARAN, and S. THIAGARAJAN
1 Introduction
For all practical purposes, dissolved organic carbon (DOC) or dissolved organic
matter (DOM) is defined operationally as the organic matter in solution that
passes a 0.45-flm filter (Thurman 1985). Some workers have used finer filter paper
(0.2flm) to separate DOC from colloidal materials which are not retained in 0.45-
flm filters (Buffle et al. 1982). In some literature the term water-soluble organic
matter (WSOM) has been used, which is the fraction of soil organic matter ex-
tracted with water or dilute salt solution that passes a 0.45-flm filter (Herbert et al.
1993).
DOC in streams and groundwater aquifers originates mainly from the
solubilization of soil organic matter accumulated through vegetation, and the
addition of biological waste materials (Tate and Meyer 1983). Addition of bio-
logical waste materials, such as poultry and animal manures and sewage sludges
increases the amount of DOC in soils, either by acting as a source of DOC or by
enhancing the solubilization of the soil organic matter (Baskaran et al. 1996).
Most biological waste materials of plant origin contain large amounts of DOC, and
the addition of certain organic manures, such as poultry manure, increases the pH
and thereby enhances the solubilization of soil organic matter (Schindler et al.
1992). DOC concentration is highly susceptible to changes induced by humans,
both directly to lakes and to other catchments, and indirectly through forest fire,
clear-cutting, wetland drainage, acidic precipitation, eutrophication and climate
change.
DOC controls a large number of important physical, chemical and biological
processes in soil and water bodies. The elevated concentrations of DOC in soils
and streams have significant effects on the quality of water and the metabolism of
aquatic populations (Boissier and Fontvieille 1993). The easily oxidisable com-
pounds in the DOC can act as chemical and biological oxygen demand com-
pounds, and thereby deplete the oxygen concentration in the aquifers (Jones 1992).
Davies-Colling and Vant (1987) have shown that DOC in fresh water lakes absorbs
light with certain wave lengths and thereby influences the plankton population.
DOC has been shown to act as a carbon source for soil organisms, and thereby
I Most of the data in this paper have already appeared in Communications in Soil Science and
Plant Analysis (1996) 27:2723-2737.
Modern Methods of Plant Analysis, Vol. 20

Analysis of Plant Waste Materials
Edited by H.-F. Linskens and J.F. Jackson
2 N.S. Bolan et al.
induces the reduction of nitrate (denitrification) in soils and effluents, resulting in

the release of greenhouse gases, such as nitrous oxide (N2 0) and nitric oxide (NO),
which are implicated in ozone depletion. It has been shown that organic pesticides
added to soil and aquifers are partitioned preferentially onto DOC, which can act
as a vehicle for the movement of pesticide residues to the groundwater (Ballard
1971). Chlorine in water reacts with DOC and forms carcinogenic compounds,
such as trihalomethanes (Oliver et al. 1983). The organic acids present in the DOC
can act as chelating agents and thereby enhance the mobilization of toxic heavy
metals (Pohlman and McColl 1988). DOC can also act as a methyl donor for the
trans-methylation of heavy metals (e.g. As, Hg and Se) resulting in the release of
poisonous methylated gases, such as dimethyl arsine and methyl mercury
(Frankenberger and Losi 1995).
DOC from environmental samples, such as soils and manures, is often
extracted with water or dilute aqueous salt solutions. Various methods have
been used to measure the concentration of DOC in soils and aquifers (Table 1).
These methods are grouped into three categories (Stewart and Wetzel 1981; De
Haan et al. 1982; Moore 1985). The most frequently used method involves the
measurement of absorption of light by the DOC, using a spectrophotometer
(Stewart and Wetzel 1981). The second method involves wet oxidation of samples
containing DOC and the subsequent measurement of either the CO 2 released or the
amount of oxidant consumed (Ciavatta et al. 1991). This method is often referred
to as chemical oxygen demand (COD). Dichromate or permanganate are the most
common oxidizing agents used in the wet oxidation of DOC, and the amount of
oxidant consumed in the oxidation of DOC is measured either by titration with a
reducing agent or by calorimetric methods. The third method involves dry oxida-
tion of DOC to CO 2 at a high temperature in the presence of a stream of oxygen.
The amount of CO 2 produced is measured either by an infrared detector, by
titration after being absorbed in an alkali, or by weight gain after being absorbed
in ascarite (Bremner and Tabatabai 1971). The most commonly used dry combus-
tion techniques include Leco combustion and the total organic carbon (TOC)
analyser.
In soils, pH and drying are considered to be the most important factors
influencing the concentration of DOC (Chittleborough et al. 1992). It has often
been observed that an increase in pH through liming and fertiliser addition to
soil increases the concentration of DOC (Smith and Willis 1985). Similarly, soil
drying has often been found to increase the concentration of DOC (Baskaran et al.
1994).
In this chapter, the various methods used to measure the DOC concentrations
of plant origin in soils, manures, sludges and stream water will be discussed. An
attempt will be made to examine the fractionation of DOC, and the factors affect-
ing the DOC concentrations in these samples.
Table 1. Methods of extraction, analysis and fractionation of dissolved organic carbon (DOC) in environmental samples s::
rt>
;.
Samples Extraction of DOC Measurement of DOC Fractionation of DOC Reference' 0
.,P-
0
....,
Sewage sludge Extraction with Wet combustion with
water (lO: 1 chromate followed by s::
rt>
liquid/solid); back titration .,III

filtration through ....ert>
47-flm membrane i3
rt>
filters a
0
....,
Volcanic ash soils Soil solutions by Dry combustion followed Chromotography (hydrophobic 2
suction lysimeters by infrared detection and hydrophilic) .,.,9-
of CO, 0
River water Natural water from Dry combustion followed 3 <"
rt>
rivers by infrared detection P-
O
....
of CO, (Oceanographic C1Cl
International carbon III
analyser) ~.
Peat water Peat water filtered Absorbance at 330 nm Molecular size fraction 4 n
III
through 47-flm and by wet oxidation d-
o
membrane filters with dichromate :::
0
....,
Lake water and hardwood Water samples and By the Oceanographic Chromotography (hydrophobic 5
and conifer forest soils porous cup soil International carbon and hydrophilic) ~
solutions filtered analyser a
through a 0.45-flm 0
....
silver membrane ~:
filter
'"
Table 1. Continued "'"
Samples Extraction of DOC Measurement of DOC Fractionation of DOC Reference'
Peat soil (typic medisaprists) Extraction with By the Oceanography 6

water, soil: water at International total C
1 : 5 ratio; filtered analyser
through Schleicher
and Schuell filter
paper No.597
Stream water from catchments Samples were filtered By absorbance at 330 nm 7
and soil water through Whatman
GF/C paper and
analysed
Soils (typic xeropsamment Extraction with 0.5 M Analysis by Dohrmann 8
and typic dystrandept) KH,P0 4 and OAM DC-80 carban analyser
K2 HP0 4 ; filtration
through OA5-~m
membrane filter
Three soils from Extraction with 1 : 2 Wet combustion Chromotography (hydrophobic 9
Mississippi soil: water ratio; persulphate digestion and hydrophilic)
delta area filtration (0045 ~m
filters)
Soil solutions from forested Samples were filtered Wet combustion Chromotography (hydrophobic 10
watersheds of North through a Whatman persulphate digestion and hydrophilic)
Carolina G/F glass filbre filter followed by TOC
analyseranalyser
z
~
Ol
o
Oi'
I:!
~
~
Soil and fertilizer aqueous Aqueous samples Wet chemical oxidation 11 s::
(C
extract with dichromate S-
followed by back o
0-
titration '"o
Liquid and solid sludge, Water extraction Dry combustion 12 ....
farm slurry, fermented followed by (Dhormann Carbon
s::
(C
straw, soil drainage centrifugation Analyser DC-80) ''"c"

(C
water (40000 x g) and "
filtration (0.45-llm S
(C
filter) ao
Soils, peat extract, sludge, Extracted with water Wet chemical oxidation 13 ....
pig and poultry manure (1 : 3 solid: solution with dichromate 9-
and mush room compost ratio); centrifugation followed by back '"'"o
(12000rpm) and titration ~
0-
filtration (0.45-llm
filter)
o
()q
"
Soil (typic haplothord) Extraction with Dry combustion (TOC Chromotography (hydrophobic 14 ::;
'"
deionized water analyser Shimadzu and hydrophilic) ;:;-
(1 : 10 solid: solution 5050) n
ratio); filtered through '"8-
o
0.45-llm polysulfore ::;
o....
membrane
"0
Pig slurry DOC by centrifugation DOC by wet dichromate 15 p)
(12000 x g) and then oxidation method a
filtration through a o
0.45-llm filter dii-
"
s-
'Reference: 1, Baham and Sposito (1983); 2, Antweiler and Drever (1983); 3, Landrum et al. (1984); 4, Moore (1985); 5, Cronan and Aiken (1985); 6, Madhun
et al. (1986); 7, Moore (1987); 8, Lee and Farmer (1989); 9, Pennington et al. (1991); 10, Qualls and Haines (1991); 11, Ciavatta et al. (1991); 12, Barriuso
et al. (1992); 13, Baskaran et al. (1996); 14, Kaiser et al. (1996); 15, Businelli (1997)_
Vl
6 N.S. Bolan et al.
2 Materials and Methods
2.1 Soils, Sludges and Stream Water
A number of environmental samples in which the dissolved organic carbon origi-

nates mainly from flora have been used in this study. Two soil samples (Patua silt
loam and Tokomaru silt loam) which differ in their organic carbon contents, three
samples of organic manures (poultry manure, pig manure and mushroom com-
post) and two sludge samples (municipal sewage sludge and an industrial paper
sludge) and a stream water originating from peat soil were used for the extraction
and measurement of DOC (Table 2).
2.2 Extraction of DOC
Distilled water and 0.5 M K2S0 4 were used for the extraction of DOC from the soil,
manures and sludge sources. Although a number of studies have used distilled
water as an extractant for the measurement of DOC (Barriuso et al. 1992), in
studies involving soil microbial biomass, O.SM K2S0 4 is used as an extractant to
measure biomass carbon (Tate et al. 1988). For the extraction of DOC, a 10 g
sample was equilibrated with 100mi solutions in 2S0-ml centrifuge tubes. The
sample was shaken in an end-over-end shaker for 1 h, and the solution was centri-
fuged at 8000rpm for 10 min. The solution was filtered through pre-washed
Millipore (OA1Ilm) HFA filters. The samples were stored at <4C before analysis.
2.3 Amendment of pH
To examine the effect of pH on the concentration of DOC, the pH of the Patua soil,
sewage sludge and poultry manure was amended to various levels. The pH was
Table 2. Characteristics of DOC sources
DOC sources pH Organic carbon CEC Total N Total P

(mgkg-I) (cmolkg-I) (mgkg-I) (mgkg-I)
Tokomaru soil 5.56 32 20 5.1 1.2

Patua soil 5.80 82 36 8.6 3.5
Poultry manure 6.8 425 58 36.5 11.6
Pig manure 7.3 296 42 28.6 12.5
Mushroom compost 6.8 385 29 38.9 16.2
Sewage sludge 7.1 321 24 1l.5 8.5
Paper sludge 8.3 281 33 6.8 5.2
Stream water 5.1 37 7.5 3.2
LSD (p = 0.05) 0.35 35 18 3.2 4.5

Methods of Measurement of Dissolved Organic Carbon of Plant Origin 7
increased to 7.5 by incubating either with Ca(OHL or 0.1 M NaOH solution and
the pH was decreased to 4.5 by the addition of 0.1 M HCl. After 4 weeks of in-
cubation, the DOC in the pH amended samples was extracted using distilled
water.
2.4 Effect of Drying
Fresh samples of the Patua soil, sewage sludge and poultry manure were used to
examine the effect of drying on DOC. One portion of the sample was allowed to air
dry at room temperature (20-25 0c) for 1 week. Another portion was oven dried at
40C for 24 h. The latter treatment was used to simulate the removal of water under
field conditions during summer months. A third portion was freeze dried. These
samples were used to examine the effect of drying on the extraction of DOC with
water.
2.5 Measurement of DOC
2.5.1 Spectrophotometric Method
The absorbance of light by DOC extracts was measured at a wavelength of 250 nm,
using a Pye-Unicam SP6-350 UV/visible spectrophotometer. A relationship was
obtained between the absorption of light and the concentration of DOC, as mea-
sured by the dry combustion method.
2.5.2 Wet Oxidation
Wet oxidation of DOC was carried out according to the method proposed
by Moore (1985). A sample of 25 ml DOC extract was evaporated to dryness at
90C, and then digested in a boiling water bath for 3 h with 25 ml of concentrated
H 2 S0 4 /H 3 P0 4 (2:1 v/v) containing 5g AgS04 dm- 3 , and 25ml 0.05M K2 Cr 2 0 7
The amount of dichromate used for the oxidation of DOC was estimated either
from back titration using 0.025 M ferrous ammonium sulphate or by calorimetric
measurements at a wavelength of 600nm (Ciavatta et al. 1991).
Chromate ions oxidise organic carbon to CO 2 (Eq. 1) and the amount of either
chromate ions consumed or chromic ions (Cr 3+) produced should be in direct
proportion to the organic carbon oxidized.
(1)
In aqueous acidic solution, Cr 3+ has absorption maxima at 450 nm and 600 nm.
Since Cr2 0 7 does not absorb at 600nm, the absorbance at this wavelength can be
used to estimate Cr 3+, and hence organic carbon (Metson et al. 1979).
8 N.S. Bolan et al.
2.5.3 Dry Combustion
Dry combustion of DOC is carried out using either the Leco combustion high
frequency induction furnace (Bremner and Tabatabai 1971) or the total organic
carbon (TOC) analyser. A high temperature (>1400C) can be produced by a high
frequency electrical flux induced in a mixture of soil sample and a conducting
matrix of iron and tin chips.
Leco Combustion. Leco combustion was originally developed to analyse carbon

and sulphur in solid samples. A known amount of aliquot is taken in aluminium
foil, placed in porcelain cups and heated at a high temperature in the presence of
an oxygen stream and the flux. The carbon dioxide released is absorbed onto
ascarite (NaOH absorbed in asbestos), and is estimated from the increase in the
weight of the ascarite bottle.
The gases produced by combustion are carried by the oxygen stream through
a dust trap, through a copper oxide catalyst to convert any CO to CO 2, through Mn
dioxide to remove halogen gases, and through Mg perchlorate to remove moisture.
From the amount of CO 2, the total dissolved carbon can be estimated:
. Amount of CO 2 (mg) x 0.2729
Percentage total dlssolved carbon = xl 00
Weight of sample
(2)
Total Organic Carbon (TOC) Analyser. The total organic carbon (TOC) analyser is
suitable for the analysis of carbon in solutions. The total carbon (TC) component
in the solution is combusted to become CO 2 , The carrier gas which contains the
combustion products is passed through a halogen scrubber and, the amount of
CO 2 present is analysed by an infrared detector. The detector is calibrated with
known concentrations of carbon, using glucose solutions. The inorganic carbon
(IC) in the form of carbonates and hydrogen carbonates can be measured by
introducing a sample of solution into an IC reactor vessel, where a carrier gas is
flowing in the form of tiny bubbles in the solution acidified by IC reagent. Total
DOC can be obtained by subtracting the IC concentration from the TC concentra-
tion. DOC can also be obtained directly if the solution has been acidified before it
is put in the instrument.
2.6 Fractionation of DOC
DOC is separated into fractions based on solubility, molecular weight and sorption
chromatography. The DOC fractionation by molecular size and sorption chroma-
tography is useful because it is based on important DOC properties (hydrophobic
and hydrophilic), which regulate DOC interaction with organic contaminants and
soil surfaces. The most common technique for the fractionation of aquatic DOC is
based on the sorption of DOC to non-ionic and ion-exchange resins (Leenheer
1981).
2.6.1 Molecular Weight Fractions
Four samples of DOC obtained from Patua soil, sewage sludge, poultry manure
and stream water were fractionated into three nominal molecular weight (Da)
fractions 5000, 5000-100000 and> 100000) using two Sephadex chromatography
gels with different exclusion limits (Sephadex G100 and GSO columns). Filtered
DOC extract (lOml) was placed on a Sephadex G100 column, eluted with Tris
buffer (pH = 9.0) and separated into >100000 and <100000Da molecular weight
fractions. The <100000 Da fraction was subsequently placed on a Sephadex GSO
column and eluted with Tris buffer to separate the organic matter into <5000 and
5000-100000 Da fractions. All the three fractions were concentrated by rotary
evaporation. The concentration of DOC in these fractions was determined follow-
ing the dry combustion method.
2.6.2 Sorption Chromatography
Briefly, the method consists of the following steps (Fig. 1). First, the DOC
sample collected is filtered through a 0.45 flm filter. Then the filtered sample
is acidified to pH 2 with HCl, and the acidified sample is successively passed
through three adsorption columns at a flow rate of 1 cm 3/min. Hydrophobic
acid (HOA), hydrophobic neutral (HON) and hydrophobic base (HOB) fractions
are retained on the first column containing the Amberlite XAD-8 hydrophobic
resin. The hydrophilic base (HIE) fraction is retained on the second column
containing cation-exchange resin (AG-MP-SO). The hydrophilic acid (HIA) frac-
tion is retained on the third column containing anion exchange resin (Duolite
A-7).
The DOC content of the effluent from the third column is measured,
which represents the fraction of hydrophilic neutral (HIN), which passes all
three columns. After removing the third column (Duolite A-7), the effluent
from the second column contains both HIA and HIN fractions. The concentration
of HIA fraction is then calculated by difference. Similarly, after removing the
second column, the effluent from the first column contains HIE, HIA and
HIN fractions. The HIE fraction can be calculated by subtracting the HIA and HIN
fractions (effluent passing through the second column contains both HIA and
HIN fractions) from the total hydrophillic DOC (effluent passing through the
first column contains total hydrophillic DOC). The fractions of HOA and
HOB are desorbed from the first column by elution with 0.1 N HCI and 0.1 N NaOH,
respectively. The HON fraction is calculated by difference between the total
DOC (having all fractions) and sum of HOA, HOB, HIA, HIB and HIN fractions
measured earlier. Thus the percentage of acidic, basic and neutral hydrophilic
and hydrophobic fractions of DOC in the extract (water or soil) can be
calculated.
lO N.S. Bolan et al.
Acidified DOC
Amberlite +-- Hydrophobic acids

+-- Hydrophobic bases
XAD-8 resin +-- Hydrophobic neutrals
AG-MP-50
cation exchange resin
+-- Hydrophilic bases
Duolite A-7 +-- Hydrophilic acids

anion exchange resin
+
Hydrophilic neutrals
in solution
Fig. 1. Analytical procedure for DOC fractionation
3 Results and Discussion
3.1 Methods of Measurement of DOC
3.1.1 Spectrophotometric Method
Absorbance oflight by DOC decreased with increasing wavelengths. Absorbance at

low wavelengths is likely to be a more sensitive indicator of DOC than absorbance
at higher wavelengths. So absorbance at 250 nm was selected in order to obtain a
relationship between absorbance and concentration of DOC, as measured by dry
combustion.
The relationship between absorbance at 250 nm and the concentration of DOC
varied between the samples (Table 3), which suggests that separate calibration
curves are required to estimate the DOC from the absorbance values. The differ-
ence in the relationship between DOC and absorbance at 250 nm between the
carbon sources has been related to the difference in the nature of organic carbon,
as measured by the relative molecular weight fractions (Stewart and Wetzel 1981),
and the presence of coloured inorganic compounds, such as soluble iron oxides
(Moore 1985). It must be pointed out that groups of organic compounds are
known to have different extinction coefficients (Oliver et al. 1983), so that differ-
ences in the relative amounts of these groups may produce differences in absor-
Table 3. Regression relationship between absorbance (A) and dissolved organic carbon (gcm-')
in various carbon sources. (Adopted from Moore 1985)
DOC samples Regression equations' Wave Cell Reference

length size
(nm) (cm)
Lake water DOM = 1O.7A - 0.98 260 10 Banoub (1973)

Lake water COD = 47.2A + 13.5 360 De Haan et al. (1982)
Lake water DOC = 160.1A + 6.9 420 Forsberg (1967)
Peat water DOC = 54.3A + 2.7Fe + 2.16 330 1.27 Moore (1985)
Poultry manure DOC = 0.0093A + 0.0008 250 1 Present study
River water DOC = 5.03A + 1.24 360 10 Lewis and Canfield (1977)
River water HAC = 0.083A + 0.00095 270 Timperley (1985)
Sewage sludges DOC = 0.0048A + 0.0056 250 Bolan et al. (1996a)
Soils DOC = 0.007A + 0.0013 250 Bolan et al. (1996a)
Stream water DOC = 0.0029A - 0.0001 250 Bolan et al. (1996a)
Stream water DOC = 142.2A - 37.2 350 Moore (1987)
Stream water DOC = 45.5A + 1.4 330 Moore (1989)
Stream water DOM = 59.6A + 1.9 360 1 Collier (1987)
Stream water DOM = 31.3A + 0.87 360 4 Grieve (1985)
Waste water TOC = 79.5A + 0.54 254 Dobbs et al. (1972)
'DOC = dissolved organic carbon; DOM = dissolved organic matter; TOC = total organic carbon;
HAC = hydroxylated aromatic compounds.
bance, independent of DOC concentration (Moore 1987). The spectrophotometric

method has been used frequently by several workers, and different relationships
have been obtained between DOC and absorbance (Table 3). As indicated by
Moore (1988), the variety of DOC sources and wavelengths used precludes the
development of an overall equation. Nevertheless, for any particular situation it
should be possible to develop a spectrophotometric method for samples with
relatively uniform organic constituents.
3.1.2 Dry Combustion
The amount of DOC, as measured by total carbon, using the Leco combustion,
TOC analyser and the wet oxidation methods, in various materials, is presented
in Table 4. The DOC values ranged from 1.02mgkg-1 in Tokomaru soil to
8.18mgkg-1 in poultry manure. Except for stream water, DOC forms only a small
fraction of the total organic carbon (1.84 to 3.8%) in other organic sources. The
DOC contributed 15% of the total organic carbon in stream water (Table 4). In
soils, it has often been observed that DOC, as measured by microbial biomass
carbon, forms only a small fraction (1.2 to 3.9%) of the total organic carbon (Bolan
et al. 1996b). The difference in the amount of DOC between the samples is related
to both the amount of total carbon, and the pH of the materials (Table 2). In
general, the amount of DOC increased with increasing total carbon and increasing
12 N.S. Bolan et al.
Table 4. Amounts of dissolved organic carbon (mgkg-l) in a range of carbon sources measured by
wet oxidation and dry combustion methods
Carbon sources Dissolved organic carbon (mg/kg) Percent wet DOC (% of total
oxidation of organic carbon)
Wet digestion Dry combustion DOC
Water 0.lMK,S04 Leco TOC analyser
Tokomaru soil 0.56 0.42 1.02 0.98 55 3.18

Patua soil 1.80 1.52 3.12 3.05 58 3.80
Poultry manure 6.46 5.59 8.18 7.65 79 1.92
Pig manure 4.53 3.96 6.13 6.02 74 2.07
Mushroom compost 5.18 4.78 7.10 6.95 73 1.84
Sewage sludges 4.08 4.01 6.00 5.96 68 1.87
Paper sludge 4.74 4.02 7.19 6.52 66 2.56
Stream water 3.40 5.58 5.49 61 15.2
LSD (p = 0.05) 0.25 0.38 0.39 0.21
pH. It has often been observed that in soils an increase in pH, due to fertilizers and
lime addition, increases the DOC (Smith and Willis 1985).
In general, the TOC analyser gave slightly lower concentrations of DOC than
Leco combustion. The difference, however, was significant only in the case of
poultry manure and paper sludge. These two samples contain significant propor-
tions of the dissolved carbon as inorganic carbon. Since Leco combustion mea-
sures the total dissolved carbon, the values are higher than those obtained by the
TOC analyser in which the DOC concentration has been corrected for the concen-
tration of inorganic carbon. It is therefore important to account for inorganic
dissolved carbon when measuring DOC.
3.1.3 Wet Oxidation
In general, water extracted higher amounts of DOC than did 0.5 M K2S04 (Table 4),
which may be attributed to the difference in the pH between these two extractants.
The pH of K2 S0 4 extract is 1-2 units less than that of water extract. 0.5 M K2SO 4 has
often been used as an extractant for measuring microbial carbon in soils. Although
a positive linear relationship between K2 S0 4 extractable carbon and microbial
carbon, as measured by respiration, has often been observed, K2 S0 4 extracted only
5-10% of the microbial carbon (Tate et al. 1988).
The wet oxidation method consistently underestimated the amount of
DOC (Table 4). Moore (1985) observed that dichromate oxidized 78% of the DOC
in peat water and called the dichromate oxidisable carbon "chemical oxygen de-
mand" (COD). In our experiment, dichromate oxidised between 55-79% of the
DOC. DOC determined by wet oxidation is dependent on the extent of oxidation
and it has often been observed that dichromate gives incomplete oxidation of
organic matter resulting in underestimation of DOC (Metson et al. 1979). In natu-
ral water, the dichromate method provides a good estimate of total organic carbon,
whereas the permanganate method is less efficient in decomposing organic
matter and usually yields lower value. Although the dry combustion method
gives an accurate estimate of DOC, it requires expensive equipment (Bremner
and Tabatabai 1971).
The percent DOC oxidized by wet oxidation increased in the order: poultry
manure> sewage sludge> stream water> Patua soil. The difference in the extent
of oxidation of DOC between the sources may be attributed to the differences in
the nature of soluble carbon, as measured by the relative molecular weight frac-
tions (see below). DOC includes microbial carbon and easily oxidisable organic
carbon (Powlson and Jenkinson 1981). DOC with low molecular weight fractions
is considered to be highly mobile and is liable for both chemical and microbial
oxidation (Boissier and Fontvieille 1993).
Meili (1992) obtained a good correlation between organic carbon as
measured by combustion and chemical oxidation methods using chromate and
permanganate. He observed that most estimates of organic carbon from wet oxida-
tion using chromate were within 80 to 90% of DOC. The permanganate method,
however, yielded only 25-60% of the total DOC in lake waters. In undiluted
samples with a high concentration of organic matter, even the dichromate
oxidation method was less efficient, due to the depletion of oxidant, but DOC
can be estimated with high reliability from chromate oxidation (CrOC) with a
correction formula accounting for the proportion of oxidant (Ox) remaining after
analysis:
DOC =CrOC/(0.9 OX027). (3)
This relationship was also valid in anoxic waters, which indicated that the interfer-
ence from inorganic reduced compounds such as Fe-hydroxides on CrOC was of
minor importance.
3.2 Molecular Weight Fractions
The percent distribution of relative molecular weight fractions in the DOC extracts
from various sources is presented in Table 5. The samples varied in the relative
distribution of molecular weight fractions. The DOC from sewage sludge and
poultry manure in general have a greater proportion of low molecular weight
fractions when compared with the DOC from Patua soil and stream water. These
results are consistent with the results for chemical oxidation which indicated that
low molecular weight fractions are more readily oxidized than the high molecular
weight fractions. The slope of the relationship between absorption at 250 nm and
DOC (absorption per unit DOC; Table 3) increased with an increase in the molecu-
lar weight of the DOC fractions. If we assume that light absorbing organic materi-
als in the DOC pool are of similar composition and roughly spherical (Schnitzer
and Khan 1972), increases in absorption per unit C would be expected to increase
Table 5. Percent distribution of molecular size fractions in

various DOC samples
DOC source Percent distribution of relative molecular

weight
(Da) fractions
<5000 5000-100000 >100000

Patua soil 52 26 22
Sewage sludge 79 15 6
Poultry manure 42 38 20
Stream water 26 32 42
LSD (p = 0.05) 11 6 9
with molecular weight of the fraction. Stewart and Wetzel (1981) have observed an
increase in the ratio between absorption at 250 nm and total C in DOC (A2S0: C)
with an increase in the relative molecular weight of DOC.
3.3 Effect of Drying on DOC
DOC was extracted from soil, manure and sludge samples after drying at various
temperatures. The amount of DOC, as measured by total C analysis using Leco
combustion, increased with drying (Table 6). There was no difference in the
amount of DOC between the field-moist and the freeze-dried samples. It has often
been observed that drying of soils increases the solubility of soil organic matter
(Haynes and Swift 1989). An increase in DOC during drying has been attributed to
the breaking of hydrogen bonds in the organic matter or to the lysis of the micro-
bial cells killed during drying. Removal of water from soil through drying will
result in the macromolecules of soil organic matter changing into a highly con-
densed state. Such shrinkage of organic compounds may result in the disruption of
organo-mineral association and subsequent release of some low molecular weight
humic components.
3.4 Effect of pH on DOC
The amounts of DOC extracted from soil, manure and sludge samples amended to
different pH values using HCI, NaOH and Ca(OH)2' are presented in Table 7. There
was an increase in the amount of DOC with increasing pH. The effect was more
pronounced with NaOH addition than with Ca(OH)2 addition. Alkaline solutions
have been used extensively to extract organic matter from soils. The solubility of
humic substances in alkaline solutions of monovalent ions, such as Na and K is
believed to be caused by the conversion of the acidic components to ions and
Table 6. Effect of drying on the amounts of dissolved organic carbon (mgkg-l) in a range of
materials as measured by the Leco combustion method
Materials Dissolved organic carbon (mgkg-l)
Field moist Air dry Oven dry Freeze dried
Patua soil 1.02 2.12 3.56 1.21

Poultry manure 8.18 10.8 19.5 7.98
Sewage sludge 6.00 9.7 17.2 6.12
LSD (p = 0.05) 0.39 0.42 0.65 0.28
Table 7. Effect of pH on the concentration of dissolved organic carbon
Carbon sources Dissolved organic carbon (mgkg-l)
Control HCl NaOH Ca(OH)2
Patua soil 1.02 0.58 3.69 2.12

Poultry manure 8.18 0.73 21.8 15.4
Sewage sludge 6.00 0.79 12.9 9.8
LSD (p = 0.05) 0.39 0.29 0.58 0.62
subsequent formation of a physical solution in aqueous solution. Various reasons

have been put forward to explain the decrease in the extractability of organic
matter in the presence of polyvalent cations. These include: formation of insoluble
compounds and subsequent flocculation of organic molecules; complexation of
organic molecules with polyvalent cations; and adsorption of organic molecules
onto clay through cation bridging (Greenland 1971). David et al. (1989) observed
that acid treatment of an organic horizon of a spodosol resulted in a decrease in
DOC concentration, which they attributed to the changes in the proportion of
hydrophobic and hydrophilic compounds in the DOC.
Previous studies have shown that the concentration of DOC or the intensity of
colour in lake waters decreases, and transparency increases as a result of acidifica-
tion (Yan 1983; Effler et al. 1985). In lakes acidification causes an increase in AI, Ca,
Fe and other metals (Nilsson 1985; Dillon et al. 1988). It has been shown that an
increase in Al input causes flocculation and subsequent removal of DOC, and thus
increases the clarity of acidic lakes. At pH > 4, DOC may also co-precipitate with
Fe(OH)3' Schindler et al. (1992) have obtained a negative correlation between DOC
and H+ concentration in lakes. The surface functional groups in DOC and the
change in surface charge with pH have indicated that DOC is likely to coagulate at
low pH values. This may be one of the reasons for the decrease in the concentra-
tions of DOC at acidic pH values. Decreasing pH also increases the lipophilic

nature of humic substances which may partly explain the decline in DOC.
4 Conclusions
1. DOC can be extracted using either water or dilute aqueous salt solutions.
2. DOC can be estimated by absorption of light at 250 nm using a spectropho-
tometer, by wet oxidation using dichromate, or by dry combustion using Leco
and the TOC analyser.
3. Absorption of light per unit DOC increases with an increase in the relative
molecular weight of the organic compound in the DOC.
4. Separate calibration curves are required to estimate the DOC from different
sources using the spectrophotometric method.
5. Dry combustion using the Leco combustion method gives the total carbon in
DOC extracts.
6. DOC can be measured accurately using the TOC analyser.
7. DOC measured by wet oxidation using potassium dichromate underestimates
DOC concentration.
8. The extent of oxidation by dichromate varied between DOC sources, which
may be attributed to the difference in the molecular fractions of DOC.
9. Both air drying and oven drying increased the concentration of DOC; whereas
freeze drying had no effect on DOC.
10. The concentration of DOC increased with an increase in pH.
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Analysis of Papermill Waste Water Treatment
Residuals and Process Residues
M.S. ERICH and P. FIRST
1 Introduction
Minimization of waste, both solid and liquid, is a current goal of the pulp and
paper industry. Canada, the United States, Sweden, Finland, Japan, Australia, and
Brazil all have relatively stringent environmental regulations governing disposal of
wastes from the pulp and paper industry (Owens 1996). The US paper industry
generated approximately 5.6 million dry tons of wastewater treatment sludge in
1995 (Lynde-Maas et al. 1997). Of the total reported, 56% was combined primary
and secondary sludge, with most of the rest being primary sludge. Primary sludge
is the gravity-settled or chemically-coagulated organic and inorganic material
derived from untreated mill waste water. Primary sludges often have a solid con-
tent of 20-45% which consists of wood fibers, clay, calcium carbonate, titanium
dioxide, and other materials used in pulp and paper production, such as inks and
dyes (National Council of the Paper Industry for Air and Stream Improvement
1993). Secondary sludges result from the biological treatment of wastewater and
contain largely microbial biomass.
In 1995 in the US most papermill sludge was disposed of in landfills (45%),
with about 21 % being incinerated, and about 14% applied to land or composted
(Lynde-Maas et al. 1997). Although some countries, states, or provinces require
leachate or extraction testing of the sludge before its disposal in landfills (Bunnage
and Imada 1996), the primary need for analysis of papermill sludge involves that
portion slated for land application since the material, and its composition, will
clearly influence terrestrial processes. The liquid waste, or effluent, from pulp and
paper mills, which is frequently discharged into rivers after treatment, has been
subject to regulation for decades, and there is a relatively large literature base on its
composition and environmental effects (e.g. Servos et al. 1996).
As the industry has explored alternatives to landfilling papermill sludge,
the beneficial uses of papermill sludge as a soil amendment have become increas-
ingly apparent (Aitken et al. 1995; Phillips et al. 1997). Field and greenhouse trials
with tree species, agronomic crops, and horticultural crops have suggested that
additions of papermill sludge can improve plant growth and increase soil organic
matter levels with concomitant improvements in soil structure, water-holding
capacity, and nutrient-holding capacity (Brockway 1983; Bellamy et al. 1995).
In addition, increases in soil organic matter can improve water infiltration rates
and decrease rates of soil erosion. The primary agronomic concern with the use

22 M.S. Erich and P. First
of papermill sludge involves the relatively high carbon (C) and low nitrogen
(N) levels associated with this amendment. Use of papermill sludge with high
C: N ratios (above 30: I) may stimulate microbial populations to immobilize avail-
able soil N, thus making it unavailable to growing plants. Yield decreases may
be associated with the use of paper mill sludge without supplemental N fertiliza-
tion (Bellamy et al. 1995; Aitken et al. 1995). In addition, high soluble salts and
relatively high levels of sodium may pose agronomic limitations, but these prob-
lems seem to occur very rarely under field conditions. Environmentally, the
major concerns with the use of papermill sludge involve trace elements and
trace levels of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans
(PCDFs).
2 The Pulping Process and the Composition of Papermill Sludge
Wood, both softwood and hardwood, is the initial raw material for the production
of pulp. Its main components are cellulose, hemicelluloses, and lignin. There
are several methods of pulping, the main purpose of which is to remove lignin
in order to facilitate wood fiber separation and improve the papermaking qualities
of the fibers (Kringstad and Lindstrom 1984). Chemical pulping entails cooking
wood chips with sodium hydroxide and other chemicals to promote cleavage of
the ether linkages in lignin. A number of wood components, including a large
fraction of the lignin, dissolves in the alkaline pulping liquid (liquor). After
separation from the pulp, the spent pulping liquor is typically evaporated and
burned for energy and the inorganic chemicals recovered (Kringstad and
Lindstrom 1984). Small amounts of lignin remaining in the pulp cause a dark
color which is removed by bleaching with molecular chlorine (Clz), chlorine
dioxide, or a bleach containing no chlorine. Bleaching and washing effluents may
be recovered or may be discharged after wastewater treatment. Lignin is an
aromatic polymer, and addition of Cl z to pulp results in the production of small
amounts of PCDDs/PCDFs (Kringstad and Lindstrom 1984), primarily 2,3,7,8-
tetrachlorinated dibenzo-p-dioxin (TCDD), 2,3,7,8 tetrachlorinated dibenzofuran
(TCDF), and 1,2,7,8 TCDF (Berry et al. 1993).
There are a number of different products produced by pulp and paper mills,
including pulp, newsprint, book and writing paper, tissue, cardboard, and coated
paper. Currently many mills use a combination of wood and secondary, or re-
cycled, fibers as raw material (Bunnage and Imada 1996). The amount of second-
ary fiber used can range from <10 to 100%. The level of secondary fiber used, the
type of secondary fiber used, any treatment of the secondary fiber such as
deinking, and the pulping and production technology will all influence the amount
and composition of the sludge produced (Bunnage and Imada 1996). In addition,
treatment of wood with polychlorophenol anti-sap stain (anti-fungal) formula-
tions has been found to influence the composition of the sludge produced during
Analysis of Papermill Waste Water Treatment Residuals and Process Residues 23
papermaking (Luthe et al. 1993). Due to the wide range of raw materials used
and pulping technologies employed, papermill sludge is a heterogeneous and
variable material, both from mill to mill and over time within a mill. This necessi-
tates both care in sampling, to obtain a representative sample, and relatively
frequent analyses.
3 Sampling and Chemical Characterization of Papermill Sludge
3.1 Sampling
The following is a brief general discussion (adapted from Erich 1988) of sampling
consideration for a waste material such as papermill sludge. In practice, frequency
of chemical characterization and, sometimes, sampling methodologies are gener-
ally dictated by federal, state, or provincial environmental regulations. Since waste
materials are generally spatially and temporally heterogeneous, their chemical
characterization must be accomplished by the analysis of "representative"
samples. In other words, a number of samples of the material must be obtained
and either thoroughly mixed together and subsampled before analysis, or analyzed
separately and the results averaged. The advantage of separate analyses is that the
amount of spatial variability existing in the sample with respect to its chemical
properties can be determined. However, in the case of papermill sludge, where
analysis for PCDDs/PCDFs, in particular, is very expensive, compositing of
subsamples before analysis is generally practiced. Analysis of a single sample,
sometimes called a grab sample, is an inappropriate way to characterize waste
materials. Maximizing the volume of samples taken during compo siting also helps
to overcome variability.
For waste materials there are at least two distinct sampling situations. One is
sampling of material which can reasonably be expected to be similar to previously
characterized material because it is produced from the same process, plant, and
very similar raw materials. Another situation is material at least part of which has
been produced from new plants, processes or raw materials. In the case of material
which is expected to be similar to previously characterized material, if past analy-
ses suggest that no regulated constituent is present at levels greater than 75% of the
maximum permissible concentration, then one composite sample (from 10 to 20
individual samples depending on the size of the batch) should adequately charac-
terize that batch.
If one or more constituents exceed the regulatory threshold, or are within
25% of it, or there is no past history to suggest what levels of constituents are
likely to be, several samples (10-20 depending on the size of the batch to
be characterized) should be taken and composited into a smaller subset for
initial analysis. For example, 12 initial samples may be divided into three groups
of four samples, and portions of the four samples within each group composited
together to obtain three samples for analysis. If the three composite samples
indicate that the waste is not very variable and is clearly below regulatory thresh-
olds, no further analysis of the material is needed. Otherwise, the initial 12 samples
may need to be analyzed separately or recomposited into more than three analyti-
cal samples. Taking large enough numbers and volumes of initial samples allows
the flexibility for several compo siting schemes if necessary.
The several samples taken for compo siting should be obtained by random
sampling, which is not the same as haphazard sampling, or by systematic sam-
pling. Generally the procedure for random sampling of a pile or container of waste
involves imposing a grid pattern over the object to be sampled in order to number
each possible sampling position. Then random numbers are chosen from a ran-
dom number table (available in most basic statistics books) to designate the indi-
vidual sampling positions. This system allows any point an equal likelihood of
being sampled and removes possible bias resulting from haphazardly chosen
samples. Systematic sampling involves imposing a grid pattern with, for example,
12 equi-distant intersections and sampling them all. Care should be taken to
sample the entire depth of a tank or pile; however, in the case oflarge tanks or piles
access to the material can be limited. In this case the easiest way to obtain samples
representative of the waste is to sample during waste removal. The number of
truck loads needed to remove the waste can be estimated and the truckloads for
sampling chosen by random numbers.
3.2 pH and Macronutrients
Since papermill sludge often has a relatively high water content, some papermill
sludge samples may be delivered to the laboratory wet enough so that pH may be
measured with no addition of water. In this case, all that is necessary is to store the
sample in the refrigerator and thoroughly homogenize it by shaking or stirring
before sub-sampling for pH analysis using single junction pH and reference elec-
trodes, or a combination pH electrode. With a drier material, pH can be obtained
from a sample mixed with de-ionized water in a 1: 1 (w:w) ratio. Samples are
generally stirred thoroughly and allowed to stand for 30 min to 1h before measure-
ment. Electrodes should be placed at the solid/solution boundary. Do not measure
pH in filtered solutions.
To obtain the solid contents, a subsample of papermill sludge should be
dried overnight at 1l0e. This subsample should not be used for any subsequent
analyses since this temperature may be high enough to volatilize some com-
ponents. C and N content of the sample may be most reliably and easily obtained
by a C/N analyzer. These analyzers combust the sample at very high temperatures
and quantify the gases produced. Before such analysis, samples should be dried
at 70C and ground to pass a 40 mesh sieve or smaller. The necessity for finer
grinding depends on the sample size to be used, which in turn depends on the
capabilities of the particular instrument. In order to remove carbonates, sample
pre-treatment with 25% HCI until no further effervescence is observed may be
necessary for some samples and some instruments (some will acidify internally in
order to determine only organic C). In the absence of the capability for instrumen-
tal analysis for C and N, N may be determined by a Kjeldahl procedure and organic
content may be estimated by loss on ignition after combustion in a muffle furnace
at 550C.
The total elemental content of calcium (Ca), magnesium (Mg), potassium
(K), sodium (Na), and phosphorus (P) may be obtained by digestion of the mate-
rial and subsequent analysis of the digest by inductively coupled plasma atomic
emission spectrometry (ICP-AES). Alternatively, flame atomic absorption (AA)
spectrometry may be used for Ca and Mg, flame emission spectrometry for K
and Na, and a colorimetric procedure for P. Digestion methods suitable for
carbonaceous material such as plant tissue would generally be suitable for
papermill sludge. Our laboratory uses a low temperature dry ashing procedure
followed by dissolution of the ash in HCI (adapted from Kalra and Maynard 1991).
Approximately 1 g of dried, ground, and homogenized material is ashed for at least
6 h at 450C and the ash dissolved in 5 ml 50% HCI plus 1 ml concentrated HN0 3
by boiling to dryness. Another 5 ml aliquot of 50% HCI is added and the samples,
after cooling, are transferred to 50ml volumetric flasks and brought to volume
with water.
3.3 Trace Elements
Microwave assisted acid digestion is a common method for sample preparation

prior to trace element analysis. The US Environmental Protection Agency (EPA)
recommends use of about 0.5 g of sludge material and 10 ml of concentrated nitric
acid (US EPA 1994a). Sample and acid are placed in a fluorocarbon microwave
digestion vessel. Samples are irradiated for about 10 min, then cooled, centrifuged,
and filtered. There are many safety considerations specific to microwave method-
ology, and the analyst must read the entire method and be familiar with microwave
technology. Most trace elements can be determined by ICP-AES. Although newer
ICP spectrometers can sensitively determine cadmium, nickel, and lead, older
instruments may not, thus requiring the analyst to use flame AA spectrometry or,
for higher sensitivity, graphite furnace AA spectrometry for these three elements.
Mercury should be analyzed by cold-vapor AA spectrometry, which uses a chemi-
cal reduction of mercury to elemental mercury vapor to achieve high sensitivity
(US EPA 1994b).
4 Polychlorinated Dibenzo-p-dioxins and Dibenzofurans

in Papermill Sludge
4.1 Chemistry and Toxicity of Polychlorinated Dibenzo-p-dioxins

and Dibenzofurans
The use of molecular chlorine in the pulp bleaching process gives rise to the
formation of chlorinated aromatic compounds. The most toxic of these are
polychlorinated dibenzo-p-dioxins ("dioxins") and dibenzofurans ("furans"), the
structures of which are shown in Fig. 1 (Clement 1991). For PCDDs, a total of 75
different chlorine-substituted compounds, referred to as congeners, are possible;
for PCDFs, there are 135 chlorine-substituted congeners.
Toxicity of the congeners varies according to the number and position of
the chlorine atoms with 2,3,7,8-TCDD being the most toxic as well as one of the
most toxic substances known. Table 1 lists the relative toxicities of the 17 conge-
ners which have been found to exhibit significant toxicity (Clement 1991). As well
as being toxic, dioxins and furans are stable and persistent in the environment.
They are lipophilic and subject to bio-accumulation. Thus, even very low levels
of these compounds, parts per trillion (ppt, 10-12 gig) or parts per quadrillion
(ppq, 10-15 gig), are potentially hazardous, and very low detection limits are
9 1
3(X-_,o~_" 2
(I
~ I
I
\
I
I
7" -- 3
Cl",
o Cly
Chlorinated dibenzo- p -dioxin
:0cJQ:
9
CI", Cly
Chlorinated dibenzofuran
No. of chlorines No. of isomers

(x + yl coo COF
1 2 4
2 10 16
3 14 28
4 22 38
5 14 28
6 10 16 Fig.!. Structures of the chlorinated dibenzo-
7 2 4 p-dioxins and chlorinated dibenzofurans.
8 _1 ..J. (Clement 1991; copyright 1991 American
Total 75 135 Chemical Society)
Table 1. International toxicity equivalency factors (1-TEF) for

2,3,7,8-substituted dioxins and furans. (Adapted from Clement
1991)
Dioxin congener I-TEF Furan congener I-TEF
2,3,7,8-TCDD 2,3,7,8-TCDF 0.1

1,2,3,7,8-PsCDD 0.5 2,3,4,7,8-P sCDF 0.5
1,2,3,4,7,8-H6 CD D 0.1 1,2,3,7,8-PsCDF 0.05
1,2,3,7,8,9-H6 CDD 0.1 1,2,3,4,7,8-H6 CDF 0.1
1,2,3,6,7,8-H6 CDD 0.1 1,2,3,7,8,9-H6 CDF 0.1
1,2,3,4,6,7,8-H,CDD 0.01 1,2,3,6,7,8-H 6 CDF 0.1
08CDD 0.001 2,3,4,6,7,8-H 6 CDF 0.1
1,2,3,4,6,7,8-H,CDF 0.01
1,2,3,4,7,8,9-H,CDF 0.01
08CDF 0.001
necessary for analysis. Due to their hydrophobicity, they tend to be associated

with the solid waste fraction rather than the aqueous fraction (Mahle et al. 1989).
They are not particularly soluble in most common solvents. Because all of the
congeners are structurally very similar, and because they are present at trace
concentrations in complex environmental matrices, extraction and separation of
the 17 toxic congeners from the other congeners and their subsequent quantifica-
tion has proved a significant analytical challenge over the past 30 years (Clement
1991).
4.2 General Analytical Considerations
Lengthy and comprehensive analysis methods are necessary to meet the challenges
described above. Most PCDD/PCDF analysis methods share the following basic
steps: extraction of organic compounds from the sample matrix (including
PCDDs/PCDFs), separation of other organic co-extractives, and finally identifica-
tion of the analyte. Quality assurance techniques are necessary to evaluate the
precision and accuracy of these basic steps. Due to the overall breadth and com-
plexity of PCDD/PCDF analysis techniques, official Canadian and US government
methods are approximately 50 and 75 pages long, respectively. The following
sections briefly summarize Environment Canada's Reference Method for the Deter-
mination of Polychlorinated Dibenzo-para-dioxins (PCDDs) and Polychlorinated
Dibenzofurans (PCDFs) in Pulp and Paper Mill Effluents (Environment Canada
1992a) and emphasize its application to the papermill sludge matrix. Figures 2 and
3 show, in schematic form, the basic steps of the extraction, cleanup, and analysis
methods recommended by Environment Canada. This method contains both
required components and recommended procedures. The recommended proce-
dures for extraction and cleanup, in particular, may be modified by the analyst
if the chosen procedures are shown to be effective by method performance tests
(see Sect. 4.4).
PAPERMILL SLUDGE Fig. 2. Extraction schematic for paper-

mill sludge. (Adapted from Environment
ij Canada 1992a)
Air Dry or Suction Filter and Discard Effluent
(grind if particles> 1 mm, U.S. EPA 1994c)
ij
Soxhlet Extraction
20 h with toluene
ij
Rotary Evaporation
to 1 to 2 mL
ij
Exchange to Hexane
ij
Rotary Evaporation to 3 to 5 mL
ij
Ready for Cleanup
Analysis of PCDDs/PCDFs should only be carried out after a thorough review

of applicable literature by trained, competent personnel thoroughly familiar with
high resolution gas chromatography (HRGC) and mass spectrometry (HRMS).
This is important due to the hazardous nature of the analytes and the environmen-
tal and economic implications of false test results.
4.3 Safety
Exposure to all of the PCDD/PCDF congeners should be minimized and all work
should be carried out in a specially designed laboratory (Environment Canada
1992a). Such a facility should include: restricted access, sufficient ventilation,
including a system to allow visual monitoring of ventilation performance, negative
pressure relative to surrounding areas, exhaust ducts routed to a common,
scrubbed outlet, and full operational capability on auxiliary power if a power
failure occurs. In addition, the facility should have an independent backup air
supply system which becomes operational if the building's air supply shuts down
and distinctive audio and visual alarm systems to alert all building personnel to
potentially hazardous conditions.
Laboratory personnel should wear protective clothing at all times including
safety glasses, disposable coveralls, and disposable gloves. Laboratory personnel
CONCENTRATED EXTRACT
ij
Acid/Base/Silver Nitrate/Silica Column
ij
Rotary Evaporation
ij
Basic Alumina Column
a) Hexane Elution (Archive)
b) 1.5% CH,CI, in Hexane Elution (Archive)
c) 50% CH,CI, in Hexane Elution (PCDD/PCDF)
ij
Rotary Evaporation
ij
Exchange to Hexane
ij
Rotary Evaporation
ij
(Supplementary Cleanup such as:
Repeat Alumina Column
Carbon/Silica Column)
ij
Concentrate to Dryness under N,
ij
Prepare to Final Volume with Recovery Standard Solution
ij
GC/MS Analysis
Fig. 3. Cleanup and analysis schematic for PCDDs/PCDFs in paper mill sludge. (Environment
Canada 1992a)
should be fully aware of the hazards associated with these chemicals and specifi-
cally trained in their handling (Environment Canada 1992a). Laboratory safety
policies must cover such topics as safe and secure handling and storage of PCDDs
and PCDFs, housekeeping, spill containment and cleanup, and handling of solid
and liquid wastes. Personnel should have copies of these policies and frequent
training and updating of skills should occur.
4.4 Method Performance Tests
Prior to the analysis of actual field samples, the laboratory must demonstrate
the accuracy and precision of the analysis procedure (Environment Canada
1992a). Three matrix blanks are spiked with known amounts of "native" and
"surrogate" PCDD/PCDF congeners according to the concentrations given for

CS2 in Table 2. The "native" spike is a substitute for the PCDDs/PCDFs found
in an actual field sample; it contains all 17 of the 2,3,7,8 chlorine substituted
dioxin and furan congeners at standard concentrations and is not isotopically
labeled. The "surrogate" spike is used to determine the efficiency of extraction
and cleanup procedures and is added to samples as well as the matrix blanks;
it contains nine isotopically labeled 2,3,7,8 chlorine substituted dioxin and
furan congeners (Environment Canada 1992b). For each test, the recovery of all
"native"spike congeners, adjusted for "surrogate" recovery, must be 80-120%
of the original "native" spike value. The recovery of all labeled "surrogate" spike
congeners must be 40-120% of their original value. Recoveries must be within
these acceptable limits before actual samples can be analyzed. Method perfor-
mance tests must be repeated at 3-month intervals, after any modification of
the procedure, and when reagents from different lot numbers are used. De-ionized
water is an appropriate matrix blank for papermill effluent. Pulp or sludge
produced entirely from primary wood fiber and not subjected to any chlorine-
containing bleaching processes could be an acceptable matrix blank for paper-
mill sludge. The matrix blank should have no PCDDs/PCDFs or a known, low
content.
4.5 Sample Preparation and Analyte Extraction
There is potential for contamination at every step of collecting, shipping and

handling of dioxin-containing samples. Amber glass bottles with Teflon-lined caps
are the best sample containers. Samples for dioxin analysis should not come into
contact with plastic other than Teflon (Environment Canada 1992b). All glassware
to be used for analysis must be rinsed with hexane and dichloromethane and the
combined solvent rinse analyzed for residual dioxin and furan. Acceptable levels
of detectable dioxins and furans are established (Hamilton et al. 1995), and glass-
ware must be re-cleaned if these levels are not attained. Solid or slurry samples
should be homogenized before subsampling.
Recommended sample size, final volume, and achievable detection limits for
various matrices are listed in Table 3. Careful records must be kept of details of
sample processing. Before processing, each sample must be spiked with known
amounts of 913 C-labeled dioxin/furan surrogates. A matrix blank (spiked with the
surrogate solution) should be processed along with each batch of ten test samples.
One sample in each ten should be processed and analyzed in duplicate to assess
reproducibility.
Effluent samples should be suction-filtered through pre-weighed glass fiber
filter paper. The filter paper and solids are dried and weighed before extraction
with toluene in a Soxhlet apparatus for 16-20h. Sample filtrate is extracted
with dichloromethane in a separatory funnel. The extraction procedure is repeated
three times. All glassware involved in liquid transfer is rinsed three times
with dichloromethane. The extracts are then combined and concentrated using
a rotary evaporator. The concentrated filtrate extract is then combined with the
Table 2. Composition of PCDD/PCDF calibration solutions.

(Environment Canada 1992a)
PCDD/PCDFs standard pg//ll (in toluene)
CS1' CS2 CS3 b CS4
Native Standards"d
2,3,7,8-TCDD 0.25 5 25
2,3,7,8-TCDF 0.25 1 5 25
1,2,3,7,8-P,CDD 0.50 2 10 50
1,2,3,7,8-P,CDF 0.50 2 10 50
2,3,4,7,8-P,CDF 0.50 2 10 50
1,2,3,4,7,8-H6CDD 0.50 2 10 50
1,2,3,6,7,8-H6CDD 0.50 2 10 50
1,2,3,7,8,9-H 6CDD 0.50 2 10 50
1,2,3,4,7,8-H6CDF 0.50 2 10 50
1,2,3,6,7,8-H6CDF 0.50 2 10 50
2,3,4,6,7,8-H 6CDF 0.50 2 10 50
1,2,3,7,8,9-H6CDF 0.50 2 10 50
1,2,3,4,6,7,8-H,CDD 0.50 2 10 50
1,2,3,4,6,7,8-H,CDF 0.50 2 10 50
1,2,3,4,7,8,9-H,CD F 0.50 2 10 50
O,CDD 4 20 100
O,CDF 4 20 100
Surrogates
13C12-2,3,7,8-TCDD 50 50 50 50
13C,2-2,3,7,8-TCDF 50 50 50 50
13C,2-1 ,2,3,7,8-P,CD D 50 50 50 50
13C,2-1,2,3,7 ,8-P,CDF 50 50 50 50
13C,2-1,2,3,6,7,8-H6CDD 50 50 50 50
13C,2-1,2,3,4,7,8-H 6CDF 50 50 50 50
13C,2-1,2,3,4,6,7,8-H,CDD 50 50 50 50
13C,2-1,2,3,4,6,7,8-H,CDF 50 50 50 50
13C,,-OCDD 100 100 100 100
Recovery standards
13C,2-1,2,3,4-TCDD' 50 50 50 50
13C,2-1 ,2,3,7,8,9-H 6CDD f 50 50 50 50
, Also used to assess detection limits.

b Used daily to verify calibration and abundance ratios.
'Penta- and hepta-CDF; hexa-CDD/CDF isomers listed in order of
elution on a DB-5 column.
dA solution of native congeners at CS2 concentrations is used as a
spiking solution for method performance tests.
'Retention time marker and recovery standard for tetra- and
penta-CDD/CDF.
fRetention time marker and recovery standard for hexa- and
hepta-CDD/CDF and OCDD.
solids extract. The transfer is completed with three hexane rinsings. The combined
extracts are then concentrated by rotary evaporation. Following extract con-
centration, the solvent is exchanged by adding hexane and repeating the concen-
tration step. The sample is then dried by passing it through hexane-rinsed sodium
Table 3. Recommended sample size, final volume, and achievable method detection limits for
various matrices. (Environment Canada 1992b)
Sediment, soil, sludge, ash Pulp mill effluent Pulp
Sample size 5g(dry) 11 12g (dry)

Final volume (111) 20 20 20
Target detection limit LRMS (HRMS) pg/g LRMS (HRMS) pg/l LRMS (HRMS) pg/g
T,CDD/F 12 (1) 60 (5) 5 (0.4)
PsCDD/F 24 (2) 120 (10) 10 (0.8)
H6CDD/F 24 (2) 120 (10) 10 (0.8)
H7 CDD/F 36 (3) 180 (15) 15 (1.2)
OCDD/F 48 (4) 240 (20) 20 (1.6)
LRMS, Low resolution mass spectroscopy; HRMS, high resolution spectroscopy. Numbers in
parentheses refer to detection limits by high resolution mass spectroscopy.
sulfate. Drying agent and flask are rinsed with three portions of hexane. The
sample is then reconcentrated by rotary evaporation.
Samples of higher solids content, such as papermill sludge, can be handled in
one of two ways. The US Environmental Protection Agency recommends filtering
the samples through a glass fiber filter paper (after spiking with labeled com-
pounds) and discarding the aqueous phase. Samples containing >1-mm particles
should be air-dried in a hood, and ground (US EPA 1994c). Alternatively, higher
solids material could be air-dried in a hood without a filtration step (Kuehl et al.
1987). Sludge samples are then subject to extraction with toluene in a Soxhlet
apparatus.
4.6 Cleanup of Sample Extracts
The use of an acid/base/silver nitrate/silica column is the first step in the recom-
mended chromatographic cleanup procedure (Fig. 4). The column is packed
according to specific method instructions. Prior to use, the packed column is pre-
washed using 2% dichloromethane in hexane. The solvent is drawn down to the
top of the packing, a collection flask is set in place, and the concentrated sample is
transferred to the column using a Pasteur pipet. The concentrated sample extract
flask and the pipet both receive three rinses of solvent to complete the transfer.
Additional 2% dichloromethane in hexane is then added to the column. After all
solvent has drained through the column, the silver nitrate/silica and acid/silica
layers are examined for color. Color indicates the unwanted saturation of these
layers and an incomplete separation. If the acid/silica layer shows signs of satura-
tion, the extract must be washed with sulfuric acid in a separatory funnel, followed
by sequential washings with deionized water and sodium hydroxide. If the silver
nitrate/silica layer shows signs of saturation, the concentrated sample extract must
be passed through an additional column of silver nitrate/silica. Additional hexane
20 mm
--,---H
E
E
E
E
'"
""
'"
1.5 i AgH0 3 / SILICA
GUSS WOOL
S mm
ACID / BASE
S mm
ALUMINA
Fig. 4. Cleanup columns. (Environment Canada 1992a)
is then added to the eluate, followed by macro-concentration by rotary evapora-

tion.
The use of an alumina column is the second step in the chromatographic
cleanup procedure recommended by Environment Canada (Fig. 4). The column is
packed according to specific method instructions. Prior to use, the packed column
is pre-washed using hexane. The solvent is drawn down to the top of the packing;
a fraction collection flask labeled #1 is set into place; and the concentrated sample
is transferred to the column using a Pasteur pipet. The concentrated sample

extract flask and the pipet both receive three rinses of solvent to complete
the transfer. Additional hexane is then added to the column, followed by 1.5%
dichloromethane in hexane. When the 1.5% dichloromethane in hexane reaches
the top of the column packing, the fraction collection flask #1 is exchanged for
fraction collection flask #2. Fifty percent dichloromethane in hexane is added
to the column and allowed to fully drain into the fraction #2 flask. This is the
fraction containing the PCDDs/PCDFs. Fraction #2 is then concentrated by rotary
evaporation.
Increased purity may be achieved by running fraction #2 through a second
alumina column. The second fraction #1 and fraction #2 are collected in the same
flasks as the previous step and then concentrated by rotary evaporation. Fraction
#1 must be clearly labeled and archived, so that it can be later referenced if
surrogate recovery is low. The concentrated fraction #2, containing the PCDDs/
PCDFs, is then transferred to a conical sample vial. The transfer is completed by
three rinses of the flask and pipet with hexane. If additional purification is needed,
an activated carbon/silica column may be added to the cleanup process (Environ-
ment Canada 1992a). The sample is then micro-concentrated to approximately
100 III using a gentle stream of pre-purified nitrogen. Samples are stored in the
refrigerator until analysis. Immediately before GC/MS analysis, the sample is
evaporated just to dryness using nitrogen blowdown and a 20 III sample of a
recovery standard solution (Table 2) is added.
4.7 Identification and Quantification of Polychlorinated

Dibenzo-p-dioxins and Dibenzofurans by High Resolution
Gas Chromatography/High Resolution Mass Spectrometry
A detailed discussion of HRGCIHRMS, even as applied to these analytes, is

beyond the scope of this chapter. The following information (drawn from Environ-
ment Canada 1992a) will give the experienced analyst an introduction to the
requirements of this specific method. The US EPA Method 1613 (US EPA 1994c)
is another source of information on this methodology. The gas chromatograph
used must exhibit temperature stability of 0.2C or better over its specified
range of operation; it must be able to accommodate at least three temperature
ramps; and it must accept a capillary column. The column is directly coupled to
a high resolution, double-focussing mass spectrometer. The mass spectrometer
is operated in the electron impact (EI) and selected ion monitoring (SIM)
modes.
The 2,3,7,8-TCDD isomer can be adequately separated from neighboring
isomers 1,2,3,7-, 1,2,3,8-, and 1,2,3,9-TCDD using a 60m DB-5 (polymethyl [5%
phenyl] silicone) column (0.25 mm ID, 0.25 11m film thickness). This column also
separates both hepta-CDD isomers and the four hepta-CDF isomers. However
2,3,7,8-TCDF cannot be resolved from its neighboring isomers on a DB-5 column.
If results from the DB-5 run indicate that 2,3,7,8-TCDF may be present at regula-
tory levels, its separation from 2,3,4,7-, and 1,2,3,9-TCDF can be achieved using a
DB-225 column.
Optimum settings for GC parameters and appropriate time windows for the
time-sequenced SIM MS analysis are extablished from analysis of window-defining
mixtures containing the first and last eluting congeners within each homologue
group. The order of elution is such that five retention windows can be defined
corresponding to the five levels of chlorine substitution (tetra-octa). Under opti-
mum conditions, the interval between the latest eluting tetra-substituted conge-
ners and the earliest eluting penta-substituted congeners is no more than 0.1 min
(Environment Canada 1992a). Parameter settings that produce a 2,3,7,8-TCDD
retention time of 25 min or more represent conditions under which the 4 ClI5 CI
gap is optimized. The following set of parameters can serve as a starting point for
parameter optimization (Environment Canada 1992a): injector temperature 300C
for split-splitless, or ambient for on-column; interface temperature of 290C; tem-
perature program: (1) initial temperature of 100C for split-splitless or 70C for
on-column and hold for 1 min, (2) 100 (or 70) to 200C at 40Cmin-1, (3) 200 to
235Cmin-1, (4) hold at 235C for 10min, (5) 235 to 310C at 8Cmin-1, and (6)
hold at 310C for 15min.
A column performance mixture containing either 2,3,7,8-TCDD or 2,3,7,8-
TCDF and its nearest neighbors at equal concentrations is used to demonstrate
acceptable chromatographic performance. On a 60m DB-5 column, the height of
the valley between the most closely eluting peaks must not exceed 25% of the
2,3,7,8-TCDD peak. On a 30m DB-255 column, neither valley must exceed 30% of
the 2,3,7,8-TCDF peak height. Acceptable chromatographic resolution must be
verified daily. Other commercially-available and well-documented columns can be
used as long as their performance is verified.
The mass spectrometer electron energy should be optimized; it is expected
to be in the range of 28 to 40eV (Environment Canada 1992a). Reference
perflurokerosene (PFK) is leaked into the reference inlet system. The instrument is
tuned to a resolution of 10000 at m/z 304.9824 and, using peak matching, the m/z
380.9760 of PFK must fall within 5 ppm of the exact value (Environment Canada
1992a). Acceptable resolution must be confirmed at appropriate points during
analytical runs. Hard copies of the measurements (peak shapes and widths) must
be available for auditing. The PFK leak is continued throughout analysis with its
level monitored and adjusted so that amplitude of the most abundant lock-mass
ion does not exceed 10% of full-scale deflection. Each lock-mass must be moni-
tored, and its intensity must not vary by more than 10% throughout its time
window. If the lock-mass does not remain constant, this may indicate the presence
of interfering ions, and the sample may require further column clean-up before
analysis.
Table 4 gives the quantification ions and chlorine isotope ratios for the analy-
sis windows. To calibrate the instrument, inject a 1-2 III sample of CS3 (Table 2).
The isotope abundance ratio of all analytes must agree with the theoretical ratio
within the control limits given (Table 4). If any ratio is outside its limits, a problem
exists which must be identified and corrected before proceeding. Any changes in
Table 4. Selected ion masses, ion type and control limits. (Environment Canada 1992a)
Window Compound Quantification ions (m/z) Ion type Control limits

number for isotope ratio
1st 2nd
TCDF 303.9016 305.8987 M/M+2 0.65-0.89

13C12-TCDF 315.9419 317.9389 M/M+2 0.65-0.89
TCDD 319.8965 321.8936 M/M+2 0.65-0.89
13C 12 -TCDD 331.9368 333.9339 M/M+2 0.65-0.89
H 6 CDPE' 375.8364 M+2
PFK 316.9824 Lock
2 P,CDF 339.8597 341.8567 M + 21M + 4 1.32-1.78
13C 12 -P,CDF 351.9000 353.8970 M + 21M + 4 1.32-1.78
P,CDD 355.8546 357.8516 M + 21M + 4 1.32-1.78
13C12-PsCDD 367.8949 369.8919 M + 21M + 4 1.32-1.78
H,CDPE' 409.7974 M+2
PFK 366.9792 Lock
3 H 6CDF 373.8208 375.8178 M + 21M + 4 1.05-1.43
13C12-H6CDF 383.8639 385.8610 M/M+2 0.43-0.59
H 6CDD 389.8157 391.8127 M + 21M + 4 1.05-1.43
13C 12 -H 6CDD 401.8559 403.8529 M + 21M + 4 1.05-1.43
sCDPE' 445.7555 M+4
PFK 380.9760 Lock
4 H,CDF 407.7818 409.7789 M + 21M + 4 0.88-1.20
llC 12 -H,CDF 419.8220 421.8191 M + 21M + 4 0.88-1.20
H,CDD 423.7766 425.7737 M + 21M + 4 0.88-1.20
13C 12 -H,CDD 435.8169 437.8140 M + 21M + 4 0.88-1.20
N,CDPE' 479.7165 M+4
PFK 430.9728 Lock
5 OCDF 441.7428 443.7398 M + 21M + 4 0.76-1.02
OCDD 457.7378 459.7348 M + 21M + 4 0.76-1.02
llC 12 -OCDD 469.7780 471.7750 M + 21M + 4 0.76-1.02
DIOCDPE' 5l3.6775 M+4 0.76-1.02
PFK 454.9728 Lock
'Response of the chlorinated diphenyl ether must be absent for PCDF determination as it has a
similar isotope ratio.
MS parameter settings require that MS resolution must be remeasured. Standard

CSl (Table 2) is used to assesss detection limits; 0.25 pg of TCDD and TCDF must
result in a peak response which is at least five times the background noise level
(SIN ~ 5) for each of the four monitored ions. If these levels cannot be met,
instrument parameters must be adjusted until they are met. Again, resolution
must be determined after any parameter adjustment. The instrument is not in
acceptable operating condition until all detection and calibration criteria are sat-
isfied simultaneously (Environment Canada 1992a).
Specific criteria must be satisfied before sample components are identified as
PCDDs/PCDFs (Environment Canada 1992a). Peak responses for each of the two
selected molecular cluster ions must have SIN ~ 3; chlorine isotope ratios must be
within control limits; peak maxima for both quantification ions must coincide
within 2 s; and peaks representing diphenyl ethers must not be present to interfere
with PCDF. In addition, for congeners for which a labeled analog is present in the
surrogate spiking mixture, all native and surrogate ion peak maxima must coin-
cide within 3 s. For congeners which do not have a labeled analog in the surrogate
spiking mixture, peaks must be within 3 s of the expected retention time.
Quantification is based upon determination of the internal calibration stan-
dard and the relative response factors (RRF). Calculation ofRRFs, limits of detec-
tion, and levels of quantification as well as data reporting, including QA/QC data,
are all discussed thoroughly in Environment Canada (l992a) and will not be
reiterated here.
4.8 Bioassays for Polychlorinated Dibenzo-p-dioxins

and Dibenzofurans Equivalents
Planar halogenated hydrocarbons (PHHs), a group which includes polychlori-

nated biphenyls (PCBs), as well as PCDDs/PCDFs, exert their toxic effects through
the same receptor (Tillitt et al. 1991a). There are strong correlations between liver
P450IA1-associated enzyme induction potency of individual congeners and their
ability to cause typical toxicological effects. This has led to the development of a
bioassay for the presence of PHHs. This bioassay is useful because it integrates the
biological effects of the many PCDD/PCDF congeners.
The bioassay typically involves exposing rat or mouse hepatoma cells to
extracts containing PCDDs/PCDFs and determining AHH (picomoles 3-0H-
benzo[a]pyrene formed per min per mg protein) or EROD (picomoles resolufin
formed per min per mg protein; Kopponen et al. 1994). Bioassay results are typi-
cally reported in terms of 2,3,7,8 TCDD equivalents (Tillitt et al. 1991b). Although
the bioassay approach has obvious abvantages, regulatory agencies have con-
tinued to require chemical analyses rather than bioassays.
5 Conclusions and Future Perspectives
Human societies have long used soils as a repository for their waste products.
Production of large amounts of many different kinds of wastes by industrial
societies has steadily increased during this century. In recent decades human
awareness of the negative environmental consequences of waste disposal has also
increased. In a few cases, including papermill sludge, the addition of waste mate-
rials to land may have beneficial, as well as negative, consequences. Most papermill
sludges contain primarily organic materials originating from wood, with only trace
amounts of potentially harmful organic and inorganic contaminants. Many soils,
particularly those used for agricultural or horticultural production, have relatively
low levels of organic matter compared to their uncultivated state. Forest land, as
well, may undergo losses of soil organic matter, especially during harvesting op-
erations (Pennock and van Kessel 1997). Soil organic matter is believed to be an
important indicator of soil resistance and resilience (Scharpenseel and Becker-
Heidmann 1994). Loss of organic matter indicates soil degradation (Oldeman
1994). Organic matter management, which may include additions of appropriate
waste materials to soils, is an important component of sustainable soil manage-
ment (Robinson et al. 1994).
Analysis of papermill sludge is crucial prior to its use as a soil amendment.
The C/N ratio must be determined and, often, supplemental N fertilization must
accompany the papermill sludge amendment. In addition, levels of trace elements
and PCDDs/PCDFs must be monitored. Research will continue on the lengthy,
exacting, and expensive analytical methodology for PCDDs/PCDFs. In the future,
bioassays for "TCDD equivalents" may prove more environmentally-relevant
than chemical analyses for PCDDs/PCDFs. The pulp and paper industry has made
great strides in reducing PCDD/PCDF production by using non-chlorine-
containing bleaching processes. However, even products produced by totally-
chlorine-free bleaching may contain PCDDs/PCDFs if recycled paper was used
as a raw material in their manufacture (Berry et al. 1993). Although paper com-
panies are pursuing technologies which minimize, or even eliminate their waste
production, papermill sludge will continue to be produced for the foreseeable
future. This is a unique industrial residual in that its responsible use as a soil
amendment has the potential to provide significant environmental benefits in the
form of improved soil quality.
References
Aitken MN, Lewis JG, Evans B (1995) Effects on soil fertility from applying paper mill sludge to
agricultural land. Soil Use Manage 11:152-153
Bellamy KL, Chong C, Cline RA (1995) Paper sludge utilization in agriculture and container
nursery culture. J Environ Qual 24:1074-1082
Berry RM, Luthe CE, Voss RH (1993) Ubiquitous nature of dioxins: a comparison of the dioxins
content of common everyday materials with that of pulps and papers. Environ Sci Technol
27:1164-1168
Brockway DG (1983) Forest floor, soil, and vegetation responses to sludge fertilization in red and
white pine plantations. Soil Sci Soc Am J 47:776-784
Bunnage W, Imada S (1996) Sludge characterization survey. Pulp Paper Can 97:33-36
Clement R (1991) Ultratrace dioxin and dibenzofuran analysis: 30 years of advances. Anal Chern
63:1130A-1138A
Environment Canada (1992a) Reference method for the determination of polychlorinated
dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in pulp and
paper mill effluents. Report EPS l/RM/19 Ottawa, Ontario
Environment Canada (1992b) Internal quality assurance requirements for the analysis of dioxins
in environmental samples. Report EPS l/RM/23 Ottawa, Ontario
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Chemical and biological 2,3,7,8 tetrachlorodibenzo-p-dioxin equivalents in fly ash from com-
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Mahle NH, Lamparski LL, Nestrick TJ (1989) A method for the determination of 2,3,7,8-
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Environmental fate and effects of pulp and paper mill effluents. St Lucie Press, Delray Beach,
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Sustainable agriculture systems. Lewis, Boca Raton, pp 109-134
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tion Agency, Washington, DC
Analysis of Sewage from Anaerobic Purification
of Effluent from a Brewery
F.ScHUR
1 Introduction
If we consider the mass balance of a brewery as a black box, it is easy to perceive

beer production as, essentially, the conversion of water into wastewater (Schur et
aL 1995, 1996). The water mass balance in a brewery can be simply written as
follows: effluent = total water consumption, minus beer, minus loss, for example
(Perry 1997) 4.5 = 6 -1- 0.5. In a brewery with an annual output of 1 million hI the
amount of effluent is about 4.5 million hI.
Wastewater means a loss of raw materials, as well as of intermediate products
and end products when manufacturing beer. To purify the sewage is to treat only
the symptoms of the problem. Getting to the actual roots of the problem means
attempting first to reduce sewage load and sewage volume.
The constituents of brewery effluent are mainly (7B%, Schur et aL 1995) of
an organic nature, resulting from process losses to drain. The COD (chemical
oxygen demand) contributions are spent grains, steep water, last runnings, trub,
wort and beer losses, surplus and storage yeast, cleaning solutions, filter residues
etc.
The liquid phase of a brewery effluent consists mainly of carbohydrates, pro-
teins, and ethanol resulting from wort and beer losses and sodium hydroxide from
washing operations. The solid components are composed of cellulose from spent
grains and silica from diatomaceous earth. Since wort, beer, trub and yeast are
concentrated forms of organic compounds, they constitute very high sources of
oxygen demand. Yeast in a fresh condition has a BOD (biological oxygen demand)
of about 17000 mg 0/1, and in a partially rotten condition up to 500000 mg 0/1
(Hoyer 1992). One litre of beer has a BOD of BOOOOmgO/L
Brewery effluent normally shows the following composition (Fang et al.
19B9; Muhlbauer 1990; Birkenstock 1991; Wildhagen et aL 1991; Hoyer 1992;
Rosenwinkel 1993; Sauer 1993; Hecht et aL 1994; Glas 1995; Hellriegel 1995;
Hoffmann and Zanker 1995; Schu and Leisinger 1995; Schur et aL 1995, 1996;
Ahrens and Schumann 1996; Perry 1997): COD, 1500-5000mgO/l; BOD, 1000-
3000mg 0/1; COD/BOD, 1.3-2.0; N tota)' 15-BOmg/l; Norganic' 1O-30mg/l; Nammonium'
2-5 mgJl; Ptotal ' 5-30 mgJl; Pphosphate' 2-5 mg/l, and settleable solids, 3-30 mllL Waste-
water from a brewery is approximately three to four times more concentrated than
effluent from a community and is therefore predestined for anaerobic treatment
(Konig 1997).

42 F. Schur
2 Anaerobic Effluent Treatment
Extensive investigations and the study of various alternatives with efficiency

analyses has proved that the optimum solution for a brewery is an anaerobic
prepurification of the more highly loaded sewage and an aerobic post-treatment of
it, together with the less loaded wastewater.
An anaerobic treatment process requires a high level of sewage load. There-
fore, it is reasonable to install in a brewery separate wastewater systems, so that the
lightly loaded wastewater is kept separate from the more highly loaded process
effluent. The application of an anaerobic process for the treatment of brewery
effluent shows, in comparison with an aerobic system, the following advantages
(Birkenstock 1991; Hofer 1991; Behmel and Meyer-Pittroff 1995; Hellriegel 1995;
Hoffmann and Zanker 1995; Kiihbeck 1995, 1997; Schur et al. 1995, 1996; Ahrens
and Schumann 1996; Perry 1997):
1. Low sewage sludge residue (one tenth)

2. Low energy demands; in particular, low electricity consumption
3. Extraction of biogas for process heat, about 0.2-0.5 m 3 biogas per kg COD
(Hecht et al. 1994; Behmel and Meyer-Pittroff 1995; Schu and Leisinger 1995;
Kiihbeck 1995,1997; Schur et al. 1995, 1996; Ahrens and Schuhmann 1996; Perry
1997)
4. Smaller space requirements
5. Suitable for effluents with a higher load (COD> 1500mg0 2/1)
6. Less additions of nutrients containing Nand P
On the other hand, the anaerobic process needs some precautions:
1. Control of a higher temperature (>25C)
2. Control of the pH between 6.5 and 7.5
3. Avoidance of odour emissions caused by sulphur-containing volatiles
Of the different anaerobic sewage treatment processes, namely the contact
sludge process (with and without base material), the fixed bed reactor process
(similar to the submerged contact bed), the flow bed process and the floating bed
process, the last achieved the best results when evaluated. This so-called up flow
anaerobic sludge blanket (UASB) process has already proved itself in various
places, and is relatively low cost (Fang et al. 1989; Hofer 1991; Hoffmann and
Zanker 1995; Behmel and Meyer Pittroff 1995; Schur et al. 1995, 1996; Kiihbeck
1995, 1997). Figure 1 shows the flow sheet of the sewage prepurification process in
a brewery (Schur et al. 1995, 1996). One can see the separate flow of the lightly
loaded wastewater, leading directly to the mixing shaft of the aerobic system of
the public sewage works, while the more heavily loaded sewage flows to the
prepurification plant.
Two further important characteristics of the anaerobic prepurification system
are biogas extraction (Fig. 2) and the treatment of the outgoing air (Fig. 3). To
avoid odour emissions it is essential to extract the outgoing air from the entire
Analysis of Sewage from Anaerobic Purification of Effluent from a Brewery 43
Production unit.
lightly loaded
Sewage
Public sewige work
Fig. 1. Flow sheet of the sewage prepurification plant
Flame
_ - - -.....A!mOSPhere
Dry
gasolfleter>-...,.,.........,..---<
Hydraulic-
safety valve
Sand
IIlter J---I~....L.-I--~oIq--.Boiler house
Gas conveyer
y
Condensate output
Collection
Bloreactor 1 channel Bloreactor 2
Acidification
Output
Fig. 2. Biogas extraction

44 F. Schur
AJr washer
Atmosphere
i:==~h;t--" Fresh waler
I ~ Soltened
~ waler
L -___. CMgolng air of\t1e
~wage prepunf\c.aUon
ptant
Waste water from

anaerobic
$Owage Irealment Waste water to public
sawage worXs
Postaerallon tank
Fig. 3. Improved treatment of outgoing air
sewage purification system (by operating in a permanent partial vacuum as

opposed to atmospheric pressure), and to use a special unit for the treatment of
outgoing air. By keeping the sewage prepurification plant strictly separate from the
works sewage network, by means of a syphon system, it is possible to avoid odour
emlSSlOns.
In anaerobic sewage treatment, for the degradation of the organic substances
to the final products carbon dioxide and methane, several metabolic pathways
have to mesh into one another, and different populations of bacteria are needed.
The micro-organisms involved consist of methanogene bacteria like methano-
bacterium, methanobrevibacter, methanothermus and methanococcus etc.; ther-
mophile archae bacteria like methanospirillum, methanococcus, thermoproteus,
pyrodictium spp. etc.; anaerobic bacteria able to form spores like clostridia,
desulfotomaculum etc. and anaerobic gram negative cocci like desulfurococcus,
thermococcus, peptococcus, thermodiscus, sarcina etc. (Rosli 1992).
The process for the degradation of the high-molecular substances can be
schematically divided into four steps (Hofer 1991; Ahrens and Schumann 1996).
Firstly, in the hydrolysis phase the high molecular, mostly undissolved substances
are degraded by exo enzymes from micro-organisms. Secondly, in the so-called
acidification phase, short-chain acids e.g. acetic acid and monomers as well as
alcohols, hydrogen and carbon dioxide are produced from the organic fragments,
like sugar, by fermentative micro-organisms. Thirdly, in the so-called acetogenic
phase, the different acids, like butanic acid, are transformed into acetic acid and
hydrogen. In the fourth step, the so-called methanogenic phase, the reactions
which are taking place are the conversion of acetic acid by acetotrophe bacteria
into methane and carbon dioxide, as well as the conversion of hydrogen and
carbon dioxide by hydrogenotrophe bacteria into methane and water.
For the chemical control of an anaerobic sewage purification system, the
following materials in particular have to be analysed: sewage before and after
treatment, biogas, air leaving the system and sewage sludge.
This chapter concentrates mainly on the methods of chemical analysis
relevant for the control of an anaerobic sewage purification system.
3 Methods of Analysis
3.1 Sewage and Sewage Sludge
3.1.1 Sampling (EDI 1983)
The definition of the places of taking samples, the frequency of sampling, the
method of sampling, the duration of sampling, the subsequent of sample treatment
and the analytical task depend on the purpose of the investigations (analysis).
Because the sample materials can change, they have to be analysed immediately
or, if possible, be preserved. Changes of sample material can take place because of
chemical reactions or biological transformations. Interpretation of the results has
to be based on more than one random sampling, particularly when the composi-
tion of the sample materials varies substantially.
The sampling is as important as the analysis itself. It is not always easy to get
a representative sample out of a heterogeneous material, such as waste with sus-
pended substances, coarse dispersed materials and particles on the surface or as
precipitates. If there is a correlation between the undissolved or inhomogeneously
distributed materials and the parameters to be investigated, each representative
substance must be included during sampling. Any heterogeneous, materials have
to be representative of the whole quantity, particularly in the sewage effluent and
sludge.
During sampling, the rate of flow of the stream, e.g. wastewater effluent,
sludge, biogas or outgoing air has to be measured or calculated. The concentration
of the entire brewery-specific materials is relevant for the evaluation.
Both the number of samples and the time of sampling have to be chosen
to represent characteristic process states. Quantitative proportional samples are
preferred, and in normal brewery practice a time proportional sampling can
be accepted.
For manual and automatic sampling, a lot of equipment, apparatus and tools
are commercially available. These always have to be cleaned before use.
46 F. Schur
If it is not possible to investigate the samples directly at the sampling site,

they have to be kept cool, and taken as quickly as possible to the laboratory.
This is also true for samples which have been preserved by the addition of suit-
able chemicals after being taken. The composition of sewage changes because
of the ongoing physical, chemical and biochemical processes. Biological pro-
cesses or biochemical oxidation can be decelerated by cooling down or adding
suitable chemicals. On the other hand, chemical processes go on until an
equilibrium state is reached, even under cooled conditions, e.g. the redox
reactions.
Correct evaluation of the analysis results is usually only possible if the sewage
flow rate is known.
3.1.2 Carbon (ED! 1983)
Principle. The total carbon (TC) comprises both the organic and the inorganic
carbon. The total organic carbon (TOC) covers the sum of the carbon of the
dissolved substances and dispersed particles in the sewage. Dissolved organic
carbon means either the dissolved part of the total or total organic carbon.
Dissolved substances are defined as those which pass a filter with a pore size of
0.45)lm.
Here, only the measuring principles, the requirements of the measuring
equipment and the sample pre-treatment are described.
In the first step, the organic substances of the sample are oxidised and sub-
sequently the resulting carbon dioxide is determined quantitatively.
Measuring Procedure. For both steps (oxidation and the determination of carbon
dioxide), several different methods and measuring apparatus are available. The
following criteria are decisive when selecting equipment: firstly, the quantitative
complete oxidation of all dissolved and dispersed organic substances. Secondly,
the quantitative determination of carbon dioxide free from interference, so that
one may distinguish between inorganic carbon, such as carbonic acid, hydrogen
carbonate and carbonate already present in the sample and the carbon dioxide
produced by oxidation.
Determination. Using the combustion method, a sample of the sewage or sludge

is placed in a preheated tube filled with a catalyser. In the incandescent tube
flushed with oxygen or synthetic air at approximately 1000C, the organic part
of the sample is catalytically oxidized, and the inorganic parts quantitatively
converted to carbon dioxide. The gas stream conducts the formed and pre-
existing liberated carbon dioxide to the detection system, e.g. an infrared
analyzer or titration system, or after reduction to methane, into a flame ionization
detector.
Differentiation between inorganic and organic carbon: all measuring proce-
dures cover the total carbon; therefore, separation is necessary. The inorganic
carbon is separated from the sample by acidification with acid and subsequent
elimination of the resulting carbon dioxide by percolation.
The detection ability of the measuring apparatus has to be 5 mg organic
carbon per 1. To prevent a blockage it is reasonable to inject only thoroughly
homogenized or filtered samples into the injection devices.
If the sample contains not easily dissolvable or volatile organic nonpolar
solvents or surface active substances, losses can occur with percolation and
perhaps during filtration. A high salt content in the samples interferes with the
catalyser in the combustion apparatus and therefore the life span is reduced.
Samples with more than 5% mineral content have to be diluted. The samples have
to be neutral or acidic, since carbon dioxide from alkaline samples is partly re-
tained as carbonate in the outlet of the combustion tube.
For sample preparation one must distinguish between the determination
of TC and DC as well as TOC and DOC. For TOC, if the sample contains solid
or emulsified particles, it has to be homogenized with an oil free blender or
ultrasonic apparatus for 1-2 min. For DOC, the sample is passed through a 0.45 ~m
filter. In order to keep the filter with the blank value negligibly low, the membrane
filters have to be washed twice by putting them into hot distilled water for several
hours and subsequently drying them. The first 30 to 50 ml of the filtrate is
discarded.
If the percolation method is used, a glass apparatus has to be installed with a
narrow vessel and a glass tube down to the bottom. The connecting tubes should
preferably be made of Teflon or metal. The sample is put into the vessel, the
pH adjusted to 2-3 with hydrochloric acid, and then it is percolated. Generally
a gas flow of approximately 200 mllmin is applied for 5 min, but the completeness
of carbon dioxide elimination has to be checked repeatedly by percolation and
measuring.
Calibration. For calibration and determination, the equipment instructions have

to be considered. The concentrations of the standard solutions for calibration have
to correspond with the concentrations of the samples. The calibration curve has to
be determined daily. If the percolation method is used, the standard solutions also
have to be acidified and percolated using purified nitrogen, synthetic air or oxygen.
To prepare the organic carbon stock solution, 2.215 g potassium hydrogen-
phthalate, CsHsK04' p.a. (analytical grade), is dissolved in 1000ml distilled water.
The solution contains 1000mg organic carbon per 1, and is stable under cool
conditions for 3 months.
To prepare the inorganic carbon stock solution, 4.404g sodium carbonate,
Na2C03 , p.a., dried at 105 DC, and 3.497 g sodium hydrogencarbonate, NaHC0 3,
p.a., dried at 105 DC are dissolved in 1000ml distilled water. The solution contains
1000mg inorganic carbon per 1. The standard solutions for calibration are freshly
prepared daily by diluting the stock solution.
Calculation. For evaluation, a calibration curve is normally used. An extrapola-

tion of the calibration curve is not allowed.
48 F. Schur
3.1.3 Organic Nitrogen (EDI 1983)
Principle. In sewage, organic bound nitrogen is mainly found in proteins and

degradation products like amines, amides, urea and amino acids. The nitrogen of
these substances is converted to ammonium during the digestion process. The so-
called kjeldahl-nitrogen includes not only the organic nitrogen, but also the origi-
nal ammonium nitrogen present in the sample. The digestion solution can also
be used for the determination of total phosphorus. During wet digestion with
sulphuric acid, the oxidation acids hydrogen peroxide and perchloric acid, as well
as selenium dioxide acting as a catalyst, the organic nitrogen forms ammonium
ions. These are determined together with the ammonia/ammonium already
present. The difference between the results of this determination and the separate
determination of ammonium leads to the quantity of organic nitrogen present.
Through choice of suitable sizes of samples for wet digestion and from the digested
solution, the method can be applied over a wide range of concentrations. Using
digestion, nearly all amines, amides, amino acids, proteins and heterocyclic
N-components can be recorded. Pyridine and some of its derivatives as well as
nitro-, nitroso-, oxime-, azo- and semi carbazide groups are in completely or not
included at all.
Determination. The sample size should be adapted to the amount of digestion

acid. On the other hand, variation in the results, particularly with samples contain-
ing suspended material, can be reduced by introducing a larger sample for analy-
sis. For example, in sedimented sewages, 20 ml samples can be used. The chosen
sample is placed in a digestion glass tube, e.g. length 30 cm, diameter 42 mm, with
a glass stopper. Two ml of the digestion acid (0.1 g selenium dioxide p.a. in a 500-
ml flask, dissolved in approximately 200ml of distilled ammonia-free water. Dur-
ing cooling, 75ml conc. sulphuric acid p.a. and 17ml60% perchloric acid, p.a., are
added. The cooled solution is made up with distilled, ammonia-free water to the
required volume), 1 ml of 30% hydrogen peroxide p.a. and some stones to prevent
boiling delay are added. The digestion tubes are put into the aluminium digestion
block (to obtain fast evaporation times, heat transfer between the digestion block
and the glass tubes should be as fast as possible; therefore, the bottoms of the
digestion tubes should be adapted to the outlets in the digestion block) and the
samples are evaporated at 160 DC and subsequently digested at 190 5 DC for
90 min. To establish the digestion blank, at least two tubes are treated in parallel
with 2 ml of the digestion acid and 1 ml of hydrogen peroxide. The slightly cooled
digested solution is dissolved with 100 ml of distilled, ammonia-free water. If at the
same time the total phosphorus has to be determined, in order to hydrolyse the
partly formed pyrophosphates, the digestion tubes are closed with a so-called
cooling finger and kept for at least 3 h or better overnight on the digestion block,
which is covered with an asbestos-like plate. In the digested solution the content
of ammonium is determined according to a method described separately. For
example, if the maximum concentration of organic nitrogen and ammonium
together in the sample is about 40 mg/l and the sample size is 20 ml, the volume of
the digestion solution should be 5 ml; if the estimated concentration is 4 mg/l and
the sample size 50 ml, the digestion solution should be 20 mI. The concentration
with strong acids should not exceed 0.5mMH+ in the introduced sample. Neutral-
ization is carried out with In sodium hydroxide solution. Ten ml of the digested
solution is mixed with one drop of nitrophenol solution (0.1 g 4-nitrophenol dis-
solved in demineralized water to make 100ml) and titrated with In sodium hy-
droxide solution, up to the point of colour change. The chosen volume of digestion
solution can be calculated from the consumption quantity of sodium hydroxide
solution necessary for neutralization.
To keep the digestion blanks low, one has to carry out the manipulation under
strict cleanliness, therefore the equipment has to be checked before use by testing
with blank series. Also, new dosing or dispensing systems for the digestion acid
can cause remarkably high blanks. The same is true for acids or hydrogen peroxide
out of flasks stored for a long time. The digestion glass tubes have to be kept closed,
except during their use in the digestion block. In the laboratory no ammonia may
be used or stored.
Calculation of the Results. The following equation is used:
mgN/1 = ([(Ilg N in B.100/B)-(llg N in ClOO/ A)l1/ A)- D,
where A is the sample volume used for digestion, B is the sample volume of the
digestion solution, C is the volume of the blank digestion solution and where D is
the ammonia/ammonium in the sample in mg/I.
3.1.4 Ammonia/Ammonium (EDI 1983)
Principle. In sewage, ammonium and ammonia are present in equilibrium. By

increasing the temperature and the pH, this equilibrium is shifted in favour of
ammonia. The method comprises the sum of ammonia and ammonium.
By the effect of active chlorine on ammonia in an aqueous solution, chloram-
ines are formed, which react in an alkaline milieu under the influence of a catalyst
to give greenish-blue indigo phenols (the so-called Berthelot reaction). Sodium
dichlorisocyanurate serves as an active chlorine compound, sodium salicylate as a
phenolic component and disodium-pentacyano-nitrosylferrate as a catalyst.
If l-cm cuvettes and an introduced sample of 50 ml are used, the range
of analysis is between 0.05 and 1.0mgN/I. At higher concentrations a smaller
sample has to be taken, so that it contains not more than 0.05 mgN/I. Undiluted
samples with more than approximately 10mgN/I can simulate a too low am-
monium content, because of the fast decline of the extinction after the maximum
wavelength. Interference by heavy metal hydroxides can be prevented by addition
of complexing agents. Strong hazes are removed by adjusting the sample to
pH 6-7 and subsequent filtration. Both types of interference can be eliminated
by distillation, if the easily destroyed organic nitrogen components are not
50 F. Schur
hydrolysed to ammonia, thus distorting the results. Strong acids and weak acids up
to 0.5 mM in the sample being analysed, as well as strong alkaline solutions, up to
0.25 mM, do not interfere with colour development. If significant amounts of
chlorine undergo reduction, e.g. more than 1 mgCl z in the analysis sample, the
determination will be adversely affected in few minutes. These effects can be
eliminated by increasing the concentration of dichlorisocyanurate. If urea in the
sewage is to be determined completely as ammonium, the urea first has to be
hydrolysed with urease.
Direct Determination. Fifty ml of the sample, which should not contain more than
0.05mgN, can, if necessary, be filtered or diluted. Step by step the following
reagents are added to 21 of solution II: 0.2 g disodium-pentacyano-nitrosylferrate
(also known as nitroprussidsodium) NazFe(CN)sNO2H 20 and 17 g sodium salicy-
late, C7Hs0 3Na, p.a., dissolved in demineralized water and made up to 100mi. The
solution, if kept dark and cool, is stable for about 1 week. Two ml of solution IV
(oxidation reagent: 100 ml solution I and 25 ml solution III) are mixed just before
use. Solution I: 100g trisodiumcitrate, C6HsNa3072HzO p.a., and 14g sodium
hydroxide, p.a. are dissolved in demineralized water and made up to 500 mi.
Solution III: 1 g dichlorisocyanuracid-sodium salt, C3N3CI 20 3Na, is dissolved in
demineralized water and made up to 100mI. The solution has to be prepared fresh
daily. After each addition the solution has to be thoroughly mixed. After a reaction
time of 120 min at room temperature, or preferably 20 min at 60C, the extinction
is measured against a blank at 690 nm. Higher temperatures lead to lower extinc-
tions. The extinction of the sample remains constant for at least 6 h. The standards
for the calibration are prepared in the same way.
Determination After Distillation. If interference cannot be eliminated by diluting

or filtering, the determination has to be carried out after distillation. One hundred
ml of the sample are placed in the distillation flask and two drops of phenolphtha-
lein solution are added. The receiver with 10 to 20 ml ammonia free water (distilled
water can be kept ammonia free by percolation over a strong acidic cation ex-
changer) is connected so that the end of the cooler dips into the water. Immedi-
ately before starting the distillation, 10 ml of a borate buffer solution (10.6 g boric
acid and 75 ml In sodium hydroxide are dissolved in ammonia-free water and
made up to 1000ml) is added to the sample. Seventy to 80ml is distilled. An
elevated temperature of the distillate has to be avoided. The apparatus used must
be checked with standard solutions (ammonium stock solution: 0.764g ammo-
nium chloride, NH 4 CI, p.a., dried over silica gel in an exsiccator, dissolved in NH 4 -
free water, and made up to 1000mI. One ml of the solution contains 0.2mg of
ammonium nitrogen. Ammonium standard solution: 10ml of the ammonium
stock solution is diluted with NH 4 -free water to 100 mi. This step is repeated, so
that 1 ml solution contains 21lg ammonium nitrogen. The solution has to be
prepared on demand in respect of the quantitative transfer. With high ammonium
contents, the ammonium can be directly titrated. In other cases, the ammonium is
determined at a suitable dilution.
Calculation. The content of free ammonia can be taken out of a sigmoid equilibra-
tion curve for a given pH value according to the following relationship:
[NH4 +].K NH .+
[NH 3 ] = [ ]
H3 0
where KNH; is the acidity constant of ammonium at 20C, which is -log KNH; =
9.37 or -log KNH + = 2706 (T is the absolute temperature in degrees Kelvin).
T+0.739
mg N in the introduced sample x 1000
Note t h at mg Nt I = - - - - = ' - - - - - - - - - - - " ' - - - - -
ml of the introduced sample
3.1.5 Phosphorus (EDI 1983)
Principle. Phosphorus occurs in sewage as ortho-phosphate, polyphosphates and

organic bound phosphorus substances. All dissolved phosphorus compounds and
undissolved phosphorus substances have to be converted to orthophosphate prior
to the actual determination. This digestion can be carried out according to the
procedure described under Section 3.1.3 for the determination of organic nitrogen,
with glass tubes in an aluminium digestion block or according to the wet mineral-
ization process described next. The solution resulting from the wet mineralization
can also be used for the determination of organic nitrogen described in Section
3.1.3.
In the wet mineralization process using sulphuric acid, hydrogen peroxide
and perchloric acid as oxidation agents, as well as selenium oxide as a catalyst, the
phosphorus compounds are transformed into ortho-phosphate and the suspended
phosphorus substances go into solution. The small quantity of acid mixture
should, at the end of the digestion, be largely eliminated, since, for the determina-
tion of phosphate, neutralization is not necessary.
An aliquot of the digestion solution is used for the determination of the ortho-
phosphate which forms, together with molybdate in an acid solution, the 12-
molybdato-phosphoric acid. One sixth of molybdenum cations are reduced by
ascorbic acid from the six- to the five-value state, to the so-called molybdenum-
blue. The addition of an antimony compound catalyses the reduction reaction and
the colour development.
By variation of the size of the sample for the analysis by wet digestion and of
the digested solution for the determination of phosphate, the method can be
adapted to a wide range of different concentrations.
Wet Mineralization. From the well mixed sample, a volume, which presumably
contains 40-200llgP, has to be transferred into a 200 to 300ml Kjeldahl flask or
digestion glass. Two ml conc. sulphuric acid is added and after addition of P-free
stones to avoid boiling delay, the solution is evaporated until all the water is
removed. To avoid losses by splashing at the end of the concentration process, the
52 F. Schur
liquid should not boil. To avoid phosphorus losses, one must not allow vapours of
sulphuric acid to develop. Also, carbonization and crust formation has to be
avoided. After a partial cooling, three drops of 30% hydrogen peroxide p.a. are
added, and the batch is heated for 10 min at about 260C. The upper part of the
flask should not overheat. The addition of hydrogen peroxide and the subsequent
heating are repeated, until the liquid is colourless and the undissolved remains
have turned white. After a second partial cooling, two drops of hydrogen peroxide
are added. Subsequently, the mixture heated for approximately 30 min to totally
remove the hydrogen peroxide. After cooling down, approximately 50ml of dem-
ineralized water is added and the mixture is heated for 15 min until it starts to boil.
The pyrophosphate eventually formed is converted to ortho-phosphate. During
boiling, the volume must not be reduced to less than 20 ml; if necessary, deminer-
alized water should be added.
Determination. The contents of the Kjeldahl flask is transferred to a rinsed 100-ml

graduated flask and under rinsing with demineralized water made up to the 100 ml
volume. The diluted solution is called the digestion solution. An aliquot (adequate)
part of the digestion solution, which should contain a P content between 5 and
50llg, is placed in a 50 ml graduated flask, and if necessary made up to about 30 ml
with demineralized water. A blank and several standards containing 5 to 50llgP
are also placed in 50 ml flasks and are made up to approximately 30 ml with
demineralized water. One drop of a p-nitrophenol solution (1 g of p-nitrophenol is
dissolved in demineralized water and made up to 100 ml) is added to the blank and
standards. Subsequently, 30% sodium hydroxide solution is added until a yellow
colour can be observed. Next, 10% sulphuric acid has to be added drop by drop
until the solution is again colourless. Following this, 5 ml of the mixed reagent
(mixed reagent: 100ml reagent I and 50ml reagent II) is transferred into a 200ml
flask, 1.69 g ascorbic acid, C6Hg0 6 is added and the volume adjusted to 200 ml with
demineralized water. The mixed solution has a yellowish colour. Reagent I: 224ml
of conc. sulphuric acid is added in a 1000-ml flask containing 500ml demineralized
water, caution!; 19.2 g ammonium molydate [(NH4)6M070244H20] is dissolved
separately in approximately 200 ml demineralized water. Both solutions are mixed
at room temperature in a 1000-ml flask and brought to volume in a 1000-ml flask
containing 500ml demineralized water. Reagent II: 400mg potassium antimonyl-
tartrate [K(SbO)C 4H40 6O, 5H20] is dissolved in demineralized water and brought
to 500 ml. The volume is made up with demineralized water to 50 ml and mixed. At
the earliest after 12 min, or at the latest after 120 min, the extinction is measured
against a blank at 700 nm or preferably at 800 nm.
To eliminate the influence of interfering substances introduced into the
analysis, e.g. nitrite> 10 Ilg N in the sample, 0.3 ml of a 5% sodium azide solution,
and 1 ml 30% acetic acid v/v, are added to the sample. In this way, interference
up to at least 30 Ilg N can be eliminated. If the active chlorine in the used quantity
of sample > 251lg C12, a sodium thiosulphate solution is added to reduce the
active chlorine. A surplus of Sp/- up to IOOllg does not interfere with the
determination.
To eliminate the effect of chromate in the sample of> 30 I-lg Cr (VI), the sample
is acidified to pH 3 and the chromate is reduced by adding a sodium thiosulphate
solution and holding a reaction time of 5min. If pyrophosphate is present, the
quantity of the sample for analysis is chosen to contain 2 to lOl-lgP in a mixture to
be measured photometrically. At the given reaction time of 12-20 min, a maximum
of 1% of the pyrophosphate present is hydrolysed.
Calculation. The following equation is used:

mgP/1 = mg P(in E2)100/E21000/E1,
where E1 is the sample volume introduced to the mineralization in ml, and
E2 is the volume of digestion solution introduced to the determination in ml.
A phosphate test solution with a defined P-concentration is prepared out of
a phosphate stock solution. The phosphate stock solution is prepared thus:
4.393 g potassium hydrogen phosphate, KH2 P0 4 , p.a., is dried at 105C, then
dissolved in a 1000-ml flask with demineralized water. One ml cone. sulphuric acid
p.a. is added for preservation, and the volume is made up to 1000 ml with deminer-
alized water. One ml of the stock solution contains 1 mg P. To make the phosphate
test solution, the stock solution is first diluted 1: 500 resp.; made up to 250 ml.
The test solution contains 2 I-lgP!ml. It is stable for approximately 2 days in a dark,
cool place.
3.1.6 Sulphate (EDI 1983)
Principle. After exchange of the cations, an adjusted barium chloride solution is

added in excess and the unreacted barium is titrated complexometrically. The
sulphate content can be calculated from the difference between the barium content
added and the amount later titrated.
Determination. One hundred and fifty to 200 ml of the sample (maximum

450mg sulphate!l) is percolated through a cation exchange column (0 15mm,
15 cm column length strong acidic cation exchange resin (e.g. Dowex 50 WX8,
20-50 or 50-100 mesh or Amberlite IR 120, preferably with indicator) brought
into the H-state with 70% hydrochloric acid, and eluted with demineralized
water) using a flow rate of 3 to 4 drops per s. The first 50 ml of the percolate
is dis cared. One hundred ml of the following percolate is pipetted into a
wide-necked 250-ml flask, and heated up to boiling point. To this, 25ml of a
0.02 M barium chloride solution (4.886 g BaC122H 20 p.a. in 11 demineralized
water) is added and kept lightly boiling for a further 2 min. The solution is
cooled down for at least 6 h, or overnight. For titration, the solution is warmed
up to 50C, 5 ml of buffer solution (5 g of disodium-magnesium-ethylene-
diamine-tetraacetate, CIOH120sNzNazMgHzO, is dissolved in 100mi of demin-
eralized water and a solution of 35 g ammonium chloride p.a. in 900 ml of
25% ammonia, p.a.) and 10 drops ofthe indicator solution (0.5 g Eriochrome black
54 F. Schur
T and 4.5 g hydroxylammonium chloride, HONH 3Cl, are dissolved together in

100 ml 95% ethanol) are added, and immediately the titration with 0.02 M EDT A
solution (7.445 g dis odium dihydrogen-ethylene-diamine-tetraacetate, EDT A,
CIOH140sN2Na22H20 dissolved in demineralized water and made up to 11) is car-
ried out as quickly as possible to the point of colour change to real blue. Illumina-
tion of the titration flask from the bottom is valuable in making the colour change
noticeable.
A blank a test solution of demineralized water, 25 ml barium chloride solution,
5 ml buffer solution and the indicator is prepared and titrated. The method is
suitable for waste water with more than 5 mg sulphate per 1. All cations able to react
with ethylediaminetetraacetate are separated by cation exchange. A haze in the
sample can prevent the detection of the exact point of colour change, so the sample
has to be filtered before analysis.
Calculation. The difference between the amounts of EDT A solution consumed, in

ml, by the test sample and the blank, is equal to the barium chloride consumed
during precipitation of the sulphates. One ml 0.02 M EDT A is equal to 1.92 mg
sulphate. If 100 ml of the percolate is used, the sulphate concentration is calculated
as follows:
mgSOt /1 = (A - B)19.2,
where A is the amount of 0.02 M EDT A consumed by the sample (in ml), and B is
the amount of 0.02 M EDT A consumed by the blank (in ml).
3.1.7 Sulphite (EDI 1983)
Principle. The sulphites in wastewater are mostly dissolved and comprise sulphite
ion SO/-, the hydrogen sulphite ion HS0 3- and sulphurous acid, H2S03' The rela-
tionship hydrogen sulphite/sulphite is given by the pH value of the waste water
(pKs at 20C is 7.3 (HS0 3- _ H+ + SO/-). Substances which liberate hydrogen
sulphite ions, like disulphite or pyrosulphite are also detectable by this method,
although substances like thiosulphate are not.
The sample is preserved by addition of sodium tetrachloromercurate. Sulphite
reacts with sodium tetrachloromercurate to produce a stable, less volatile complex:
The coloured complex is measured photometrically.
Determination. Normally, preserved samples are tested. Samples with a pH of

less than 5 have to be neutralized by adding 0.1 n sodium hydroxide immediately
after sampling. The resulting solution is subsequently mixed with TCM-
amidosulphonic acid solution (54.4g mercuri-II-chloride, p.a., and 23.4g sodium
chloride, p.a., dissolved in 800 ml of demineralized water. Separately, 5 g
amidosulphonic acid, p.a., is dissolved in demineralized water and neutralized to

pH 7 with In sodium hydroxide solution - theoretically 51.5ml is necessary. Both
solutions are combined, and brought to 1000ml with demineralized water) in the
ratio 5: 1 v/v, sample to 10 ml TCM -amidosulphonic acid.
If thiosulphate is present or if, during addition of the TCM-amidosuphonic
acid solution, a precipitation or colour change is observed, the determination has
to be carried out immediately after sampling.
For determination at the place of sampling, the sample size has to be chosen
so that 3 to 30 ~g S03 2- is present. One to 20 ml of the pretreated sample is placed
in a 25-ml flask, depending on the sulphite concentration. If necessary, the sample
is diluted with demineralized water to a volume of about 15 ml. Two ml of
amidosulphonic acid solution [0.5 g amido-sulphonic acid, p.a. (= sulfamine acid,
NH 2S0 3 H, p.a.)] is dissolved in demineralized water and brought to 100ml
(the solution is stable in a closed flask for a few days at least). One ml of EDTA
solution is added (4g of the disodium salt of ethylene-diamine-tetraacetic acid,
CIOHI40sN2Na22H20, p.a. (dissolved in demineralized water and made up to
200ml) is added to the sample and to the blank (lOml demineralized water).
After 10 min 1 ml para-rosanilin test solution (stock solution: 0.2 g para-rosanilin,
e.g. para-fuchsin standard Fluka, for microscopy, suspended in a mixture of
10ml methanol and 10ml demineralized water). The solution is stable for 1
week. Test solution: 4 ml of the stock solution is mixed with 6 ml conc. hydrochlo-
ric acid p.a. and is made up to 100ml with demineralized water. The mixture has
to be kept still before use for 30 min. The red colour becomes lighter immediately
after mixing; the remaining colour should be at most a light yellow-brown. The
solution remains stable for about 1 day. One ml 0.2% formaldehyde solution (l ml
40% formaldehyde p.a.) is diluted with demineralized water up to 200ml. The
solution is stable for several weeks). These recipes are thoroughly mixed, and
added to each sample and blank. The volume is always made up with demineral-
ized water, mixed and kept in the dark for 30 min. The colour intensity is measured
at 560 nm against a blank. The blank, according to the quality of the para-rosanilin,
can have a light pinky colour.
For determination in the laboratory without distillation, the determina-
tion has to be carried out at the latest 24h after sampling. The sample for
analysis should contain 3 to 30 ~g SO/-. According to the estimated concen-
tration of SO/-, 1 ml to at most 12 ml of the preserved sample is pipetted into
a 25-ml flask. Two ml TCM-amidosuphonic acid solution has to be present in
the test batch. If necessary, the sample size has to be completed as shown in
Table 1.
One also adds 2 ml TeM -amidosulphonic acid solution to a blank (10 ml
demineralized water). Each sample and blank batch is combined with 1 ml
of EDT A solution, 1 ml para-rosanilin test solution and 1 ml formaldehyde solu-
tion, thoroughly mixed, and then made up to 25 ml with demineralized water,
mixed once again and kept in the dark for 30 min. The extinction is measured
at 560nm.
56 F. Schur
Table 1. Quantities for completion of TeM solution
Size of preserved sample Sample contains TeM Necessary completion of TeM

(ml) solution (ml) solution (ml)
12 2.00 0
10 1.65 0.35
5 0.85 1.15
3 0.50 1.50
2 0.35 1.65
0.15 1.85
Calibration. The sulphite stock solution and particularly the sulphite test solution
are unstable. Therefore the following procedure is recommended. Pipette 2 ml
of TCM-amidosulphonic acid solution into each of several 2S-ml flasks. The
exact content of the sulphite stock solution (119 mg sodium disulphite = sodium
pyrosulphite, NazSzOs' p.a. dissolved in 100ml demineralized water; 1 ml of
this solution containing approximately ImgS0 3 2- and prepared fresh daily) has
to be determined by titration (Sml O.ln potassium jodate is mixed with ISml
demineralized water in a flask). Next, 0.5 g of potassium jodate, 30 ml 2 n hydro-
chloric acid and 5.0 ml sulphite stock solution are added. The titration is carried
out until decolouring with sodium thiosulphate. Just before reaching the end
point, starch solution is added. A blank without sulphite is prepared and titrated
in the same way. The difference in thiosulphate consumption between the test
samples and the blank determines the calculation: 1 ml 0.1 n sodium thiosulphate
- difference - is equal to 4.003 mg 50 32 -. Subsequently, the stock solution is diluted
with demineralized water 1: 200 (1 ml of the test solution contains approximately
S!lg S03 2-)
Immediately after, 0.5 to 6 ml of the test solution is pipetted into the prepared
2S-ml flasks. Each is rinsed with approximately Sml of demineralized water and
mixed with the TCMM amidosulphonic acid solution already present in the flask.
After preparing the different batches, the content of the sulphite stock solution is
again determined by titration. From the results of the first and second titrations,
the average value is calculated and then the exact content of the sulphite test
solution and of the standards is calculated. To each of the blank and the standard
batches, 1 ml EDT A solution, 1 ml para-rosanilin test solution and 1 ml formalde-
hyde solution are added step by step, and continually mixed. The batches are made
up with demineralized water, mixed and kept in the dark for 30 min. The colour
intensity is measured at 560 nm.
The calibration curve has to be checked periodically.
Calculation. After determination at the sampling place:
mg S03
2-/I (samp I)e = mg S032-(sample in the analysis) x 1000
--'''----=----'----''---------'--'-----
ml sample in the analysis
After determination in the laboratory without distillation:
z-/l ( 1) mg S03Z -(preserved sample in the analysis) x 1000

mg S03 samp e = .
1.2ml preserved sample in the analysis
3.1.8 Sulphide (EDI 1983)
Princicple. Sulphide is dissolved in waste water as hydrogen sulphide, HzS, as

hydrogen sulphide ions, HS-, and as sulphide ions, Sz- and in bound complex form
as complex ions e.g. as a silver sulphide complex. Furthermore, undissolved
sulphides can occur.
The ratio hydrogen sulphide/hydrogen sulphide ions/sulphide (sulphide here
means the sum of the dissolved sulphides) is mainly given by the pH of the waste
water. Sulphide ions are present at a pH above 12. The dissociation of hydrogen
sulphide is defined with the pKa values as follows:
pKaJ at 20C = 7.1 :HzS r= H+ + HS-
pKaZ at 20C = 12.4: HS- r= H+ + Sz-
pKatotal at 20C = 19.5:H zS r= 2H+ + SZ-.
Sulphide is unstable, therefore preservation immediately after sampling

is necessary. Sulphide is precipitated as zinc sulphide by addition of zinc acetate
and an alkaline buffer solution. Zinc sulphide is distributed in a precipitate
of zinc hydroxide formed in parallel. Sulphide forms with N,N-dimethyl-
p-phenylenediamine leucomethylene blue (III), which can be oxidized by
three-valued iron to methylene blue IV. The resulting colour is measured
photometrically.
At high sulphide concentrations, the described reaction can be drastically
affected and the result at a few hundred mg S/l can be negative. Therefore, a pretest
with lead nitrate has to be carried out at the place of sampling.
The detection limit of the described method is approximately 0.02 mg S/l, the
optimum concentration is 0.05 to 1 mg S/l of the sample used for the analysis.
Normally, the dissolved sulphides are to be determined and therefore the sewage
has to be filtered using a filter paper. If the undissolved acid-soluble sulphides need
to be determined too, interference from suspended material cannot be eliminated
by filtration. However, in this case, the absorption capacity of the suspended
substances for methylene blue in most cases can be fully removed by addition
of 0.8ml methylbenzethonium chloride (1 g methylbenzethonium chloride = N-
benzyl-N ,N -dimethyl-N -4-( 1,1 ,3,3-tetramenthylbutyl)-tolyloxyethoxyethyl-
ammonium-chloride, CzsH44ClNOz, e.g. from Merck, dissolved in demineralized
water and made up to 100ml) to the precipitate. During filtration of sewage at
a pH lower than 6, high losses of sulphide can occur. Interference from dissolved
substances, e.g. sulfite, can be eliminated by separating sulphides such as zinc
58 F. Schur
sulphide prior to the determination. The preserved sample has to be stored in a

cool place.
Pretest. The sample is to be filtered using filter paper immediately after sampling.
The first 20 ml of the filtrate is discarded. If the pH of the sample is below 7, prior
to filtration it has to be increased to about 8 using sodium hydroxide.
Fifty ml of the filtrate is put into a glass cylinder and 1 ml of lead nitrate
solution is added while stirring (3.2 g lead nitrate, Pb(N0 3)2, p.a. is dissolved in
demineralized water, 2ml conc. acetic acid p.a. is added and made up to 200ml
with demineralized water). In parallel, 50ml of the same filtrate, without lead
nitrate, is pipetted into a second glass cylinder. After some minutes the resulting
colours of the two batches are compared. In bright and clean samples, 0.05 mg S/l
can be detected. In coloured sewage samples the detection limit is higher. With
sulphide concentrations of a few mgll a brownish-black colour of lead sulphide
can be observed; at higher sulphide contents a black precipitate can be seen.
Sulphate can interfere in the pretest if the concentration is above 50mg S042-/1
because haze is formed by lead sulphate. This, combined with a low sulphide
content, can impair the visual detection of the brownish-black colour.
Determination of the Dissolved Sulphide. Fifty ml of the filtered sample should

be measured. If, according to the results of the pretest with lead sulphide, a con-
tent of more than 1 mg S/l is expected, a smaller sample has to be taken and diluted
with demineralized water and made up to 50 ml. Immediately, 2 ml of zinc
acetate solution (2g zinc acetate, Zn(CH3COO)22H20, p.a., dissolved in 100ml
demineralized water) and lOml of borate buffer solution, pH 9 are mixed (l0.6g
boric acid, H3B0 3, p.a. with the addition of 75 ml 1 n sodium hydroxide solution,
dissolved in demineralized water and made up to 1000 ml). The preserved sample
has to be kept cool until determination, which has to be carried out as quickly
as possible.
In the laboratory, a blank with 50 ml demineralized water, 2 ml zinc solution
and 10 ml borate buffer solution has to be prepared. The preserved samples and the
blank are centrifuged and the upper phase liquid is decanted from the precipitate
and discarded.
In each of several 50-ml graduated flasks, each 5 ml of the dimethyl-p-
phenylenediamine solution (1 g N,N-dimethyl-p-phenylenediammoniumchloride,
CsH14N2C12) is placed in a 1000-ml flask and dissolved in 500ml demineralized
water. Next, 200ml conc. sulphuric acid, p.a., is added and, after cooling, the
volume is made up to 1000ml with demineralized water. The solution is stable for
several months. One ml of the ammonium ferri (III) sulphate is mixed (25 g
ammonium ferri (III) sulphate, FeNH4(S04)212H20, p.a., are dissolved in a 200-ml
graduated flask in about 150 ml of demineralized water. Next,S ml conc. sulphuric
acid, p.a. is added and made up to volume with demineralized water. The precipi-
tates of the blank and the samples are suspended in approximately 20 ml of dem-
ineralized water and are added to the prepared mixtures in the 50-ml flasks while
mixing continually. The centrifuged precipitates are dissolved. The centrifuge
glasses are immediately rinsed with approximately 15ml of demineralized water

and made up to the volume of the flasks (50 ml). Even in sulphide-free batches, a
red colour can temporarily be observed. In the batches containing sulphide, a blue
colour develops within a few minutes. The blue colour should be measured in a 40-
or 50-mm cuvette against the blank at 670nm, at the earliest after 30min, and at
the latest after 120 min.
Calibration. As the sulphide stock solution and particularly the sulphide test
solution are unstable, the following procedure is recommended for calibration.
First, the exact content of the sulphide stock solution (use sodium sulphide,
NazS9HzO, p.a., which shows no decomposition) has to be determined by titration.
Colourless pieces are washed with some water, dried with paper and subsequently
reduced to small pieces. Next, 0.75 g of this material is dissolved in demineralized
water and made up to 100 ml. One ml of the sulphide stock solution contains
approximately 1 mgS. The sulphide stock solution is stable for 2 days at best. For
titration,S ml 0.1 n potassium jodate is placed in an Erlenmeyer flask and mixed
with 15 ml demineralized water. Next, 0.5 g of potassium jodide, 30 ml 2 n hydro-
chloric acid and 5 ml sulphide stock solution are added. Because of the precipita-
tion of sulphur, a white coloured haze is observed. The titration is carried out with
0.1 n sodium thiosulphate to the point of decolourization, and just before getting
to the end a starch solution is added. A blank is prepared and titrated in the same
way. The difference between the consumption of thiosulphate by the blank and by
the sample is used for the calculation. One ml 0.1 n sodium thiosulphate is equal to
1.603mgS.
Secondly, the stock solution is diluted 1: 100 with demineralized water. The
resulting test solution contains approximately 10)lgS/ml. Immediately, standard
batches with 0.5 to 5.0 ml of the test solution and a blank are prepared. The
introduced sample with a defined amount of S is completed to 50 ml with deminer-
alized water. Subsequently, 2 ml of zinc acetate solution and 10 ml borate buffer
solution are added to each 50 ml bath just created and mixed.
After the preparation of these batches, the content of the sulphide stock
solution is determined again by titration. From the results of the first and second
titrations, the average is calculated and with this value the exact content of the
sulphide test solution is calculated. The further treatment of the standard batches
is done in the same manner as with the samples. The calibration curve has to be
checked periodically.
3.2 Air and Biogas
3.2.1 Hydrocarbons (VDI 1975)
Principle. The carbon atoms bound in organic molecules are ionised in a hydro-
gen flame. This results in a flow of ions in an electrical field which is amplified and
monitored. The current is proportional to the number of organically bound carbon
60 F. Schur
1 G
~T'"J
p7 8
111
6 7J
2 T
8
S
Fig. 4. Gas analyser with flame-ionization detector. 1 Burning air intake; 2 burning gas intake;
3 trial gas intake; 4 pump (heatable); 5 trial gas surplus; 6 bypass; 7 pressure gauge; 8 capillaries;
9 burner with mixing chamber; 10 flame; 11 trap electrode; 12 amplifier; 13 instrument display;
14 measuring-signal exit
atoms in the sampled air. The proportion factor depends on the kind of binding,
the binding partners of the carbon atoms as well as the apparatus and the measur-
ing conditions.
Sampling. Figure 4 shows the gas analyser schematically, and Fig. 5 shows the
measuring equipment. To create the flame, hydrogen or hydrogen mixtures and
air free of organic substances are used. During the analysis, the burner's gas and
the sample gas are mixed before reaching the flame. The flame is also fed with air
separately. The flow rate of the burner's gas has to be adjusted so that the flame
burns steadily and the dilution of the burning gas needs to be kept low enough so
that no undesired condensation occurs in the gas outlet.
The sampling sonde is made of stainless steel, can be heated up to 200 C, and
has a diameter of 4 mm. The glass or ceramic filter should also be able to be heated
up to 200 C.
Determination. The kind of sonde to be used and its installation depends on the
measuring purpose. To avoid long adjustment times, the sampling sonde and
tubes for transport of the samples have to be kept as short as possible. Sampling
sonde, filter and the tube for the sampled gas should be able to be heated up to
200 C, depending on the required test, and they should not chemically react with
the material being measured.
The actual determination comprises the following steps: feeding the gas
sample into the measuring equipment, the control of the flow rate and the pres-
sure in the apparatus and the registration of the results. The gas sample flows
through the sampling sonde (Fig. 5). During this step with the throttle valve,
the flow rate and calibration pressure are adjusted, depending on the require-
I
5---~1
'1----1
Fig. S. Example for the construction of the measuring apparatus. 1 Withdrawal sonde (heatable);
2 trial gas conduit (heatable); 3 filter (heatable); 4 burning gas intake; 5 burning air intake; 6 gas
analyser with FID; 7 registering apparatus; 8 exhaust
ments of the test. The adjusted parameters have to remain constant during the
measurement.
Calibration. The flame ionization detectors in the gas analyser have several ranges
of measurement. The range used has to be calibrated with commercially available
test gases like propane in purified air. For the preparation of a calibration diagram
for each range of measurement, at least four test gases with an adequate graduation
of concentration is necessary. The test gases for the calibration can also be pre-
pared using suitable gas mixing equipment.
The results are presented in the form of a calibration diagram, e.g. as the sum
of carbon atoms in ml per m 3C,. For example, with a reference gas of propane
(C3HS) and a measured value of 100mllm3, the concentration of C, is 300mllm3.
Condensation in the sample gas flow leads to incorrect results. The results can also
be affected by changing the relative amounts of oxygen and inert gases and by
different structures of the hydrocarbons in the gas sample.
3.2.2 Ammonia (VDI 1974)
Principle. During sampling, the air to be investigated is passed through diluted

sulphuric acid to bind ammonia as ammonium sulphate. Subsequently, this is
transformed to a blue indigo colour in an alkaline solution of phenol and sodium
hypochlorite, under the catalytic effect of di-sodiumpentacyano-nitrosylferrate
(nitroprusside sodium). The concentration of the colour complex solution is mea-
sured at a wavelength of approximately 630 nm. Ammonia reacts with phenol in a
mol ration of 1 to 2 and with the catalyser in a mol ratio of 1 to 1.
Sampling. This is carried out according to the scheme shown in Fig. 6. Using a
Muencke washing flask (Fig. 7), 20ml, or using an Impinger flask, 100ml, of 0,1 n
sulphuric acid p.a. is used as an absorption solution. The absorption flask is then
62 F. Schur
Fig. 6. Muencke washing flask sampling system. 1 Induction sonde; 2 absorbtion vessel; 3 drop
eilminator; 4 gas meter with thermometer and pressure gauge; 5 bypass; 6 pump; 7 barometer;
8 outside thermometer
Fig. 7. Muencke washing flask
connected to a gas flow meter, a liquid separator and a sucking pump (flow effect
1501lh using a washing flask or 2 m 3/h using an Impinger). After reading the value
at the flow meter, the sucking pump is started and the flow rate is adjusted so that
during the sampling time of approximately 30 min, 50 I of air is sucked through the
washing flask. If the Impinger flask is used, 1 m 3 of air has to be passed through.
The ammonia absorbed should not be greater than 200llg per 25 ml of the absorp-
tion solution.
At the manometer connected to the flow meter, the under pressure (lower
than atmospheric pressure) has to be recorded. The pump is then switched off.
After equilibration of pressure, the pressure at the flow meter is registered once
more. The efficiency, 11, of the absorption depends on the type of absorption vessel
and the given NH3 concentration. 11 normally achieves 90-95% and has to be
considered in the calculation.
Determination. With a pipette, a defined volume (~30 ml, containing less than
50 flg NH 3) is transferred from the absorption vessel to a 50-ml flask. Sub-
sequently, step by step 2 ml of a 0.003 M disodium-pentacyano-nitrosylferrate
solution (89.4mg/l00ml, prepared fresh daily) 8ml of phenolic solution (62.4g
phenol p.a. in ammonia-free water, dissolved and adjusted to 1000ml, and kept
in brown glass) and 2 ml of a sodium hypochlorite solution are added and the
flask with bidistilled water is adjusted to 50 m!. The sodium hypochlorite solu-
tion consists of 200ml 10% NaOH p.a. placed in a washing flask with a screw
stopper, cooled with ice, saturated with chlorine gas and then mixed with
a solution containing 100 g sodium hydroxide. The mixture is adjusted with
ammonia-free distilled water to 2000 m!. It is easier to prepare the sodium
hypochlorite solution by mixing 50 g calcium hypochlorite or Ca( OCI)2 with
600 ml of a 20% sodium carbonate solution. After thorough mixing and sub-
sequent sedimentation, the undissolved material is separated by filtration.
The solution should contain about 1% active chlorine and 1.5% free alkali. The
chlorine has to be determined jodometrically. To determine the free alkali, a
defined volume of the solution is mixed with a 3% hydrogen peroxide solution.
The surplus hydrogen peroxide is boiled out and the remaining solution is titrated
with 0.1 n hydrochloric acid against methyl red. The solution is stable for several
weeks if kept away from light. After immediate closing, the flask has to be kept
in a water bath (50C) for 20 min. Subsequently, the extinction of the solution
is measured photometrically in l-cm cuvettes against water at a wavelength
of630nm.
Sulphur dioxide and nitrogen dioxide normally do not interfere with the
determination, but sulphur hydrogen in a concentration of more than 30 flg/20 ml
of the sample reaction solution leads to lower results. A similar effect is also true
for substances like formaldehyde, pyridine, piperidine, diethylamine and cyclo-
hexylamine. The presence of aliphatic monoamines, e.g. methylamine, ethylamine
as well as aniline and aniline derivatives can lead to higher results.
The method described is applicable for amounts of ammonia between 1 and
50flg
Calibration. The ammonium chloride content of the reference solution is

8.5flgNH/m!. Into each ofa series of five 50-ml flasks, 15ml ofO.Oln sulphuric
acid is added. After the addition of 1.0; 2.0; 3.0; 4.0 or 5.0 ml of the reference
solution to each flask, 2.0 ml disodium-pentacyano-nitrosylferrate solution, 8 ml
phenol solution, 2ml sodium hypochlorite solution and ammonia-free distilled
water up to 50 ml are added step by step.
64 F. Schur
After a holding time for the closed flask at 50C for 20 min, the extinction of
the mixed solution is measured in 1-cm cuvettes against water as a blank at a
wavelength of 630 nm.
The calibration curve is generally linear up to 150llg ammonia.
Calculation of the Results. The result is generally calculated as mass concentration

in Ilg per m 3
(1)
C NH3 = l/n: v/x(E- EJk/V, (2)
CNH3 =l/C;n .1/Ylv/x(E-EJk/Vn , and (3)
Vn = V -Tn (b - Pu)/b n(Tn +t);c;n = Mrel./VMil '

where CNH3 is the concentration of ammonia according to Eqs. (1) and (2) as mass
concentration, and according to Eq. (3) as volume content in ppm; E is the mea-
sured extinction; Eo is the average of the extinctions of the blanks; K is the recip-
rocal slope of the calibration curve in Ilg; v is the volume of the absorption solution
in the absorption vessel in ml; X is the volume of the used sample reaction solution
in ml; Yl is the efficiency of the absorption in mllml; v is the sample volume in 1; vn
is the normal volume (1013 mbar; 273 K) in 1; b is the barometer pressure at the
place of measuring in mbar; b n is the normal pressure of the atmosphere
(1013 mbar); Pu is the lower pressure in the gas flow meter in mbar; t is the
temperature in the gas flow meter in DC; ta is the temperature of the atmosphere in
DC; Tn is the normal temperature (273 K); c;n is the normal mass per volume of the
ammonia (0.7714g/l); VM,n is the mol volume of the ammonia in the normal
condition (22.0711mol); and Mrel is the relative mol mass of the ammonia (mole-
cular weight 17.031 g/mol).
3.2.3 Hydrogen Sulphide (VDI 1982)
Principle. For sampling, the air to be analysed has to be sucked at high

speed through a so-called Impinger (Figs. 8 and 9), containing 50ml absorp-
tion solution consisting of 25 ml cadmium sulfate solution with 8.6 g 3CdS048H2 0
in 1000 ml bidistilled water and 25 ml of 0.1 M sodium hydroxide solution.
The hydrogen sulphide in the air sampled is converted to cadmium sulphide.
For the analytical determination, the solution is decanted from the precipitate.
The cadmium sulphide of the precipitate will, in a sulphuric acid solution,
be transformed with N,N-dimethyl-p-phenyldiammonium dichloride and
Fe(III)chloride into methylene blue. The colour of the resulting solution is mea-
sured photometrically.
6
2 J
Fig. 8. Sampling equipment. 1 Induction sonde; 2 special impinger (see Fig. 9); 3 safety
washing flask; 4 pump; 5 heat exchanger; 6 gas meter with thermometer; 7 barometer; 8 outside
thermometer
NSn,5
NS1V,5
u...#55
Impinger system
~NSqs
~
I
multipurpose vessel
Fig. 9. Impinger system for sampling for H,S

nozzle 2.5 inside determination
Sampling. The volume stream has to be adjusted so that during a sampling time of
30 min, 1000 I 10% of air is sucked through. The air pressure at the barometer, the
temperature outside and the air temperature in the aerometer are read. After
the pump has been stopped, the gas flow meter is checked. The precipitate in
the Impinger has to be kept away from light during and after sampling. The
66 F. Schur
samples can be stored closed and in the dark at room temperature, without spoil-
ing the results of the analysis. The inlet and outlet of the Impinger have to be kept
closed in any case.
Determination. The main part of the Impinger apparatus has to be centrifuged for
20 min at 3000 rpm. After careful decantation of the liquid phase, 2 ml of the
Fe(III)chloride solution (8 g FeC1 3 6H 20 in 100 ml bidistilled water) is added and
quickly mixed to the residue resuspended in 20 to 30 ml of bidistilled water.
Immediately, 5ml of the reagent is added (2.88g N,N-dimethyl-p-phenylene-
diammoniumdichloride in 1000ml of diluted sulphuric acid, 1: 1 with bidistilled
water). Thereafter the mixture in the Impinger with the closed upper part is shaken
thoroughly for at least 1 min.
After 30 min, the solution is transferred into a 100 ml flask and filled up
to the mark with bidestilled water. The extinction of the methylene blue solu-
tion is measured at 660nm in a 50-mm cuvette against bidestilled water as a
reference.
Calibration. Immediately before preparing solutions with different dilutions, the

real sulphide content of the reference solution is determined by at least three
potentiometric titrations with silver nitrate solution (0.1 mol AgNOil). Ten ml of
the reference solution is put in a flask and brought to 100ml with sodium hydrox-
ide solution (0.1 mol NaOH/l). Of the freshly prepared standard solution, 0.1, 0.25,
0.5, 0.75 or 1.0 is added to 50 ml of the absorption solution and the analytical
determination is carried out in this way several times.
Calculation of the Results. The results are calculated as mass concentration in mg

per m 3
where Cmis the H2S mass concentration in mg/m 3; E is the measured extinction; Eo
is the average of the extinctions of the blanks; k is the reciprocal slope of the
calibration function in Ilg; V is the sample volume sucked through the Impinger
in 1; t is the temperature in the flow meter in C; ta is the temperature of the
atmospheric air at the sampling place in C; and Tn is the normal temperature (=
273 K).
The temperature and the pressure of the atmosphere are noted with the results
since they are crucial to them.
Under normal conditions (273 K, 1013 bar) and supposing that hydrogen
sulphide in a diluted condition behaves as an ideal gas, a mass concentration of
H2S of 11lg/m3 is equal to a content in volume of 0.658 ppm v/v. The described
procedure is suitable for concentrations :::::lOllg/m3. The relative detection limit is
0.3 mg/m 3 for a sample volume of 1 m 3 The coefficient of variation of the results is
about 20%.
3.2.4 Mercaptanes (Meier 1975)
Principle. For sampling, the air to be analysed is sucked through two washing
glasses, each containing 50 ml of mercury acetate solution. The mercury salts of the
mercaptanes form a coloured complex with N,N-dimethyl-p-phenylenediamine,
which is measured photometrically.
Sampling. Two washing glasses (each 50ml) with a fritte at the end of the inlet
placed near the glass bottom are filled with 25 ml of the absorption solution con-
sisting of 50 g mercury(II)acetate [Hg(00CH 3)2] and 25 ml acetic acid in 1000 ml
distilled water, and placed in series. For sampling over 20 h, about 11 of air per min
is sucked through the apparatus.
Determination. The two washing glasses are treated separately. While

mixing thoroughly 5 ml of the reagent (0.7 g N,N-dimethyl-p-phenyldiamine-
hydrochloride brought to 100 ml with concentrated hydrochloric acid) and 5 ml
of the so-called Reissner solution (3.6g Fe-(III)-chioride (FeCI 2 6H2 0) dissolved
in 80ml distilled water and 80ml of nitric acid of a concentration of 10%) are
added.
After a total holding time of 1 h in diffuse light, the mixture in the washing
glass is transferred to a flask and adjusted with distilled water to 100 m!.
The extinction of this solution is measured at a wavelength of 490 nm in a
l-cm cuvette against a blank prepared with the same chemicals used in the
determination.
Calibration. A suitable calibration diagram from different solutions containing

several known concentrations of a mercury salt of methylmercaptane is obtained
using photometric determination. The detection limit is 81lg methylmercaptane
per m 3, if the reaction solutions are measured in l-cm cuvettes. Higher quantities
of sulphur dioxide can interfere with the results.
4 Mass Balances (Schur et al. 1995, 1996)
From Fig. lOwe can see that in the anaerobic treatment 76% of carbon is converted
into biogas, only 4% remains as sludge and about 20% is in the output. The
corresponding values for the aerobic process are 42% as carbon dioxide entering
the atmosphere and 48% in the sludge.
With anaerobic treatment the greater part of nitrogen in the sewage, namely
70%, is in the output; around 30% remains in the sludge and only a trace is
converted to biogas (Fig. 11). In contrast, the share of nitrogen remaining in the
sludge of the aerobic method is, as a result of nutrient additives, three times as high
as the nitrogen in the original wastewater.
Anaerobic Biogas Fig. 10. Carbon balance
Sewage 100 %
Output
Sludge
Aerobic C02
Sewage 100 % ~D Output
Sludge
Anaerobic Fig. 11. Nitrogen balance
Output
Sewage 100 %
Aerobic Addit ives
=m~$ Output
Sludge
Anaerobic
Sewage 100 % Output
Aerobio Add itives

20
Output
Sewage
Sludge
Fig. 12. Phosphorus balance
Of the phosphorus content in the sewage, 90% is in the output and only 10%
is retained in the sludge with the anaerobic method (Fig. 12). In the case of aerobic
purification, the sludge contains the same percentage share of phosphorus as the
wastewater when it enters the plant, and the share of phosphorus in the nutrient
additives corresponds to that of the output.
Figure 13 shows, in the case of anaerobic prepurification, that only small
quantities of the sewage's sulphur content enter the biogas, outgoing air and
sludge. Eighty-one percent of the sulphur in the form of sulphate reaches the
output directly, and 13% indirectly via the outgoing air treatment; in total 94%
reaches the output. Of the sulphur present in the sewage after the bioreactor, only
20% was sulphate, 61% was sulphide and about 19% was hydrogen sulphide and
other volatile sulphur compounds. In the post-aeration tank, the sewage's sulphate
share increased from 20% to 81 % at the expense of the sulphide share.
Only by building in a washer, could the very high share of hydrogen sul-
phide in the outgoing air be sufficiently contained, converted into sulphate and
directed to the output. The elimination of hydrogen sulphide in the biofilter
from a level of 1% - relatively little - is nevertheless of decisive importance.
Topping with an active carbon filter is only an additional security. Higher HzS
concentrations lead to the death of the microorganisms in the biofilter (H 2S is
oxidized to S03-)'
70 F. Schur
A n a ~obl c
A lmO l pt" , .
~
ca. O %
Carbo" IIIler
Blo-
4 ppm S
IUler
1%
14ppm 5
100 %
SO~
47ppm S
PO~I ""l lo"

u nit
Fig. 13. Sulphur balance
Sewage from anaerobic reactors contains a relatively large number of sulphur

compounds and with the secondary formation of sulphurous acid it is very de-
structive to concrete. It is therefore not permitted to discharge the prepurified
sewage directly into the public sewage works without post-treatment. Sulphur
compounds are particularly destructive to concrete when the redox value exceeds
-200mV. Due to strong calcification of the trickling filter, the decomposition of
sulphur by way of a biological solution is not feasible. A reduction in destructive
capability was achieved by blowing air into the sewage.
The avoidance of odour emissions is one of the biggest problems in planning
and executing anaerobic sewage purification. A completely satisfactory solution to
the problem can only be obtained by extracting the outgoing air from the entire
sewage purification system, by operating in a permanent partial vacuum as op-
posed to atmospheric pressure, as well as by installing a special unit for the
treatment of outgoing air. In keeping the sewage prepurification plant strictly
separate from the works sewage network, by means of a syphon system, it was
possible to avoid odour emissions.
When feeding the biofilters, which are exposed to the atmosphere, from below
with outgoing air that is virtually saturated with humidity, considerable distur-
bances can occur. Instead of the escaping air having the expected fresh smell of
wood peat, it can give off an unpleasant odour. The humidity in the air can also
condense in the biofilter, causing a loss in performance. The biofilter normally
consists of a concrete chamber with an acid-proof cladding and a concrete grate
Table 2. Analysis of biogas in comparison with petroleum gas
Parameter Symbol Unit Biogas Petroleum gas
Oxygen 0, Vol% 0.03 0.01

Nitrogen N, Vol% 1.42 3.23
Methane CH. Vol% 79.79 90.25
Carbon dioxide CO, Vol% 18.75 1.24
Hydrogen sulphide H,S Vol% 0.97 0
Hydrogen sulphide H,S g/m3 13.80 0
Gross calorific value Ho kWh/m 3 8.83 11.15
Calorific value Hu kWh/m 3 7.95 10.05
Standard density pN kg/m 3 0.96 0.79
Analysis conducted by EGO-Laboratorium in CH-801O Ziirich.
within, on which layers of scrap foamglass (6 x 6 x 6 cm) swelling clay pellets (0 1-

2 cm), pine brushwood, compost soil, another layer each of pine brushwood and
compost soil, and then wood shavings are piled.
New plans are to hermetically sealed off the biofilter from the atmosphere. To
prevent an increase in the humidity of the biofilter pulp, the outgoing air is to be
fed in from the top instead of from the bottom. On entering, a partial vacuum of
1.5 mbar, and on exiting a partial vacuum of 9 mbar, prevails. Furthermore, a
medium pressure suction fan is to be placed behind the biofilter, which propels the
outgoing air through an active carbon filter. The flow rate is to be automatically
regulated through the difference in pressure between the two filters by means of
variable speeds of rotation. If the ventilator is installed behind the active carbon
filter, its performance could be reduced by almost two thirds due to the relatively
high humidity of the outgoing air and condensation. By positioning the ventilator
in front of the filter, the dew point is not reached.
Even the smallest traces of hydrogen sulphide in the atmosphere are cor-
rosive, particularly for copper parts, and would destroy the control system.
The electricity room of the central control system has to be sealed off hermet-
ically and given a slight overpressure. An active carbon filter provides treat-
ment for the room's atmosphere. Copper plates have to be hung in the room for
monitoring.
The exploitation of biogas for heating purposes requires that specific
measures be taken for the relatively high quantities of carbon dioxide and
sulphur hydrides. The burner of the steam boiler needs to be modified to guarantee
a support flame in view of the fluctuations in the carbon dioxide levels in
the biogas, to ensure that gas handling meets all legal and safety requirements
(pressure and leakage control), and to avoid falling short of the acid dew point of
140C.
Biogas from the anaerobic prepurification of sewage is composed largely of
methane and carbon dioxide and traces of nitrogen and hydrogen sulphide, which
must be taken into account for thermal exploitation (Table 2). The calorific value
72 F. Schur
Table 3. Sewage sludge analysis
Properties Actual Standard
pH value (1: 2) 7.8 7

Dry matter (dm) % 12.1 5
Organic substances %dm 77.9 46
Ash %dm 22.1 54
Nutrients Actual Standard

kg/tdm kg/tdm
Total nitrogen N 107.3 34

Ammonium nitrogen NH4 23.4 12
Active nitrogen N 42.0 16
Carbon C 355
Phosphorus P 18.6 10
Sulphur S 2.5
Potassium K 4.8 3
Calcium Ca 14.7 60
Magnesium Mg 2.0 8
Heavy metals Actual Limit

g/tdm g/tdm
Molybdenum Mo 3'798 20
Zinc Zn 3'033 2000
Copper Cu 1'178 600
Cadmium Cd 1.59 5
Cobalt Co 4.93 60
Nickel Ni 45.70 80
Chrome Cr 32.50 500
Lead Pb 41.20 500
Mercury Hg 0.41 5
Analysis conducted by UFAG-Laboratories CH-621O Sursee.

Standard and limit values upon the judgement of animal food.
of biogas is 7.95kWh/m3 which is 21% lower than the corresponding value of

petroleum gas.
With the anaerobic process almost nine times more biogas energy is
produced than electric current is consumed by pumps and machinery for operat-
ing the plant. There results on average from one kg COD 0.45m3 biogas or
3.6kWh calorific value. In comparison, the aerobic process has a significantly
high specific electricity consumption, and this without any gain in the form of
biogas.
The sewage sludge displays, for example, 12.1% solid matter, of which 77.9%
is organic substance and 22.1 % mineral aggregate (Table 3). Among these sub-
stances, when designated as nutrients according to fodder criteria, nitrogen domi-
nated, followed by phosphorus and calcium. But heavy metals, like molybdenum,
zinc or copper also have to be taken into consideration.
5 Conclusions
After a short explanation about the origin type and composition of brewery sew-
age, one can see why and under what conditions brewery sewage is predestined for
anaerobic treatment. A modern system is described for anaerobic sewage prepro-
cessing and the resulting processes. In the main, one is concentrating on the
methods for the examination of sewage and sludge as well as of the outgoing air
and biogas. In this way, the priority is given to especially important parameters of
the anaerobic system. The escaping sulphur compounds are particularly impor-
tant. In conclusion, the mass balances of carbon, nitrogen, phosphorus and above
all sulphur are shown and special technological problems with anaerobic sewage
preprocessing systems are presented.
References
Ahrens A, Schumann G (1996) Grundlagen der anaeroben Abwasserreinigung und ihre

Vmsetzung am Beispiel einer VLB-Modellanlage. Brauwelt 136(3):84-89
Behmel V, Meyer-Pittroff R (1995) Anaerobe Abwasserreinigung im Brauereibetrieb:
Technologien - Chancen - Risiken. Der Weihenstephaner 1:86-90
Birkenstock B (1991) Halbtechnische Versuchsanlage zur anaeroben Vor- und aeroben
Nachreinigung von Brauereiabwasser. Brauwelt 131(10):330,332-333,336,345-348
Bruckner H (1995) Vergleich verschiedener Abwasseranalysenmethoden. Wasser Abwasser gwf
136(9):474-477
ED! (Eidgenossisches Departement des Innern) (1983) Richtlinien fUr die Vntersuchung von
Abwasser und OberfHichenwasser (Allgemeine Hinweise und Analysenmethoden) 1. Tei!
Abwasser. ED I, Bern
Fang HHP, Jinfu Z, Guohua L (1989) Anaerobe Behandlung von Brauereiabwasser. Biotechnol
Lett 11(9):673-678
Glas K (1995) Der Einsatz von Reinigungs- und Desinfektionsmitteln und deren Auswirkungen
auf die Abwassersituation. Seminar Weihenstephan, pp 16/1-16/4
Hecht S, Knorie V, Neugebauer A (1994) Betriebserfahrungen mit der anaeroben
Abwasserbehandlung. Brauwelt 134(26):1265-1268
Hellriegel K (1995) Anaerobe, aerobe oder kombinierte Abwasserbehandlung unter
wirtschaftlichen Gesichtspunkten. Brauwelt 135{1I2):24-25
Hofer H (1991) Anaerobe Industrieabwasserreinigung - Ein neues Konzept? Gas-Wasser-
Abwasser 71(7):470-476
Hoffmann S, Zanker G (1995) Versuche mit einer anaeroben Pi!otanlage auf VASB-Basis fUr
Abwasser. Brauwelt 13545:2310-2316
Hoyer S (1992) Brauereiabwasser in bezug auf die Wasserrechtsnovelle 1990. Brauwelt 132(49):
2527-2529
Konig E (1997) Auftei!ung der Kosten der Abwasserentsorgung. Brauindustrie 3:145-150
Kuhbeck G (1995) Praxiserfahrungen mit der anaerob-aeroben Reinigung von Brauereia-
bwiissern mit Denitrifikation und P-Eliminierung. Proc 25th Congr, Brussels, 1995, European
Brewery Convention. Oxford Vniversity Press, New York, pp 751-960
Kuhbeck G (1997) Praxiserfahrungen der Bitburger Brauerei mit der anaerob-aeroben Reinigung
von Brauereiabwiissern mit Denitrifikation und P-Eliminierung. Brauwelt 137(24125):959,
962,964,965,966
Meier W (1975) Bestimmung von Merkaptanen in der Luft. Ciby-Geigy Zentrale. Analytik,
Vmwelt 1-2
74 F. Schur: Analysis of Sewage from Anaerobic Purification of Effluent from a Brewery
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Perry M (1997) The handbook of brewery effluent. Print Point, Dunkeld West, Johannesburg
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Brauereien. Abwasser- und Umweltseminar des BBB im Dezember 1993 in Beilngries (D)
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vom 28.2. 1992. Mikrobiologische Laboranalysen
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Apple Pomace and Products Derived from Apple
Pomace: Uses, Composition and Analysis
M. KENNEDY, D. LIST, Y. Lu, L. Y. Foo, R. H. NEWMAN, I. M. SIMS, P. J. S. BAIN,
B. HAMILTON, and G. FENTON
1 Introduction
Apple pomace is the press cake resulting from pressing apples for juice (see Fig. 1).
This chapter is not an all-encompassing review of apple pomace, but rather high-
lights areas the authors view as significant and with which they have expertise.
Production figures for apple pomace are shown in Table 1.
The composition of the final pomace is linked to the morphology of the
original feed stock and the extraction technique used. With respect to extraction,
the aim of the apple juice manufacturer is relatively simple:
1. Maximise the extraction yield of the juice from the original apple mash.
2. Minimise the Brix value of the moisture in the final pomace (as this represents
the cost associated with not retrieving discarded/unextracted sugars).
3. Minimise the extraction of any compounds that may cause problems with juice
quality (unless they can be degraded or separated later in the process).
4. Maximise the throughput capacity of the extraction device to maximise the
financial return on capital invested (in some scenarios this criterion will mean
that the processor will forego yield).
Despite a simple aim, achieving these objectives is dependent on many interac-
tions between processing steps. Apple juice extraction may contain the following
steps: variety selection; fruit grading; milling; primary mash enzymation; primary
extraction; leaching, heating and secondary enzymation (liquefaction); secondary
extraction. If a peeler line is operating adjacent to the juicing plant, the pomace will
comprise a higher ratio of peel and core by-product. A host of other juice process-
ing steps follow the extraction. However, those steps do not influence the pomace
composition.
Without even considering compositional differences within specific apple
varieties (resulting from such conditions as natural variation, husbandry practices,
fruit maturity and post harvest management) there are significant differences in
apple composition between varieties. Fruit physiology plays a large part in the
quality of the extraction and interacts with pre-extraction processes such as mash
enzymation or pomace liquefaction. As an apple ripens, the ratio of soluble pectin
to insoluble pectin in the mash increases. This has a negative effect on the physical
structure of the mash, making juice extraction by conventional methods more
difficult.

76 M. Kennedy et al.
Fig. l. Piles of apple pomace produced during the manufacture of apple juice
Table l. Production of apple pomace (AP) or apples
Region covered Material Year Production Reference

(wet tonnes
per annum)
Global apple pomace AP 1981-1985 38 x 106 Jarosz (1988)

production
USA AP 1984 1.3 x 106 Hang (1985), Jewell and
Cummings (1984)
North America AP 1992 1.5 x 106 Chong (1992)
Argentina AP 1985 1 x 10' Hours et al. (1985)
Spain apples 1984 1 x 106 Alibes et al. (1984)
NZ AP 1982 5.5 x 106 Davies (1982)
NZ AP 1997 2 x 106 -4 X 106 List (1997, pers. comm.)
UK AP 1987 1.5 x 104 Givens and Barber (1987)
India apples 1976 3.94 x lOs Murti et al. (1976)
The mash enzymation or pomace liquefaction regime chosen may utilise dif-
ferent enzymatic activities, time/temperature profiles etc. In as much as mash
enzymation has an impact on the composition of the juice by changing carbohy-
drate and acid profiles, enzyme activity also affects the composition of the residual
pomace. For example, it is accepted that mash treated with pectolytic enzymes
Apple Pomace and Products Derived from Apple Pomace 77
liberates pomace that is less suitable for commercial pectin extraction. Other
compounds of potential interest may be degraded during mash treatment whilst
the extraction of some other recoverable compounds may increase. In addition,
leaching of pomace (either in association with or independently of secondary
enzymatic treatment) strongly affects the composition of the pomace. Added water
dilutes the soluble solids in the pomace moisture to the same level as that con-
tained in the secondary-extracted juice. Leaching improves extracted sugar yield
but consequently lowers the soluble solids and metabolizable energy of the pomace
(per unit dry matter).
The liberation of juice and dietary fibre from the mash is also influenced by
various "mechanical"interactions. The size distribution of mash particles, attribut-
able to fruit morphology and milling technique strongly influences extraction. The
extraction technique may utilise straight compression, compression interacting
with sheer forces, centrifugal forces or simple counter-current dilution. Since these
techniques liberate different chemical fractions into the juice, they influence the
composition of the final pomace.
Pomace is not homogenous. Upon dissection it visibly contains discrete tissue
attributable to peel, core, seed, calyx, stem and soft tissue. Using a finisher, the soft
tissue can be separated from the other, harder tissues. This can be done prior to an
extraction if desired or after the final extraction if there is a customer requirement.
The composition differences between tissue types is significant.
2 What Use Is Apple Pomace?
Considering the importance of the final use of the apple pomace in determining
the analyses performed on it, it is pertinent to review the use to which apple
pomace has been put. Finding uses for apple pomace has been a favourite topic for
researchers for a large number of years, starting with Hills' (1902) studies on
animal feeding. A literature search of Chemical Abstracts and Biosis databases
(Fig. 2) reveals that research on apple pomace has increased since 1971 with 1988
and 1989 being the most popular years for apple pomace research publication. The
ongoing effort indicates that the ideal use for apple pomace has yet to be found.
Although finding a high value product in apple pomace is profitable, these com-
pounds are usually only present in small amounts, e.g. aromas or apple seed oil.
Thus the producer is still left with the problem of what to do with the rest of the
apple pomace. As long as apple juice is still made, the problem of what to do with
the pomace will remain.
The literature summarising the uses of apple pomace can be seen in Table 2.
Table 2 reveals some interesting trends. The uses of apple pomace can be
broadly classified as either a waste reduction strategy e.g. animal feed, fuel use or
composting; or obtaining a high value product e.g. aroma or pectin production; or
preferably both. In terms of getting the apple pomace off site and away from the
78 M, Kennedy et al.
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Fig. 2. Historical data on the number of papers published on apple pomace
Table 2. The literature summarising the uses or potential uses of apple pomace
Use of Apple Pomace References
Review articles Kiviat (1982)

Hang and Walter (1989)
Kennedy (1994)
Animal feed Hills (1902)

Knott et al. (1932)
Walter et al. (1975a)
Agricultural Research Council (1976)
Wolter et al. (1980)
Rumsey and Lindahl (1982)
Alibes et al. (1984)
Heinemann (1984)
Sharma and Sharma (1984)
Bath et al. (1986)
Nikolic and Jovanovic (1986)
van der Merwe et al. (1986, 1987)
Givens and Barber (1987)
Gasa et al. (1989)
Singhal et al. (1991)
Gasa et al. (1992)
Singh and Narang (1992)
Garrity (1994)
Modification and/or incorporation Kitson et al. (1972)

in human food stuffs Walter et al. (1977)
Dreyer and van der Walt (1979)
Table 2. Continued
Waugh (1981)
Walter et al. (1985, 1986)
Caprez et al. (1987)
Sly et al. (1989)
Wang and Thomas (1989)
Renard and Thibault (1991)
Carson et al. (1994)
Joshi et al. (1996)
Walter (1985)
Wine Gitenshtein et al. (1975)

Muraki and Masuda (1976)
Compostlfertilizer Stapleton et al. (1984)

van de Kamp (1986)
Emmaus (1991)
Jones (1992)
Chong (1992)
Gies (1995)
Schaub and Leonard (1996)
Fuel use Sargent and Tennes (1982), Sargent and

Pierson (1983), Sargent (1985)
Swanson et al. (1983)
Jewell and Cummings (1984)
Mason et al. (1985)
Sargent et al. (1986)
Hang (1987)
Pectin Bosak (1969)

Malhotra and Miller (1973)
CSIR India (1975)
Marinescu et al. (1976)
Moiseeva et al. (1976)
Zaiko et al. 1978
Davies (1982)
Voragen et al. (1986)
Lopez et al. (1990)
Methane/biogas from waste treatment Lane (1979)

Etheridge and Jewell (1988)
White et al. (1989)
Kalia et al. (1992)
Ethanol production via fermentation Hang et al. (1981, 1982)

Miller et al. (1982)
Jarosz (1988)
Gupta et al. (1990)
Ngadi and Correia (1992a,b)
Table 2. Continued
Citric acid production via fermentation Hang and Woodams (1984)

Hang (1985, 1988a,b), Hang and
Woodams (1986)
Hang and Woodams (1989)
Butanol production via Fermentation Voget et al. (1985)
High protein feedstock (SCP) via fermentation Hours et al. (1985)

Gunde-Cimerman and Cimerman (1986)
Fellows and Worgan (1987)
Hang (1988a)
Williams and Henry (1989)
Nigam and Singh (1996)
Enzymes
Pectinase Hours et al. (1988a,b)
Polygalacturonase Hang and Woodams (1994a)
f3-Glucosidase Hang and Woodams (1994b)
13- Fructofuranosidase Hang and Woodams (1995)
Apple seed oil Morice et al. (1971)

Bain (1988, 1989)
Apple vinegar Malhotra and Miller (1973)
Apple wax: Kiviat (1982)
Aroma compounds/flavouring Shaudys (1981)

A1mosnino and Belin (1991)
Almosnino et al. (1996)
Oxalic acid Mane et al. (1987)
Xyloglucan extraction Renard et al. (1995)

Sims et al. (1998)
Insect bait Smock and Neubert (1950)

Cone (1969)
Archer and Musick (1977)
Sechriest and Sherrod (1977)
Activated carbon Kiviat (1982)
Ion exchange resin Kiviat (1982)
Furfural Kiviat (1982)
processor, human food incorporation, animal feeding, compo sting, landfill, and
fuel use have been most popular. Apple pomace has been fed to cattle, sheep, pigs,
horses, deer, bighorn sheep, rats, rabbits, geese and insects (Kiviat 1982). Cost
of disposal is the driving force for seeking new pomace uses, see Table 3. A
Table 3. Disposal cost of apple pomace reported in the literature
Method of disposal Year Cost US $/tonne Reference

wet weight
Transport to farmer 1994 25 Garrity (1994)

Trucking to land for 1982 10-20 Hang et al. (1982), Kaviat (1982)
application to soil
Animal feed 1984 7 Jewell and Cummings (1984)
Land fill tiping fees 1991 9.5 Emmaus (1991)
Food use 1980 64 Waugh (1981)
problem arises with transport costs. It is uneconomic to transport the pomace far.
Garrity (1994) shows nicely the trade-off between transport costs and utility of the
product. Transport costs would be much reduced if the pomace was first dried, but
this is uneconomic. Fermentation of apple pomace has been popular. Products
such as single cell protein (SCP), ethanol, citric acid, butanol and enzyme produc-
tion have been attempted. All of these, with the possible exception of enzymes,
have been low value commodity products making profitable processes difficult to
achieve. The same applies to the extractive processes of pectin and oxalic acid
production. The higher value products, apple seed oil, aromas and xyloglucan
production offer some hope, but the problem remains of what to do with the waste
after these products have been made.
What is the best thing to do to obtain value from apple pomace? While there
is no right answer to this question, as each situation is unique, the authors' pick is
as follows:
Mushroom Compost. Apple pomace makes an ideal mushroom substrate when

mixed in with the normal forms of mushroom compost e.g. hay (Kennedy 1994;
Upadjhyay and Sohi 1988). The apple pomace provides much needed nitrogen for
the mushroom farmer, and after use it can be bagged up for sale to the home
gardener. The only problem is that the mushroom farm must be close to the
processor to avoid prohibitive transport costs.
Apple Seed Oil. Apple seed oil is high in 18: 2 and 18: 1 fatty acids (see Table 6). It
thus has value for nutritional and cosmetic usages. The problem is the small
volumes produced, as apple seeds form only 2-3% of the total weight of pomace
(see Table 8). Residue from the oil expression will have value as an animal feed-
stuff.
Separation of pips from pomace can either be done using flotation or sieving.
Apple oil has connotations of good health and, although the oil has no special
composition, it should have a good market among health conscious people. It
could be used as a health supplement or as a salad oil. It could also be used in
cosmetics (even possibly for making paint!). Depending on its use, it could vary in
value from $1000 to $10000/tonne. Since apple juicing goes on for most of the year,
apple pips can be stored if they can be economically separated from the pomace.
Consideration should be given to using the same plant to process other seeds and
so improve its economics. The chosen method for pip drying depends on the
production rate. Air drying is suitable for small quantities but hot air drying would
be needed for any large amount. Residual moisture of 15%-20% is acceptable for
storage and oil expression. The pips cannot be stored damp or fungal growth will
spoil them.
Solvent extraction or expression can be used to extract the oil. Solvent extrac-
tion involves flammable hexane with storage tanks and solvent recovery units as
well as a facility for flaking the pips. This would involve considerable capital outlay
and planning consent. It would have to be on a site remote from the apple juicing
plant. It has the merit that essentially all of the oil is recovered.
Expression of the oil using a screw expeller gives a solvent free oil with a better
sales prospect since it is "clean". From 5-10% of the oil is retained in the spent
seed, according to the make of press. Expression would more easily gain planning
approval than solvent extraction and it can be on the juicing site. Unless the oil is
sold as crude, it would need refining to remove free fatty acids and phospholipids.
This is usually done by extraction with caustic soda (centrifugal separator) fol-
lowed by treatment with bleaching earth to remove traces of soap. The oil can just
be water-washed if a "natural product"is required.
Aroma Compounds. Apple aroma is used in cosmetics e.g. in shampoos, and as a

natural food flavouring. Again, the yields from apple pomace are extremely small.
The main volatile components of apple juice are n-butanol (40mgt 1), hexanol
(22mgtl), 2-methyl butanol (9mgtl), 3-methyl butanol (9mgt\ n-propanol
(7mgtl) and ethanol (5mgt 1) (Acree and McLellan 1989; Maarse and Visscher
1989).
Xyloglucan Production. Xyloglucan has unique properties due to its ability to

interact with cellulose (Hayashi et al. 1994; Carpita and Gibeaut 1993). Potential
applications include the ability to coat and suspend particulate matter in cloudy
juice preparations, thus increasing consumer acceptance of such products which
would no longer have sludge layers in the bottom. Xyloglucan can be used as a
surface treatment chemical for cellulosic materials with potential applications
in dyeing, and predominantly as thickeners. Tamarind seed gum is the only
xyloglucan exploited commercially at the moment. It has food approval in Japan,
where it is used as a thickening, stabilising and gelling agent (Glicksman 1986). In
Europe and the USA, the use of tamarind seed xyloglucan has lapsed due to the
production of galactomannan gums (guar and locust bean; Reid and Edwards
1995).
The viscosity of xyloglucan from apple pomace was approximately 70 cps
(centipoise) at 2% w/v, compared with 800 cps for tamarind seed xyloglucan at the
same concentration (Sims et al. 1998). Tamarind seed xyloglucan probably has
similar properties to galactomannan gums, and can be similarly modified by
removing galactose residues present in the side chains. Reid et al. (1988) have
shown that tamarind xyloglucan gradually reduces in viscosity as galactose is
removed, until a critical point is reached at which it gels. The degree of galactose
substitution at which gelling occurs is similar to that for galactomannan to gel.
Thus, there would appear to be potential uses of xyloglucans in food.
Through modifications (i.e. carboxylation, amination etc.) xyloglucan may
have other potential industrial uses. The galactose side chains are particularly
good for specific modification following oxidation with galactose oxidase (Lang
et al. 1992).
Antioxidant Production. Antioxidants are a very big seller in the nutritional

supplement market. Extraction of antioxidants from apple pomace has been devel-
oped by Lu and Foo at Industrial Research Limited. The key question is whether
apple pomace antioxidants are more biologically efficacious than the many other
antioxidants on the market.
In the future, researchers will quantify the minor components of apple pom-
ace e.g. unusual carbohydrates to an even greater extent in the never ending search
for a good use for apple pomace.
Apple Pomace Contaminants and Safety of Derived Products. Apple pomace can
potentially contain either natural toxins or chemical residues. Kiviat (1982) warns
of amygdalin in apple seeds, patulin and other mycotoxins from mold contam-
ination, pesticide residues, insecticide residues, and oil contamination from
leaky hydraulic presses. Processors should ensure that the pomace or pomace-
derived products meets local food standards regulations e.g. solvent residue types
and concentrations should a food product be contemplated.
3 The Goal of Apple Pomace Analysis
The exact type of analytical procedures performed on apple pomace depend upon
the use to which the data will be put. To the industrialist the goal will be to either
find a way of getting rid of the vast amount of waste apple pomace produced, or
find a high value product from apple pomace. The analytical requirements in the
first case are likely to be engineering or physical property related. Variables such
as the water content, bulk density, viscosity, and residual juice content will be
important. These analyses are usually simple and robust, and are of great value in
designing and operating a process involving apple pomace. If a high value product
is the goal, the industrialist will primarily be interested in quantifying the yield of
the high value product and in measuring variables that affect this yield. Examples
of this type of analysis may be enzyme concentration, cell biomass content, or
pectin measurement. As one of the most important ways of utilizing apple pomace
is as an animal feed supplement, the variables measured for this application will be
protein, fiber, mineral and most importantly its digestible energy content. If fuel
use of apple pomace is desired, the combustible energy level and moisture content
are of prime importance. If apple pomace is going to be profitably disposed of by
Table 4. The hierarchy of analytical requirements when analysing apple pomace
Person Goal of Example Example Assay Data utility

using data pomace application variables sophistication
use measured and cost
Industrialist Waste Fuel use Water content Low "Need to know

utilization Animal feed Digestible energy basis"
Mushroom Kjeldahl nitrogen
compost
High Enzyme Enzyme units
value Fermentation Pectin content
product product Aroma profile
isolated Seed oil content
or made
Scientist None Fundamental Cell wall High Of fundamental
science study components importance,
Carbohydrates may not be of
short-term use
using it as a mushroom compost, then Kjeldahl nitrogen content will be relevant.

In the industrial situation, cost of analysis is of central consideration and so simple
in-house analytical techniques are desired, or else low cost laboratory tests pre-
ferred. Rarely will the industrialist invest in "nice to know" type of information,
but instead only wants "need to know" information.
To the scientist whose goal may be a greater fundamental understanding of
the apple pomace substrate, the number of components measured and the sophis-
tication of the analytical techniques increases dramatically. Novel analytical tests
will often be developed. This is an evolving situation. New analytical developments
often mean that new techniques can be applied from other disciplines or else what
was extremely sophisticated, costly and of dubious accuracy one year can be
turned into a very cheap and reliable technique within a very few years. Also, the
ever-increasing number of analytical techniques becoming available means that
detection levels are reducing and many new components can be measured that
were not previously available. Table 4 summarises a hierarchy of analytical
requirements.
4 The Composition of Apple Pomace
Many published compositional analyses of apple pomace exist. Usually the com-
position published is geared toward one particular application, so it can be difficult
to find an all-inclusive composition in one place. Data on the composition of whole
apples themselves are also a valuable source of data about apple pomace, as the
pomace is simply whole apples minus the juice. The main source of composition
data can be found in animal feed and nutrition books or else in the scientific
literature. Tables 5-9 and Figs. 3-5 present a collation of all the composition data
Table 5. A collection of published apple pomace (AP) composition data
Variable Material Value Reference
Fraction of apple crop Apples 30 Walter et al. (1977)

used for juice and
hence for apple
pomace production
(% of apple crop)
Fraction of fruit fed to Apples 25 Walter and Sherman

press during (1976)
juicing which ends Apples 25-30 Kiviat (1982)
up as pomace Apples 25 Sargent and Pierson (1983)
(% offed apples) Apples 25-30 Hang (1985)
Apples 20-25 Jarosz (1988)
Press aid Rice hulls 0.5-1.0 Walter et al. (1975c)

concentrations Rice hulls 5-6% Kiviat (1982)
(% of wet AP) Paper 4.7-6.6 Heinemann (1984)
Rice hulls 3.9-4.1 Heinemann (1984)
Silvacel 9.9 Heinemann (1984)
(alderchips)
Rice hulls 2-6 Bump (1989)
Kraft fiber 1-3 Bump (1989)
Diatomaceous Emmaus (1991)
earth
Rice hulls 20.1, 21.6, 26.4 Carson et al. (1994)
Dry matter content WetAP 48 Kertesz and Green (1931)

(% of total weight) WetAP 21.1 Morrison (1942)
WetAP 21.85-33.61 Smock and Neubert (1950)
WetAP 24 Walter and Sherman
(1976)
WetAP 20.8 Toyokawa et al. (1980)
WetAP 28-32 Kiviat (1982)
WetAP 35 Sharma and Sharma
(1984)
WetAP 27 Hang and Woodams (1984)
WetAP 21.0 Kennedy and Powlesland
(1984)
WetAP 19.5 Hours et al. (1985)
WetAP 20.7 Voget et al. (1985)
WetAP 28.0, 23.2, 23.9 Hang and W oodams (1986)
WetAP 35 Sargent et al. (1986)
WetAP 19.37-27.39 Nikolic and Jovanovic
(1986)
WetAP 19.3-28.6 Givens and Barber (1987)
WetAP 23.3 Givens and Barber (1987)
WetAP 26.5-29.0 Jarosz (1988)
WetAP 14.8 Hours et al. (1988a)
WetAP 16.9, 11.0, 19.4, 9.5 Hours et al. (1988b)
WetAP 20.5 Gasa et al. (1989)
WetAP 20-30 Lopez et al. (1990)
WetAP 20 Preston (1991)
WetAP 21.5-22.7 Ngadi and Correia (1992a)
Table 5. Continued
WetAP 18.9 Gasa et al. (1992)

WetAP 18.0 Garrity (1994)
WetAP 24.4 Hang and Woodams
(1994a,b)
WetAP 27.72-27.77 Joshi et al. (1996)
WetAP 24 Almosnino et al. (1996)
Dried AP 88.49 Knott et al. (1932)
Dried AP 89.4 Morrison (1942)
Dried AP 89.0-87.5 Smock and Neubert (1950)
DriedAP 98 Kitson et al. (1972)
DriedAP 93 Walter et al. (1975b)
Dried AP 94.6 Dreyer and van der Walt
(1979)
Dried AP 91.23 Wolter et al. (1980)
Dried AP 94.6 Waugh (1981)
Dried AP 89.0 Bath et al. (1986)
Dried AP 89.2 Van der Merwe et al.
(1986)
Freeze dried AP 92.7 Hang (1988a)
DriedAP 93 Hang and Woodams (1989)
Dried AP 89 Preston (1991)
Dried AP 98.5, 98.8, 98.7 Carson et al. (1994)
Ensiled AP 25.5-31.0 Fontenot et al. (1977)
Ensiled AP 14.7-21.5 Alibes et al. (1984)
Ensiled AP 21.4 Bath et al. (1986)
Ensiled AP 18.9 Gasa et al. (1989)
Ensiled AP 18.9, 16.6 Gasa et al. (1992)
Apples 16.1 Gebhardt et al. (1982)
Titratable acidity Apples 0.55,0.4 Withy et al. (1978)

(total as malic acid) Apple juice 0.48 McLellan et al. (1991)
(% of wet material)
pH AP 3.2 Agricultural Research

Council (1976)
AP 3.4 Hang et al. (1982)
AP 3.1-3.8 Jewell and Cummings
(1984)
AP 3.6 Hang and Woodams (1984)
AP 3.5 Alibes et al. (1984)
AP 3.5 Kennedy and Powlesland
(1984)
AP 4.1 Hours et al. (1985)
AP 3.62-3.95 Nikolic and Jovanovic
(1986)
AP 3.5, 3.5, 3.6 Hang and Woodams (1986)
AP 3.5 Jarosz (1988)
AP 4.1 Hours et al. (1988a)
AP 3.3, 3.3, 4.1, 4.1 Hours et al. (1988b)
AP 3.5-3.7 Ngadi and Correia (1992a)
Ensiled AP 3.8 Agricultural Research
Council (1976)
Ensiled AP 3.6-4.0 Rumsey (1978)
Table 5. Continued
Ensiled AP 3.6 Alibes et al. (1984)

Apple juice 3.54 McLellan et al. (1991)
Crude protein (% of AP 4.56 Knott et al. (1932)

dried material) AP 4.5 Morrison (1942)
AP 6 Walter et al. (1975b)
AP 6.5 Agricultural Research
Council (1976)
AP 4.1 Dreyer and van der Walt
(1979), Waugh (1981)
AP 5.3 Toyokawa et al. (1980)
AP 3.6-3.9 Kiviat (1982)
AP 5.03 Sharma and Sharma
(1984)
(1984)
AP 4.05-4.95 Heinemann (1984)
AP 5.4-8.0 Nikolic and Jovanovic
(1986)
AP 4.9 Bath et al. (1986)
AP 4.4 Arrigoni et al. (1986)
AP 3.87 Van der Merwe et al.
(1986)
AP 6.7 Givens and Barber (1987)
AP 6.0 Hang (1988a)
AP 4.5 Gasa et al. (1989)
AP 5-6 Preston (1991)
AP 5.17 Singhal et al. (1991)
AP 4.7 Singh and Narang (1992)
AP 1.9, 2.5, 2.2 Carson et al. (1994)
AP' 3.5 Hours et al. (1985)
AP 4.4-4.9 Alibes et al. (1984)
AP' 3.0 Hours et al. (1988a)
AP' 1.8, 2.2, 4.0, 4.1 Hours et al. (1988b)
AP 5.86 Wolter et al. (1980)
AP' 3.0 Voget et al. (1985)
Dried AP 4.45-5.67 Smock and Neubert (1950)
Ensiled AP 8.1 Garrity (1994)
Ensiled AP 6.7,9.0 Gasa et al. (1992)
Apples 2.8 Bath et al. (1986)
Fat, lipid or ether AP 6.15 Knott et al. (1932)

extract (% of dried AP 5.0 Morrison (1942)
material) AP 3.75-4.65 Smock and Neubert (1950)
Council (1976)
(1979), Waugh (1981)
Table 5. Continued

(1984)
AP 3.70-4.55 Heinemann (1984)
AP' 2.05 Hours et al. (1985)
AP' 4.0-7.0 Nikolic and Jovanovic
(1986)
AP 3.25 van der Merwe et al.
(1986)
AP 6.6 Hang (1988a)
AP' 4.1, 8.2, 12.6, 14.4 Hours et al. (1988b)
AP 5.2-5.6 Preston (1991)
AP 1.0, 1.1 Carson et al. (1994)
Council (1976)
Apple wax (% of AP 1.2 Kiviat (1982)

dried material)
Crude fibre (% of AP 14.25 Knott et al. (1932)

dried material) AP 15.6 Morrison (1942)
AP 15.3-20.6 Smock and Neubert (1950)
AP 26 Walter and Sherman
(1976)
Council (1976)
(1979)
AP 14-30 Kiviat (1982)
(1984)
(1986)
AP 17 Bath et al. (1986)
(1986)
AP 42-44.5 Heinemann (1984)
AP 14.2-14.4 Alibes et al. (1984)
Table 5. Continued
AP' 20.0 Hours et al. (1985)

AP' 21.7 V oget et al. (1985)
AP 19.8 Hang (1988a)
AP" 11.6, 19.4,20.5,29.6 Hours et al. (1988b)
AP 18.5 Gasa et al. (1989)
AP 17-19 Preston (1991)
Freeze dried AP 35.29 Wang and Thomas (1989)
Drum dried AP 33.24 Wang and Thomas (1989)
Council (1976)
Acid detergent fibre AP 42.04 Wolter et al. (1980)

(% of dried AP 31.52 Sharma and Sharma
material) (1984)
AP 53.9-55.2 Heinemann (1984)
AP 24.5,25.1 Alibes et al. (1984)
(1986)
AP 26 Bath et al. (1986)
AP 25.3 Gasa et al. (1989)
AP 31-32 Preston (1991)
AP freeze dried 24.39 Wang and Thomas (1989)
AP drum dried 25.11 Wang and Thomas (1989)
Ensiled AP 35.3,34.8 Alibes et al. (1984)
Ensiled AP 26 Bath et al. (1986)
Apples 9 Bath et al. (1986)
Neutral detergent fibre AP 48.20 Wolter et al. (1980)

material) (1984)
AP 33.9,33.8 Alibes et al. (1984)
AP" 53.6-56.4 Nikolic and Jovanovic
(1986)
AP 30.1 Gasa et al. (1989)
AP 42-45 Preston (1991)
Table 5. Continued

Ash (% of dried AP 2.13 Knott et al. (1932)

material) AP 2.2 Morrison (1942)
AP 2.11-3.50 Smock and Neubert (1950)
AP Walter and Sherman
(1976)
Council (1976)
(1979), Waugh (1981)
(1984)
AP 1.45-5.25 Heinemann (1984)
AP 2.6,3.7,2.5 Alibes et al. (1984)
AP' 2.1 Hours et al. (1985)
AP 4.01 Mason et al. (1985)
(1986)
(1986)
AP 1.3 Hang (1988a)
AP' lA, 1.7, 1.9,2.5 Hours et al. (1 988b)
AP 3-4 Preston (1991)
AP 5.5, 6.2, 4.8 Carson et al. (1994)
Council (1976)
Ensiled AP 2.9, 4.5, 3.0 Alibes et al. (1984)
Apples' 1.7, 1.3 Withyet al. (1978)
Potassium AP 0043 Morrison (1942)

(% of dried AP 0.81 Toyokawa et al. (1980)
material) AP 0.55-0.69 Kiviat (1982)
(1984)
AP 0048 Bath et al. (1986)
AP 0.5-0.6 Preston (1991)
Table 5. Continued
Apples" 0.66,0.47 Withy et al. (1978)

Sodium AP 0.03 Toyokawa et al. (1980)

(% of dried AP 0.0025-0.0029 Kiviat (1982)
material) AP 0.04 Kennedy and Powlesland
(1984)
Calcium AP 0.10 Morrison (1942)

(% of dried AP 0.2 Alibes et al. (1974)
material) AP 0.08 Toyokawa et al. (1980)
AP 0.07-0.10 Kiviat (1982)
AP 0.16 Heinemann (1984)
(1984)
AP 0.13 Bath et al. (1986)
(1986)
AP 0.13 Preston (1991)
Apples' 0.04,0.03 Withy et al. (1978)
Magnesium AP 0.1 Alibes et al. (1974)

material) AP 0.05-0.07 Kiviat (1982)
(1984)
AP 0.07 Bath et al. (1986)
Apples" 0.29,0.D25 Withy et al. (1975)
Manganese AP 0.7 x 10-4 _1.1 X 10-4 Kiviat (1982)

(% of dried AP 2 x 10-4 Alibes et al. (1984)
material) Ensiled AP 1.6 x 10-4 Alibes et al. (1984)
Phosphorus AP 0.09 Morrison (1942)

material) AP 0.105 Wolter et al. (1980)
AP 0.08-0.09 Kiviat (1982)
AP 0.06-0.07 Heinemann (1984)
AP 0.01 Alibes et al. (1984)
(1984)
AP 0.12 Bath et al. (1986)
AP 0.12 van der Merwe et al. (1986)
Table 5. Continued

AP 0.12 Preston (1991)
Zinc (% of dried AP 6 x 10-4 _8 X 10-4 Kiviat (1982)

material) AP 2.1 x 10-4 Alibes et al. (1984)
AP 1.3 x 10-3 Preston (1991)
Ensiled AP 2.1 x 10-4 Alibes et al. (1984)
Sulphur (% of dried AP 0.007 Kennedy and

material) Powlesland (1984)
AP 0.02-0.06 Preston (1991)
Oxygen (% of dried AP 44.78 Mason et al. (1985)

material)
Carbon (% of dried AP 44.56 Mason et al.(1985)

material)
Copper (% of dried AP 5 x 10-4 _6 X 10-4 Kiviat (1982)

Ensiled AP 1.4 x 10-4 Alibes et al. (1984)
Molecular weight AP 22.80 Mason et al. (1985)

(g of dried AP
mol-I)
Hydrogen (% of AP 6.18 Mason et al. (1985)

dried material)
Nitrogen (% of dried AP 0.72 Morrison (1942)

(1984)
Chlorine (% of dried AP 1.02 Kennedy and Powlesland

material) (1984)
Aluminium (% of AP 0.003 Kennedy and Powlesland

dried material) (1984)
Iron (% of dried AP 3.6 x 10-3 -5.7 X 10-3 Kiviat (1982)

AP 5.0 x 10-3 Kennedy and Powlesland
(1984)
Ensiled AP 6.9 x 10-3, Alibes et al. (1984)
4.2 x 10-3
Organic matter AP 97.75 Wolter et al. (1980)

material) (1984)
Table 5. Continued
AP 97.5 Gasa et al. (1989)

Soluble solids AP first press 60 Kitson et al. (1972)

(% of dried AP second press 39.4 Kitson et al. (1972)
(1984)
Apples 75.7,76.4 Withy et al. (1975)
Total carbohydrate AP' 52.2 Voget et al. (1985)

(% of dried AP freeze dried 46.62 Wang and Thomas (1989)
material) AP drum dried 36.71 Wang and Thomas (1989)
AP concentrated 54.34 Wang and Thomas (1989)
AP 56.1, 48.8, 58.5 Carson et al. (1994)
Sugar content AP 29 Almosnino et al. (1996)

(% of dried
material)
Sugar content (Brix) Apple juice 13.7 McLellan et al. (1991)
Fermentable sugar WetAP 9-22 Smock and Neubert (1950)

(% of wet material) WetAP 12.3 Hang and Woodams (1984)
WetAP 12.3, 11.3, 10.6 Hang and Woodams (1986)
WetAP 7.0-7.9 Jarosz (1988)
Total sugar WetAP 12 Stapleton et al. (1984)

(% of wet material) WetAP 3.7 Hours et al. (1988a)
WetAP 1.4,2.1,9.1, 9.1 Hours et al. (1988b)
WetAP 5.7 Hang and Woodams (1994)
Apples 8.9, 10.5 Withy et al. (1978)
Reducing sugars Apples 6.9,9.1 Withy et al. (1978)

(% of wet material)
Sucrose (% of dried AP' 5.2 Voget et al. (1985)

material) AP 5.31 van der Merwe et al.
(1986)
Fructose (% of dried AP 25.72-27.94 Ngadi and Correia (1992a)

material) AP 13.6 Dreyer and van der Walt
(1979), Waugh (1981)
AP' 35.0 Voget et al. (1985)
(1986)
Table 5. Continued

AP 17.9-22.5 Ngadi and Correia (1992)
Glucose (% of dried AP 6.1 Dryer and van der Walt

material) (1979), Waugh (1981)
AP 12.3 Wang and Woodams
(1984)
AP' 12.0 Voget et al. (1985)
(1986)
AP 34.84-50.76 Ngadi and Correia (1992a)
Xylose (% of dried AP 0.06 Dreyer and van der Walt

material) (1979), Waugh (1981)
Starch (% of dried AP 17.89 Singhal et al. (1991)

material)
Cellulose (% of AP 22.94 Wolter et al. (1980)

dried material) AP 12-16 Kiviat (1982)
Apples 5.3,6.2 Withy et al. (1978)
Hemicellulose (% of AP 6.16 Wolter et al. (1980)

dried material) AP 5.01 Singhal et al. (1991)
Lignin (% of dried AP 19.10 Wolter et al. (1980)

material) AP' 16.1-18.5 Nikolic and Jovanovic
(1986)
Ensiled AP 8.3,8.6 Gasa et a!. (1992)
Pectin (% of dried AP 22-25 Kertesz and Green (1931)

material) AP 15.0-18.0 Smock and Neubert (1950)
Table 5. Continued

AP 3.5 Waugh (1981)
AP 14.5 Voget et al. (1985)
AP' 6.7,7.1,9.1,10.9 Hours et al. (1988a)
AP 18 Lopez et al. (1990)
Apples' 4.67,4.43 Withy et al. (1978)
Tannin (% of dried Apples 0.28,0.22 Withy et al. (1978)

material)
Phenolics AP 0.99 Dreyer and van der Walt

(% of dried (1979), Waugh (1981)
material) AP 0.72 Lu and Foo (1997)
Ascorbic acid Apples 0.043, 0.064 Withy et al. (1978)

(% of dried
material)
Bulk Density (kg/m') WetAP 935 Jewell and Cummings

(1984)
WetAP 385-526 Caprez et al. (1987)
Apples 1048, 1054 Withy et al. (1978)
Water binding AP 5.1 Arrigoni et al. (1986)

capacity (g/g of AP 3.9 Caprez et al. (1987)
dried material)
Oil absorption AP 1.9 Arrigoni et al. (1986)

capacity (g/g of AP 2.0 Caprez et al. (1987)
dried material)
Chemical oxygen AP 250-300 Hours et al. (1985)

demand - COD AP 282 Voget et al. (1985)
(g/kg wet material) AP 250-400 White et al. (1989)
Volatile fatty acids AP 1.59 Alibes et al. (1984)

(% of dried Ensiled AP 3.16 Alibes et al. (1984)
material)
Lactic acid AP 1.14 Alibes et al. (1984)

(% of dried Ensiled AP 5.84 Alibes et al. (1984)
material)
Ethanol yield from WetAP 43 Hang et al. (1981)

fermentation WetAP 51 Miller et al. (1982)
(gkg~l of wet apple WetAP 29-40 Hang et al. (1982)
pomace) WetAP 50.5 Alibes et al. (1984)
WetAP 44 Jarosz (1988)
WetAP 57 Gupta et al. (1990)
Table 5. Continued
Citric acid yield from WetAP 90 Hang and Woodams

fermentation (1986), Hang (1988a)
(gkg- 1 of wet apple WetAP 85.4-93.6 Hang and Woodams (1989)
pomace) 5% dry material 130 Hang (1988b)
AP
Butanol yield from WetAP 19-22 Voget et al. (1985)

fermentation
(gkg- 1 of wet apple
pomace)
Energy content AP 17.196 Stapleton et al. (1984)

(MJkg-l of dried AP 18.100 Sargent et al. (1983, 1986)
material) AP 19.9 Givens and Barber (1987)
Digestible energy AP 14.3 van der Merwe et al.

(MJkg- 1 of dried (1986)
material) AP 12.7-13.3 Preston (1991)
Total digestible AP 71.6 Knott et al. (1932)

nutrients (% of dry AP 60.5 Morrison (1942)
material) AP 69.0 Bath et al. (1986)
AP 69-72 Preston (1991)
Nitrogen -free extract AP 72.91 Knott et al. (1932)

(% of dried AP 62.1 Morrison (1942)
material) AP dried 54.8-59.3 Smock and Neubert (1950)
Council (1976)
(1984)
(1986)
AP 59.0 Hang (1988a)
Council (1976)
Oil content of apple Apple seed 22-25 Truelle (1926)

seeds(% of dry seed Apple seed 19.2 Pritzker and Jungkunz
weight) (1935)
Apple seed 19-23 Bertrand (1942)
Apple seed 13-15 Wakayama et al. (1956)
Apple seed 24.5 Amelotti et al. (1967)
Apple seed 22.1-31.0 Morice et al. (1971)
Apple seed 23.5 Bain (1988, 1989)
a Calculated from wet basis data and assumes 0% moisture in dried material.
40 .--------------------------------------------------,
35
o

1:
Cl
'iii
30 ? 0
3:0;:- ~ .;

~~ 25
o<tl .; 0
~~
20 II :
0
0"0
<tI::
0

EO
oC/l
D.. ~ 15
a.a.
~~
10

<{
o L-----~~----~------~------~------~----~
1938 1948 1958 1968 1978 1988 1998
Year
(0-'-0 data pWlished as range )
Fig. 3. Published data on apple pomace solid matter (wet weight basis) versus year
Table 6. Fatty acid composition of apple seed oil (% of total acids)
Material Value Reference

(%)
16:1 16:0 18:3 18:2 18:1 18:0 19:0 20 : 2 20: 1 20:0 21 :0 22:0 24:0
Granny Smith 0.2 7.0 0.3 62.9 25.8 1.5 0.3 0.4 0.2 0.8 0.1 0.1 Morice et
apple seed al. (1971)
oil
Stur mer 0.1 5.5 0.2 54.8 34.9 2.0 0.3 0.3 0.3 1.1 0.1 0.2 Morice et
oil
Dougherty 0.1 6.2 0.5 51.7 38.4 1.6 0.1 0.3 0.2 0.7 0.1 Morice et
oil
Apple pips 0.07 7.22 0.49 62.84 24.76 1.99 - 1.13 - 0.17 0.03 Bain (1989)
on apple pomace known to the authors. The dry matter content of wet pomace in
Table 5 is particularly interesting because it shows the efficiency of commercial
presses over time. With the increase in performance of presses it would be ex-
pected that the dry matter of the wet pomace would increase with time. However,
Smock and Neubert's best reading of 33.8% dry matter in 1950 is very close to the
best numbers to date of 35% dry matter by Sharma and Sharma (1984) and Sargent
et al. (1986; see Fig. 3).
List (1997 pers. comm.) notes that the best dry matter figures he has seen
have been, where extreme liquefaction followed by Bucher pressing gave 45% dry
Table 7. Gross amino acid profile of apple pomace protein (Data

from Patchell 1984)
Fraction of dried Fraction of total measured

material (0/0) amino acids (0/0)
ASP 0.71 11.6

THR 0.3 4.9
SER 0.37 6.1
GLU 1.24 20.3
GLY 0.39 6.4
ALA 0.37 6.1
VAL 0.35 5.7
MET 0.Q7 1.1
ISO 0.3 4.9
LEU 0.54 8.8
TYR 0.24 3.9
PHE 0.33 5.4
HIS 0.27 4.4
LYS 0.43 7.0
ARG 0.2 3.3
Total 6.11 100.0
Table 8. Physiological composition of apple pomace (0/0 of dried

material; data from Carson et al. 1994)
Flesh Seed Stem Press aid (rice hull)
70.0 3.2 0.4 26.4

75.7 3.3 0.9 20.1
75.7 2.2 0.5 21.6
matter. If older style decanters are used, then 15-16% dry matter is probable.
Normal Bucher press apple pomace with light enzyme maceration gives apple
pomace between 25-30% dry matter. There is a trade-off between press yield
and throughput when limited hardware is available, so maximising the dry
matter content of apple pomace can be of only academic interest. The advent of
enzyme use in apple juice pressing has had a dramatic impact on the use of
press aids. Historically, rice hulls, defribrated bleached Kraft fiber, paper ground
wood pulp and diatomaceous earth have been used as press aids (Bump 1989).
Now many apple processors do not use press aids, the exception being those
who do not use pectinases because they want to use the apple pomace for pectin
extraction.
As expressed in the introduction, the apple pomace user does not have the
luxury of having a constant composition material, and Table 5 acutely reflects
apple crop 70% direct consumption
30%
apples for 76% apple juice

juice extraction
24%
wet apple pomace
flesh and skin seeds stem

94.5% 4.1% 1.1%
Fig. 4. Mass flows of components from the apple crop based on data from Tables 5 and 8. All
data on wet weight basis
this with many components varying over wide limits. With many of the compo-
nents in Table 5, care must be taken with interpreting the data from the literature.
For example, component analyses can either be reported on a dry or wet basis.
As Table 5 shows, dried apple pomace contains about 10% water. If a paper reports
data on a dry basis, is it the concentration in dried material (10% water) or back
calculated to a bone dry basis (0% water)? Often researchers take little care with
specifying the correct basis and there can be a subtle difference between the
phrases "% dried material" and "% dry matter".
wet apple pomace ------+ water 76.3;;,
1
dry material 24.0%
I
l r 1 l'l! ! !
Polyphenols Vitamins Crude Protein Fat Wax Carbohydrate Simple Ash
4.5% 4.9% 1.7'/;, Polymers Carbohydrates 2.6%
Tannin Vitamin C
I 1
0.25% 0.054% 1 I I 1 1 I I 1
1
Pectin Lignin Hemicellulose Cellulose Starch Xylose Sucrose Fructose Glucose
12.7';10 12.8% 5.0% 17.6% 17.9% 0.06% 5.4% 22.8% 16.8%
Fig. 5. Summary of average composition of apple pomace based on data in Table 5. The total comes to 125%! The most likely source of error is the starch, fructose
and glucose contents which vary widely depending on apple ripeness and cultivar. Another potential source of error is confusion related to reporting data on an
"as driedl' basis (24% water) or a "bone dry" basis (0% water). The data above is, however, a useful proximate summary of apple pomace composition
Table 9. Elemental analysis of apple pomace. Summary of data

in Table 5
Element Dried material (0/0)
Oxygen 44.78
Carbon 44.56
Hydrogen 6.18
Chlorine 1.02
Potassium 0.61
Nitrogen 0.57
Calcium 0.14
Phosphorus 0.09
Magnesium 0.Q7
Sulphur 0.03
Sodium 0.02
Iron 5.25 x 10-3
Aluminium 3 X 10-3
Zinc 7.3 X 10--4
Manganese 1.27 X 10-4
Copper 4.2 X 10--4
Other elements 1.91
Total 100.0
Another confusion in the analytical literature is the misuse of similar terms

for the same component. A typical example is water insoluble components.
Should this part be called "ether extract", "lipid fraction" or "fat content"?
"Ether extract", "lipid fraction" and "fat content" are not synonymous - each
applies to specific procedures, e.g. "ether extract" is obviously used when the
sample is extracted with either anhydrous diethyl ether or petroleum ether, ac-
cording to the procedure. "Lipid fraction" usually refers to the fraction which has
been separated from the "non-lipid fraction", often with a water-immiscible sol-
vent mixture such as chloroform-methanol. "Fat content" is based on the details of
specific methods and generally refers to the triglycerides and other ether-soluble
materials. Some methods may use an enzyme solution treatment before solvent
extraction.
Similarly, some authors use the terms "total solids", "total sugars" and
"total carbohydrates" with rather more poetic licence than a strict scientific
definition would allow. Another example is ash and organic matter. Ash +
organic matter should not equal 100%, and these variables are not reporting
the same information. The inorganic material can be present as chlorides,
sulphates or phosphates. Some inorganic metals are also volatile and can be lost
unless a sulphated ash technique is used. Crude protein and Kjeldahl nitrogen
also represent the same information. Often the apple pomace analysis does
not indicate if the pomace contains press aid. The use of significant figures
in reporting data in the literature is haphazard, as Table 5 shows. Table 5 should
thus be used with care but is useful for identifying papers which contain methods
for analysing particular components.
5 Analytical Techniques
This review does not concentrate on all analyses which are routine when dealing
with plant material, but rather on techniques which are crucial to the use of apple
pomace, those techniques in which conducting analyses on apple pomace requires
some modification of standard methods, or else techniques that have only recently
been applied to apple pomace.
5.1 Routine Methods of Analysis
The standard methods used for pomace analysis are usually taken from the US
Association of Official Analytical Chemists (1990) who publish Official Methods
of Analysis. In particular, Chapter 37 deals with "Fruits and Fruit Products".
Table 10 lists some of the more relevant analyses.
5.2 Water ContentlDry Matter Content
Various moisture meters and methods for measuring moisture are available com-
mercially. The most common methods involve deriving a dry matter value by
drying a pomace sample from a wet basis. The final weight over the original weight
multiplied by 100 is the dry matter percentage. The difference between the dry
weight and the original weight multiplied by 100, over the original weight is the
moisture percentage. The measurement is important as dry matter content can be
linked to stability. Dry matter is often used as basis to describe the percentage
composition of other compounds in pomace (such as pectin). The moisture con-
tent of pomace and consequent bulk of pomace is so variable that references to
chemical composition on a wet basis are often oflittle use. Dried apple pomace can
reabsorb moisture in storage (Kiviat 1982; Kertesz and Green 1931).
Although oven drying is a standard method, microwave drying has been used
for fruit products in recent years. This has the advantage that it is quick (minutes
compared to overnight for oven drying), but suffers from the disadvantage that
often localised heating occurs, and that heating time is dependent on sample size.
As a constant microwave drying time is often used, under drying or charring can
occur depending on sample size. The disadvantages of microwave drying are
overcome with the use of modern infrared drying techniques (Kennedy and
Phillips 1996). The infrared dryer heats the sample by transmission of electromag-
netic radiation which penetrates and heats the sample from the bottom, giving
uniform drying. Sample size and temperature can be easily varied, and drying
progress monitored to obtain rapid drying times of approximately 30 min, without
compromising the accuracy of the determination. A study by Fenton and Kennedy
(1997) comparing the traditional oven drying technique with the infrared drying
technique using apple pomace showed that the infrared dryer could be used to
accurately measure the dry weight of samples with a wide range of moisture
Table 10. Analyses of fruit and fruit products from the US Association of Official Analytical
Chemists Official Methods of Analysis
Number Title Method
920.151 Solids (total) in fruit products Vacuum oven

922.10 Solids (water insoluble) in fruit Vacuum oven
products
932.12 Solids (soluble) in fruit products Vacuum oven
940.26 Ash of fruit and fruit products Vacuum oven
929.05 Potassium in fruits and fruit products Chloroplatinate method
929.06 Potassium in fruits and fruit products Cobaltinitrite method
965.30 Potassium in fruits and fruit products Flame photometric method
966.16 Sodium in fruits and fruit products Flame spectrophotometric method
931.09 Manganese in fruits and fruit products Flame spectrophotometric method
929.07 Calcium in fruits and fruit products Flame spectrophotometric method
931.10 Magnesium in fruits and fruit products Flame spectrophotometric method
942.14 Phosphorus in fruits and fruit products Volumetric method
935.45 Phosphorus in fruits and fruit products Colorimetric method
970.39 Phosphorus in fruits and fruit products Spectrophotometric method
970.40 Phosphorus in fruits and fruit products Quinoline molybdate method
924.06 Sulfur in ash of fruit products Gravimetric
924.07 Sulfur (total) in fruit products Gravimetric
928.06 Chlorine (total) in fruit products Gravimetric
920.152 Protein in fruit products Kjeldahl method
942.15 Acidity (titratable) of fruit products Both indicator and pH electronic
methods
925.34 Acidity (volatile) of fruit products Steam distillation
943.03 Citric acid in fruits and fruit products Pentabromacetone method
954.07 Malic acid (laevo and inactive) in Titrimetric method
fruits and fruit products
968.19 Laevo-malic acid in fruits and fruit Polarisation method
products
986.13 Quinic, malic, and citric acids in Liquid chromatography
cranberry juice cocktail and apple
juice
969.30 Organic acids (foreign) in fruit juices Paper chromatography
971.18 Carbohydrates in fruit juices Gas chromatography
925.35 Sucrose in fruit and fruit Products Optical rotation
968.20 Oil (recoverable) in fruit and fruit Titrimetric method
products
contents. Also, there was shown to be excellent agreement between the two meth-
ods for the dry weight data obtained, indicating that the much quicker infrared
method could produce reliable data, see Fig. 6.
5.3 Bulk Density
Bulk density (kg/m3) is an important parameter to know if an apple pomace

processing plant is being designed e.g. a fermentation plant, or a pectin extraction
plant. It is one of the key parameters used in sizing the reactor. Its value varies
widely with water content (Fig. 7). The usual method of measurement is by filling
a volumetric cylinder and weighing its contents. Simple but effective.
5.4 Crude Protein
Crude protein is estimated by measuring the Kieldahl nitrogen content of the

sample and multiplying by the "magic number" 6.25 (Davidson 1980). However,
many people do not realise that this number, which assumes that all protein
30
.
..... - PredIcted % dry wt

.. I
25
I
~ Oven %dry w\
Inhaled %dty w1
'
'"
.
~ 20 it.
'0
'"
i!
.,
'tI 15
....
'2'1"
.
:;
.,'"
E 10
.~ ' .
'. "
...... .

5 ........
'!
0
-50 0 50 100 150 200 250 300
% dilution 01 apple pomace
Fig. 6. A comparison of oven and infrared drying techniques to estimate the water content of
apple pomace. (Data from Fenton and Kennedy 1998)
1200
1000
.!.:..' # . ,
ien
800 ..... '
':!. ... .J
. .. ....... ..... ..., .'.

600
i:' ."
.
Iii
c
.....-... .... ..;

0
400
~
200
0
0 10 20 30 40 50 60 70 80 90 100
Moisture Content (percent water)
Fig. 7. Apple pomace density as a function of moisture content

contains 16% nitrogen, is actually an average for many types of protein. This
"magic number" can vary from 5.30 for most nuts and seeds, through to 6.38 for
most milk products. The "magic number" can be simply calculated once the amino
acid profile of the protein is known. The nitrogen content of each amino acid is
easily found from its empirical formula. Based on the data reported by Patchell
(1984; see Table 7) a protein conversion number of 6.42 is obtained for apple
pomace. This is quite high, and by using 6.25 an under reporting error of 3% can
be introduced into crude protein measurements.
5.5 Apple Pomace Buffering Capacity
The pH of apple pomace can vary from 3.2 to 4.1, see Table 5. For fermentation
purposes this pH may be too low for many micro-organisms and may have to
be adjusted up to 6 or higher to get good microbial growth. Similarly, enzyme
processes may require high pHs. This means that the amount of base to be
added is an important economic consideration. The buffering capacity of apple
pomace is therefore a valuable measurement. This is determined by taking
apple pomace and adding a known amount of base until the pH rises, followed
by adding a known amount of acid until the pH falls, (see Fig. 8) or vice versa.
5.6 Bioavailable Energy
The most popular sales opportunity for apple pomace, either wet or dried, is
within the animal feed market. Apple pomace does not have an especially high
14 ,--------------------------------------------------------,
12
. .. e.. 8 ..l::~ ' '' ci ., .... .... ..., ....
. 8
.. 0' . 9
.
. - . . - . ... .. .
I. NaOH 1M
o HCIIM
I
0
~:
10

Jo
o
o
8
xQ,
.~
6
'. 0
o '~ 0
o "~~ 0
4 o " 0 0
o
..g.. a..e'9 ' e e',9. ',9 ' '<9 ' '6" ' 6"
" 0 ., 0
o 0 ~ l
2 0
o ~------------------------------------------------------~
o 0 .2 0 .4 0 .6 0.8 1.2
mL added I 9 Apple Pomace
Fig. 8. Buffering capacity of apple pomace. Addition of sodium hydroxide followed by hydro-
chloric acid
lO6 M. Kennedy et al.
protein component and consequently does not represent a good stand-alone

feed for many animals. However, alongside a traditional fodder such as grass,
apple pomace acts as a very' good supplement to stimulate weight gain or lactation.
The pomace can be sold wet if a local feed lot can use the product quickly.
Frequently the pomace is dried to reduce the cost of freight to farms and to
increase stability.
When making least cost buying decisions, it is important that the farmer
compares the efficacy of various feed supplements. This data can then be related to
the price of the feed-stock (usually on a dry matter basis). A common way to assess
efficacy is to run energy balances on specific animals of interest consuming a
specific diet. For example, metabolism trials for pigs may be conducted as follows
(Smith et al. 1987):
1. Select several animals from a weaner pool.
2. De-worm the animals and pen them singly for several days.
3. Transfer animals to metabolism crates where they are kept in a temperature
controlled environment.
4. Daily feed animals a restricted and constant level of gross energy and water.
5. Collect a representative sample of each animal's faeces, freeze dry, grind and
store.
6. Collect urine samples and freeze.
7. Determine dry matter of faeces.
8. Using adiabatic bomb calorimetry (ABC), determine gross energy of sub
samples of faeces, urine and air dried sample of the diet.
The metabolizable energy unit is normally described in terms of MJkg-l
dry matter. A result is calculated by subtracting the calorimetric value (as
determined by ABC) of the faeces and urine from the calorimetric value of the
feed (a correction for analysed dry matter content is made). The measurement
is frequently described as apparent metabolizable energy (AME), as the ex-
perimental method does not determine energy that is utilised by intestinal
microbes during digestion (as opposed to that used by the animal). In some
cases researchers seek to analyse the energy left in the faeces of the animal
separately (and not the urine). In that case, calorific energy calculated to be ab-
sorbed by the animal is referred to as apparent digestible energy (ADE). Clearly,
for trial stock such as chickens, which do not excrete urine, only AME may be
calculated.
The metabolizable energy of various pomace streams usually ranges
between 7.5-1O.2MJ/kg dry matter. The actual energy is dependent on a host of
interactions.
5.7 Polyphenol Components
The polyphenol fraction is predominantly concentrated in the skin of apples.

While collectively the polyphenols amount to only a small fraction of the pomace,
they nevertheless are of interest as a potential source of nutraceuticals because

they possess free radical scavenging and antioxidant activities. They consist of
different classes of compounds and in apple pomace the major polyphenols consist
of chlorogenic acid, epicatechin and their oligomers, quercetin glycosides and
phloridzins (Lu and Foo 1997). For analysis, these compounds are best extracted
with aqueous organic solvent mixture, the most common being 70% aqueous
acetone. Good resolution of these compounds can be obtained by HPLC using
LiChroCART 125-4, Lichrospher 100 RV-18 (Sj.lm) column set at 30C. Solvents
are (A) acetic acid/water (2/98) and (B) acetic acid/acetonitrile/water (2120178)
with solvent programming starting with 80% A and 20% B to give 40% A and 60%
B at 40 min and 100% B at 60 min with flow rate of 0.5 ml per min. Detection is by
UV absorption at 280 nm for all phenols and 350 nm for flavonoids. The HPLC
chromatogram of a 70% aqueous acetone extract of apple pomace is reproduced in
Fig. 9, and shows good resolution of the flavans and their oligomers as well as the
various quercetin glycosides.
Where the identity of individual compounds is not required, the total
polyphenol content can be determined by a variety of assays. The preferred assay
today is the Folin-Ciocalteu method which is based on the reduction of a
phosphotungistic-phosphomolybolic reagent in a slightly alkaline medium. This
colorimetric assay has been evaluated by Scalbert (1992) to be the most appropri-
ate for the determination of total phenol in plant extracts. However, several sub-
stances including ascorbic acid and excess sugars which are present in the pomace
cause some interference with the analysis and this can be avoided by passing the
extracts through a column of hydrophobic resin such as XAD-2 or HP20, and then
washing the resin with water to remove the ascorbic acid and sugars before eluting
the polyphenols with methanol. The residue, after removal of methanol, is then
taken up to volume in acidified methanol (0.3% HCl) and water (3: 2 v/v). Extract
aliquots of 100 j.ll are dissolved in 2.0ml of 2% Na2C0 3 solution, and after 2min,
100j.ll of a 50% Folin-Ciocalteu reagent is added and left to stand at room tempera-
ture for 30 min. The absorbance of the solution is measured at 750 nm against a
blank consisting of all the reagents and solvents minus the test sample. The stan-
dards commonly used are catechin or gallic acid, and they are prepared at concen-
trations from 0.05 to 0.5 mg/ml. The results are expressed as catechin or gallic acid
equivalent.
5.8 Antioxidant Analysis
To assess the antioxidant activity of the pomace extracts (or purified compounds
derived from them) the relative free radical scavenging activity (compared with
ascorbic acid or a-tocopherol) is measured using 2,2-diphenyl-1-picrylhydrazyl
(DPPH) as the radical species. This is carried out by adding 0.1 ml of a polyphenol
solution (0.1 mg/ml in 95% ethanol) to 2.0ml of a DPPH solution (0.1 mM in 95%
ethanol). The mixture is kept at room temperature for 60 min and at the end of this
NOr DAD1 A, Sig=280,8 Ref=590,1 15
80-
60
40
20
o
o 10 20 30 40 50 60 70 min
Fig. 9. HPLC chromatogram of 70% acetone extract of apple pomace
Peak identification
Peak RT compound
1 25.20 Catechin
2 29.01 ChI orogenic acid
3 33.lO Pro cyanidin B,
4 35.18 Unknown
5 38.46 Epicatechin
6 44.27 Epicatechin trimer
7 47.22 Epicatechin tetramer
8 61.64 3-Hydroxyphloridzin and quercetin-3-rutinoside
9 62.21 Quercetin -3 -galactoside
lO 63.19 Quercetin-3-g1ucoside
11 65.11 Phloretin -2' -xyloglucoside
12 65.70 Quercetin -3-xyloside
13 68.77 Quercetin-3-arabinoside
14 70.76 Quercetin-3-rhamnoside
15 72.08 Phloridzin
period the absorbance is measured at 517 nm. The radical scavenging activity
(RSA) is calculated from the formula:
where Ao is the absorbance of the blank, and As is the absorbance of the test sample.
The relative free radical scavenging activity is the ratio obtained by dividing the
RSA of sample by the RSA of ascorbic acid or a-tocopherol.
Another method which is popular is the superoxide dis mutase (SOD)

method. This assay is based on the generation of O~ by the xanthine oxidase
system, which then reduces nitro blue tetrazolium (NBT). Essentially, a 0.1 ml
solution of various concentrations (O to 100 units/ml) of SOD (Sigma, S-2515) or
test samples dissolved in DMSO (0.1 mg/ml) are added to a 1 ml mixture consisting
ofO.4mM xanthine and 0.24mM NBT in 0.1 M phosphate buffer (pH 8.0). Xanthin
oxidase (0.049 unit in 1 ml of 0.1 M phosphate buffer) is added to the samples and
the mixtures incubated at 37C for 20 min. After this period, 2 ml of 69 mM sodium
dodecylsulfate is added to terminate the reaction and the absorbance of the solu-
tions is measured at 560 nm. The SOD activity is calculated from the standard
curve as SOD equivalent per mg of extract.
5.9 NMR Analysis: A Novel Method of Characterising Apple Pomace
Nuclear Magnetic Resonance (NMR) is a powerful tool for characterising apple

pomace. As it is an expensive research tool, its use on apple pomace to date has not
been extensive, although a study of apple cell walls is relevant (Newman et al. 1994).
As some readers may not be familiar with NMR, and since its application to fruit
wastes is relatively recent, a brief outline of the NMR technique is provided here.
About 99% of carbon atoms have carbon-12 nuclei, with no magnetic proper-
ties. About 1% have carbon-13 nuclei which behave like weak magnets. They can
be aligned in a strong magnetic field imposed, for example, by a superconducting
solenoid. The alignment can be disturbed by applying an intense pulse of radio
frequency energy through a wire coil wound around the sample. This provokes
a response which can be detected by a very sensitive radio frequency receiver.
Carbon-13 nuclei in different chemical environments respond at different frequen-
cies. A plot of signal strength against frequency is called a "spectrum". The "chemi-
cal shift" of a signal is defined as the deviation of the resonance frequency from
that observed for a reference standard, namely, carbon-13 in tetramethylsilane.
Chemical shifts are expressed in parts per million (ppm).
Chemical shifts for liquid samples can provide information about chemical
structures of molecules. Chemical shifts for solids can provide additional informa-
tion about how the molecules are packed together. The information content of
solid-state NMR spectra is far higher than that ofliquid-state NMR spectra, but the
problem is extracting that information. Harris (l993) describes developments that
have unlocked the information content of solid-state NMR spectra. In particular,
"magic-angle spinning" (MAS) collapses broad patterns of related signals to single
frequency, while "cross-polarisation" (CP) enhances signal strengths and shortens
the timescale for accumulation of data. The spectra described in this report were
obtained with a combination of these two techniques, i.e., by CPMAS NMR
spectroscopy.
Jarvis and Apperley (l990) have surveyed applications of CPMAS NMR in
studies of plant cell walls. The obvious advantage of the technique is that it allows
us to study cell wall polymers in situ, rather than requiring selective extraction and
chemical characterisation in solution. A further crucial advantage is that the rigid-

ity or mobility of individual cell wall components can be assessed from "relaxation
time constants", i.e., timescales over which signals decay or recover following
pulses of radio frequency energy. There is, however, a serious problem. Magic-
angle spinning imposes high centrifugal forces that can crush normal living plant
tissues. Current research efforts are therefore mostly confined to isolated cell
walls, partly-dried material or intact seeds (Haw and Maciel 1983). The problem
of centrifugal force also limits the size of the sample to dimensions of a few
millimetres.
Applications of NMR are illustrated (Figs. 10 and 11) by spectra obtained on
a Varian Inova-200 spectrometer operating at a carbon-13 NMR frequency of
50.3 MHz with MAS at 4kHz. Samples were air-dry, and weighed between 0.2 and
0.3 g. Each 5lls proton preparation pulse was followed by a 2 ms CP contact timed,
30 ms of data acquisition, and a recovery delay of at least 1 s. Signals were averaged
overnight (Fig. 10) or for periods of 1 or 2h (Fig. 11).
The NMR spectrum of the pomace (Fig. 10) is dominated by signals assigned
to cellulose and pectin, as confirmed by comparison with the spectra of related
model substance (Fig. 11, a and b). Relative signal strengths do not match those
observed in the spectra of the models. In the case of pomace cellulose, the crystal-
lites have such narrow cross-sectional dimensions that most of the cellulose chains
are exposed on the crystallite surfaces (Newman et al. 1994). The spectrum of
Avicel microcrystalline cellulose shows signals assigned to crystallite surface
chains (labelled s) weaker than signals assigned to crystallite interior chains (la-
belled i) because the crystallite cross-sectional dimensions in this commercial
200 180 160 140 120 100 80 60 40 20 o

Chemical shift (ppm)
Fig. 10. Carbon-13 solid-state NMR spectrum of apple pomace. c Cellulose; p pectin
Apple Pomace and Products Derived from Apple Pomace III
C-2 ,3,5
cellulose
pectin
starch
20 o
Fig. 11. Carbon-13 solid-state NMR spectra of three carbohydrate polymers related to those
found in apple pomace: i crystallite interior, s crystallite surface
product are sufficiently large for most chains to be enclosed in crystallite interiors.
Signal assignments for cellulose (Fig. 11a) are based on Newman and Hemmingson
(1995). In the case of pomace pectin, the signal assigned to C-6 (170 to lS0ppm)
shows an asymmetric line shape with the maximum at 171 ppm assigned to galac-
turonic ester units and a shoulder on the left-hand side assigned to galacturonic
acid units (Jarvis and Apperley (1995). The model selected to produce Fig. llb, a
citrus pectin (Sigma P-9596), is clearly more highly esterified. The peak at 171 ppm
shows little sign of a shoulder, and the methoxyl signal at 54 ppm is relatively
strong.
The spectrum ofM&B maize starch (Fig. lIe) is shown to illustrate NMR peaks
that can appear if pomace is prepared from fruit that is not fully ripe. Pectins and
carbohydrate oligomers or monomers can account for signal strength in the
chemical shift range 100 to 103 ppm in Fig. 10, so there is no evidence for a high
starch content in this particular pomace. Minor components of the pomace iden-
tified by NMR include tannin, wax, cutin and protein. The condensed tannins can
be identified by characteristic signals at 155 and 145 ppm, assigned to oxygen-
substituted aromatic carbon in the A and B rings, respectively (Newman and
Porter 1992). Wax and cutin can be identified by signals at 33 and 30ppm, respec-
tively (Pacchiano et al. 1993). Protein cannot be so positively identified by specific
characteristic signals, but a band between IS and 35 ppm is consistent with side
chain carbon in protein. Signals from secondary amide carbon (173 ppm) and C-2
of each amino acid (typically 55 ppm) coincide with other signals in pomace. Other
minor signals, e.g., those associated with fruit acids, remain unidentified at this
stage.
Perhaps the most useful contribution of NMR spectroscopy to pomace chem-
istry is in the area of clarification of terminology. In particular, the word "lignin"
is commonly used in describing the composition of pomace. This word is also used
in wood chemistry to label polymeric matter constructed from certain phenolic
monomers. The carbon-13 NMR spectra of softwood and hardwood lignins in-
clude signals at 149 ppm and 153 ppm, respectively, assigned to oxygen-substituted
aromatic carbon (Leary et al. 1986). The spectrum of apple pomace (Fig. 10) shows
no detectable signal in that chemical shift range. It seems that the word "lignin" is
used in a looser sense in analysis of pomace. An operational definition on "lignin"
can cover any substance that is left in an insoluble residue following specified
chemical treatment. That residue could contain cutin, tannins or other matter that
is distinct from the lignin analysed by wood chemists. Incorporation of tannin in
a "lignin" residue has been described by Leary et al. (1986) in the context of wood
chemistry. Cutin has been identified as remaining in the acid residue that is
conventionally termed "lignin" in analysis of cereal brans (Ha et al. 1997). It has
been suggested that cutin, rather than lignin, plays an important role in protecting
wheat bran from microbial degradation in the rumen and in the human gut (Ha
et al. 1997).
The signals at 155 and 145ppm in Fig. 10 correspond to just five carbon
atoms out of 15 in each structural unit of a condensed tannin (Newman and
Porter 1992). After allowance for the other carbon atoms in the structure of a
condensed tannin, it becomes clear that the tannin content of pomace amounts
to a few percent by weight. Lower values in Table 5 probably refer to water-soluble
tannin. Benner et al. (1990) have drawn attention to the inaccuracy of colorimetirc
methods for tannin analysis in senescent plant tissue. They found that carbon-
13 NMR spectra indicated a tannin content of about 20% by weight in mangrove
leaves, unchanged during senescence and decomposition, yet a colorimetric
assay indicated an initial tannin content of 10% and a rapid decrease until the
level dropped below the detection limit. They concluded that the colorimetric
method provides a good measure of leachable tannin, but not of the total tannin
content.
Many of the solubility-based gravimetric techniques used to determine the
biochemical components of plant tissue were developed primarily for quantifica-
tion of compositions of fodder and wood. Problems arise when the same methods
are applied beyond their intended use. In addition to problems in distinguishing
between "lignin", "tannin" and "cutin", Benner et al. (1990) suggest that complex
polysaccharides are not accurately resolved into cellulose and hemicellulose.
The most important advantage of solid-state carbon-13 NMR over standard
degradative methods is the ability of NMR to provide a measure of the relative
contents of each of the major classes of structural polymers in a single, non-
destructive analysis (Benner et al. 1990).
Additional information can be gained from NMR experiments which deter-
mine nuclear spin relaxation time constants, i.e., the timescales for signal decay
or recovery following specified perturbations induced by pulsed radio frequency

energy. The time constants can be sensitive to differences in molecular rigidity,
so it can be helpful to study samples with a moisture content adequate to plasticise
domains containing non-cellulosic polysaccharides. Newman et al. (1994) used
this approach to edit the carbon-13 NMR spectrum of Braeburn apple cell
walls, separating subspectra assigned to relatively rigid matter (primarily cellu-
lose) and less rigid matter (pectins, xyloglucan, etc.). Such editing procedures
allow more detailed characterisation of selected components, e.g., estimation
of relative proportions of different crystalline forms of cellulose, the number
of cellulose chains in each microfibrillar bundle, etc. (Newman et al. 1994).
Solid-state NMR also shows potential for exploring molecular interactions,
e.g., between cellulose and xyloglucan (Whitney et al. 1995) although further
work on signal assignments seems desirable before this work is extended to
pomace.
6 Discussion
This review highlights the highly variable composition of apple pomace. The user
has little ability to control the composition of this product, and simply takes it as
it comes from the processing plant. There have been many uses investigated for
apple pomace but few show significant economic potential.
One of the issues which quickly faces the analyst (and user) of apple pomace
is that it quickly ferments with subsequent production of an ethanolic odour.
Thus, if an accurate analysis is required, the samples must be quickly refrigerated
or frozen. The analyses of apple pomace conducted by industry are often simple
e.g. quantification of dry material, or sugar content, but are widely used. The more
sophisticated analyses tend to be used during the search for high value products
from apple pomace. This trend will continue with each more complex (and minor)
chemical separated from pomace. Of all the tools used to analyse apple pomace,
NMR offers a high degree of molecular information and to date has been under
utilized.
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Kiwifruit Waste and Novel Products Made from
Kiwifruit Waste: Uses, Composition and Analysis
M. KENNEDY, D. LIST, Y. Lv, L.Y. Foo, A. ROBERTSON, R.H. NEWMAN and G. FENTON
1 Introduction
Kiwifruit is known worldwide as an attractive green fleshed fruit. This chapter

is not an all-encompassing review of kiwifruit. Articles such as that of
Warrington and Weston (1990) provide an excellent status of the industry. This
review highlights areas the authors view as significant and with which they have
expertise.
Kiwifruit waste comes in two forms. The first is whole fruit that has not made
the grade for direct sale. These so-called reject kiwifruit can comprise up to 30% of
the total kiwifruit crop grown. The second is kiwifruit pomace (see Fig. 1), which
is the presscake residue left after kiwifruit juice manufacture. These two wastes
have different compositions and often the processor does not have the luxury of
determining which waste will be dealt with.
The production of kiwifruit in New Zealand began to increase in the 1970s
(see Fig. 2). Kiwifruit processing is conducted at several locations in New
Zealand (Anonymous 1997). In the Southern Hemisphere the major producers are
New Zealand, Australia, Chile and South Africa. In the Northern Hemisphere the
USA, Italy, Japan, France and Greece are involved in production (Warrington
1990).
2 What Use Is Kiwifruit?
Finding uses for fruit wastes has been a preoccupation of fruit growers and proces-
sors for at least 100 years. Whereas a large amount of effort has been expended in
finding alternative uses for wastes such as apple pomace, comparatively little work
has been done in finding uses for reject kiwifruit and kiwifruit pomace. The reason
for this is the young nature of the industry. Figure 3 shows, from a literature search
of the Commonwealth Agricultural Bureau (CAB) database, that kiwifruit re-
search, as reflected by a number of publications, peaked in 1992. Few publications
deal with kiwifruit pomace or waste processing. Large-scale commercial growing
began in New Zealand, and as the fruit transitioned from a high value item to a
more common commodity, more effort went into recovering value from the waste
(or co-products). New Zealand in particular has been keenly involved in the hunt
Fig. 1. Kiwifruit pomace produced from a rotary vacuum filter at Industrial Research Limited
300
oS
E 250
";,
CI>
U
"~
0
-
200
~
.~ 150
:;;:
..
'0
CI>
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100
c
...
0
.g 50
0;
:::;:
o L.--~----~--~--~~~~--~----~--~--~
1952 1957 1962 1967 1972 1977 1982 1987 1992 1997
Year
Fig. 2. Kiwifruit production in New Zealand. (Data from Earp 1990 and the Zespri (kiwifruit)
Marketing Board of New Zealand)
Kiwifruit Waste and Novel Products Made from Kiwifruit Waste 123
~
250
<.>
~
iii
.0
ct 200
CD
ct
()
.~
-g> .,oj 150

;;;~
8.. ~
a.",
ct C 100
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CD
en
CD
en en en
... ...
en en en en ~ ...
en en en ~
Year
Fig. 3. Historical data on the number of papers published on kiwifruit
for added value uses for kiwifruit. The uses of kiwifruit are crucial in determining
the assays to be performed in the fruit. The uses, potential uses and ideas that have
been considered for using kiwifruit are listed in Table 1. The grower and processor
has one urgent aim; to get the waste dealt with as soon as possible and preferably
off site. Thus the aim of finding uses is to reduce waste volumes or to find a high
value product, preferably both.
Finding a new high value product is good for economics, but if it is only
present in low concentrations, large amounts of waste are still left to be dealt with
after recovery of the high value product.
One of the first efforts in using reject kiwifruit went into producing kiwi-
fruit juice. This highlighted the problem that treating kiwifruit juice at high
temperature and under the acid conditions of the juice meant that the kiwi-
fruit chlorophyll degraded to pheophytin. Thus the kiwifruit juice and canned
products were olive green/brown instead of bright green. Much effort then
went into retaining the chlorophyll and hence bright green colour, which was
successful.
Following this hurdle, the next breakthrough was the development of kiwifruit
wine, popular at tourist outlets in New Zealand. This was followed by distilled
kiwifruit liqueur and kiwifruit vinegar. The only kiwifruit vinegar production
facility known to the authors was started in Tauranga, New Zealand by Preston
Group Ltd. This produced vinegar with excellent flavour characteristics which is
innovatively used by New Zealand food manufacturers. Kiwifruit vinegar must
rank as one of the most underutilized uses for kiwifruit wastes.
One of the most intensely studied kiwifruit products is the proteolytic enzyme
actinidin which has been promoted as a meat tenderiser. For many years the
Table 1. Uses and potential uses of kiwifruit pomace and waste kiwifruit
Use of product References
Review articles Warrington and Weston (1990)

Kennedy (1994)
Human food incorporation:

Puree Dalla Rosa et al. (1990)
Castaldo et al. (1992)
Juice and concentrate Davies (1982)
Wilson et al. (1983)
Wilson and Burns (1983)
Palmieri et al. (1990)
Crivelli et al. (1990)
Dawes et al. (1991)
Taylor (1992)
Anonymous (1992b)
Smith (1994)
Minimally processed kiwifruit, "fourth generation" Lodge and Perera (1992)
foods
Nektalite Anonymous (1993d)
Retention of green colour of kiwifruit Robertson (1985)

products/chlorophyll production Lawes (1989)
Johnson et al. (1990)
Townsend (1991)
Johnson and Steel (1991)
Perera and Lodge (1992)
Cano and Marin (1992)
Anonymous (1991, 1992a)
Steele and Johnson (1995)
Distilled kiwifruit liqueur Grace and Logan (1989)
Kiwifruit wine Heatherbell et al. (1980)

Lodge (1981)
Withy and Lodge (1982)
Lodge et al. (1986)
Craig (1988)
Kiwifruit vinegar Preston (1997, pers. comm.)
Aroma and volatile compounds Young et al. (1983)

Young and Paterson (1985)
Takeoka (1987)
FischbOck et al. (1988)
Bartley and Schwede (1989)
Tatsuka et al. (1990)
Young et al. (1995)
Citric acid via fermentation Hang et al. (1987)

Table 1. Continued
Use of product References
Enzymes:
Actinidin Boland and Hardman (1972, 1973)
Boland (1973)
Baker et al. (1980)
Yamaguchi et aI. (1982)
Lewis and Luh (1988)
Otter et al. (1989)
Pn!stamo (1995)
Paul et al. (1995)
Lipoxygenases Boyes et al. (1992)
Xyloglucan endotransglycosylase Percy et al. (1996)
a- Mannosidase Ogawa et al. (1990b)
p-Galadosidase Ogawa et al. (1990a), Ross et aI.
(1993)
Peroxidase Prestamo (1989), Soda et al. (1991)
Lysozyme Lynn (1989)
Polyphenol oxidase Park and Luh (1985)
Enzyme inhibitors:
Pectin methylesterase inhibitor Balestrieri et aI. (1990)
Pectinesterase inhibitor Balestrieri et aI. (1991)
Cell wall components Redgwell et al. (1988, 1991a,b)

Dawson and Melton (1991)
Landfill Anonymous (1993a-c)
Mucilage from pruning wounds Redgwell (1983)

Redgwell et al. (1986)
Clark et al. (1986)
Peteriunger et aI. (1990)
Galletti et al. (1993)
Kiwifruit pollen Clark and Lintas (1992)

Matsunaga et al. (1996)
Vitamin C Burns (1985)
Pectin Burns (1985)

Lodge et al. (1987)
Animal feed Kennedy (1994)
Mushroom compost yet to be tested
Kiwifruit antioxidants Industrial Research Ltd

economics of production of actinidin were not considered favourable. Part of this

pessimism was due to strong competition from widely available products such
as papain, which one author estimated is available for one third to one half the
cost of producing actinidin (Strachan 1974). Despite this pessimism, actinidin is
manufactured in New Zealand. A host of other enzymes from kiwifruit have been
studied, mainly in relation to post harvest treatment. None have received the
commercial attention that actinidin has.
Pectin and vitamin C production from kiwifruit have also been con-
sidered, but the pectin was not sufficiently distinct from other fruit pectin to
warrant development. Animal feed and landfill must surely be the methods of
last resort for disposing of kiwifruit wastes. Minimally processed food provides
a new paradigm for kiwifruit processing and this offers a new avenue for fruit
producers.
One use not considered to date but which may be worth investigating is the use
of kiwifruit pomace as a mushroom compost ingredient. Trials on apple pomace
have proved successful (Kennedy 1994). Minor products such as aroma products
(Maarse and Visscher 1989), enzyme inhibitors and cell wall components are of
research interest currently but may expand in the future.
Two other kiwifruit vine products deserve attention although these are not
related to the fruit. These are kiwifruit mucilage and kiwifruit pollen. Kiwifruit
mucilage is the polysaccharide exudate produced from pruning wounds. Its use as
a lubricant has been touted. Kiwifruit pollen also offers potential as a novel natural
health product related to its free radical scavenging and other activities. Commer-
cial development is still awaited.
3 The Composition of Kiwifruit
The composition data on kiwifruit products has largely been conducted on whole
fruit and most of this data reported on a wet weight basis. Very little data has been
reported on kiwifruit pomace. A summary of the data from the literature can be
seen in Tables 2-3, and Fig. 4.
4 Analytical Techniques
This review does not concentrate on all analyses which are routine when dealing
with fruit material (see Kennedy et al., this Vol., for a discussion of routine meth-
ods), but rather on techniques which are crucial to the use of reject kiwifruit or
kiwifruit pomace, those techniques in which conducting analyses on reject kiwi-
fruit or kiwifruit pomace requires some modification of standard methods, or else
techniques that have only recently been applied to kiwifruit.
Table 2. A summary of the literature data on kiwifruit and kiwifruit product composition
Energy content Material Value Reference
Fraction of Kiwifruit 25 Heatherbell et al.

kiwifruit crop (1980)
available for Kiwifruit 30 McArthur (1980)
processing Kiwifruit 25 Park and Luh (1985)
(% of kiwifruit
grown)
Fraction of Kiwifruit 16-23 Heatherbell et al.

kiwifruit (1980)
weight fed to Kiwifruit 15-25 Withy and Lodge
press ending (1982)
up as pomace Kiwifruit 15-20 Martin-Cabrejas et
(% of wet fruit al. (1995)
weight)
Press aid Cellulose 2 Heatherbell et al.

concentration (1980)
(% of wet
weight
processed)
Dry matter (% of Kiwifruit 17.0 Dawes (1972)

wet weight) Kiwifruit 18.8 Beutel et al. (1976)
Kiwifruit 19.8-18.1 Wehmeyer and Von
Staden (1984)
Kiwifruit 15-22 Ferguson (1984)
Kiwifruit 16.8,17.7 Wills et al. (1986)
Kiwifruit l3.0-18.5 Spraggon (1988)
Kiwifruit 16.2-20.3 MacRae et al.
(1989a,b)
Kiwifruit l3.5-20.4 Lintas et al. (1991)
Kiwifruit 17.18-19.24 Fourie and
Hansmann (1992)
Kiwifruit 16.6-17.4 Smith et al. (1994)
Kiwifruit l3.5-21.0 Scott et al. (1986)
Kiwifruit edible portion 15-17 Visser et al. (1990)
Peeled kiwifruit 12.2-15.2 Cotter et al. (1991)
Total soluble Kiwifruit 8.53 Matsumoto et al.

solids (DB) (1983)
Kiwifruit 14.64-17.32 Fourie and
Hansmann (1992)
Kiwifruit juice 12.8-13.9 Heatherbell et al.
(1980)
Stored kiwifruit 14.8-16.2 Testoni et al. (1990)
Soluble solids Kiwifruit 14.16 Dawes (1972)

concentration Hopkirk et al. (1986)
(% wet weight) Kiwifruit 5.8-8.8 MacRae et al.
(1989a,b)
Kiwifruit 4.4-8.9 MacRae and
Redgwell (1992)
Kiwifruit 3.9-9.2 Smith et al. (1994)
Kiwifruit 12.8-l3.1 Gorini et al. (1990)
Table 2. Continued
Kiwifruit at harvest 8.22-lO.50 Cotter et al. (1991)

Kiwifruit at harvest 4.9-9.2 Snelgar et al. (1993)
Kiwifruit at harvest 6.2 Gorini et al. (1990)
Kiwifruit after storage 13.05-15.15 Cotter et al. (1991)
Kiwifruit after 16 weeks lO.0-l3.1 Heatherbell et al.
storage (1980)
Kiwifruit at maturity 12-15
pH Kiwifruit 3.38 Dawes (1972)

Kiwifruit 3.26 Matsumoto et al.
(1983)
Kiwifruit 3.3-3.8 Ferguson (1984)
Kiwifruit 3.36,3.30 MacRae et al.
(1989a,b)
Kiwifruit juice 3.24-3.38 Heatherbell et al.
(1980)
Kiwifruit juice 3.4 Lodge et al. (1986)
Kiwifruit juice 3.53 Palmieri et al.
(1990)
Kiwifruit juice 4.35 Pes is et al. (1991)
Kiwifruit juice 3.2-3.5 Perera and Lodge
(1992)
Kiwifruit puree 3.31 Castaldo et al.
(1992)
Kiwifruit pulp 3.36 Dalla Rosa et al.
(1990)
Protein (% of Kiwifruit 7.1,8.4 Wills et al. (1986)

dried material) Kiwifruit 2.5-2.9 Fourie and
Hansmann (1992)
Kiwifruit pomace 5.4 Martin-Cabrejas et
al. (1995)
Kiwifruit puree b 3.6 Castaldo et al.
(1992)
Kiwifruit edible 4.2 Beutel et al. (1976)
portion'
Kiwifruit edible portion 5.1-6.3 Wehmeyer and von
Staden (1984)
Kiwifruit edible 4.4,5.3 Visser et al. (1990)
portion'
Kiwifruit at maturityb 2.9-8.8 Ferguson (1984)
Lipid or fat Kiwifruit 1.2, 1.2 Wills et al. (1986)

(% of dried Kiwifruit 5.0 Walton and de Jong
material) (1990)
Kiwifruit edible portion' 0.4 Beutel et al. (1976)
Kiwifruit edible portion 3.0-3.7 Wehmeyer and Von
Staden (1984)
Kiwifruit edible portion' 5.3, 4.7 Visser et al. (1990)
Kiwifruit at maturitl 1.8-5.3 Ferguson (1984)
Dietary fibre (% Kiwifruit edible portion 11.7-20.4 Wehmeyer and Von

of dried Staden (1984)
material)
Table 2. Continued
Kiwifruit edible portion 3.0-3.7 Lintas et al. (1991)

Ferguson (1984)
Kiwifruit' 13.4-18.1
Kiwifruitb 6.4-17.1
Kiwifruit 22.6, 15.8 Wills et al. (1986)
Kiwifruit' 11.5-19.7 Fourie and
Hansmann (1992)
Kiwifruit pomace 20.1,23.2,25.8 Martfn-Cabrejas
et al. (1995)
Soluble dietary Kiwifruit pomace 7.1,7.1 Martin-Cabrejas

fibre (% of et al. (1995)
dried material) Kiwifruit edible portion 4.1-7.4 Wehmeyer and Von
Staden (1984)
Insoluble dietary Kiwifruit edible portion 7.6-7.4 Wehmeyer and Von

fibre (% of Staden (1984)
dried material) Kiwifruit pomace 13.0-18.7 Martin-Cabrejas et
al. (1995)
Total amino acids Kiwifruit 0.0154-0.0484 MacRae and

(% wet weight) Redgwell (1992)
Kiwifruit puree b 0.14-0.92 Castaldo et al.
(1992)
Pectin (% of Kiwifruit' 4.4 Dawes (1972)

dried material) Kiwifruit' 2.3-3.4 Lintas et al. (1991)
Kiwifruit puree' 3.5 Castaldo et al.
(1992)
Kiwifruit at maturitl 1.8-6.5 Ferguson (1984)
Tannin (% of Kiwifruit' 0.29 Dawes (1972)

dried material)
Lignin (% of Kiwifruit pomace 3.2 Martin-Cabrejas et

dried material) al. (1995)
Cellulose (% of Kiwifruit pomace 6.8 Martin-Cabrejas et

dried material) al. (1995)
Citric acid (% of Kiwifruitb 5.8,5.9 Heatherbell (1975)

dried material Kiwifruit 5.1,6.9 Wills et al. (1986)
Kiwifruit 5.9-12.4 Lintas et al. (1991)
Kiwifruit' 2.8-9.1 McMath et al.
(1991), Paterson
et al. (1991)
Kiwifruit 2.5-4.5 Walton and de Jong
(1990)
Kiwifruit at harvest 5.2,5.1 MacRae et al.
(1989a,b)
Kiwifruit maximum 7 Okuse and Ryugo
during growth season (1981)
Table 2. Continued
Glucuronic and Kiwifruitb 0.47,0.57 Heatherbell (1975)

galacturonic
acid (% of
dried material)
Malic acid Kiwifruitb 3.1-2.8 Heatherbell (1975)

(% dried Kiwifruit 0.77,0.79 Wills et al. (1986)
material) Kiwifruit 1.5-2.7 Lintas et al. (1991)
Kiwifruit" 0.5-1.6 McMath et al.
(1991), Paterson
et al. (1991)
Kiwifruit 1.0-1.5 Walton and de long
(1990)
Kiwifruit at harvestb 0.7-0.6 MacRae et al.
(1989a,b)
Kiwifruitb 5.4-5.8 Heatherbell (1975)
QuinicAcid Kiwifruit" 2.2-6.4 McMath et al.

(% dried (1991), Paterson
material) et al. (1991)
Kiwifruit 3-4 Walton and de long
(1992)
Kiwifruit at harvestb 3.8,3.8 MacRae et al.
(1989a,b)
Kiwifruit at harvest 2 Given (1993)
Kiwifruit range during 3-30 Okuse and Ryugo
seasonal development (1981)
Total acid (% of Kiwifruit 11.1-19.3 Lintas et al. (1991)

dried material) Kiwifruitb 15.4, 15.5 Heatherbell (1975)
Uronic acids (% Kiwifruit pomace 6.9 Martin-Cabrejas et

of dried al. (1995)
material)
Sugar Kiwifruit 6.2 Clark and Smith

concentration (1988)
(Brix) Kiwifruit 6.5 Dawes et al. (1991)
Total soluble Kiwifruit 55.3 Dawes (1972)

sugars (% of Kiwifruit 53-85 Lintas et al. (1991)
dried material) Kiwifruit edible portion 46.4-57.0 Wehmeyer and Von
Staden (1984)
Kiwifruit 6.9 Walton and de long
(1990)
Kiwifruit pomace 57.7 Martin-Cabrejas
et al. (1995)
Kiwifruit edible 40.7,47.1 Visser et al. (1990)
portion"
Kiwifruit at maturityb 35.3, -70.6 Ferguson (1984)
Table 2. Continued
Reducing sugars Kiwifruit 47.6 Dawes (1972)

(% dried Kiwifruit edible portion' 47.1,40.7 Visser et al. (1990)
material) Kiwifruit at maturity" 35.3,70.6 Ferguson (1984)
Starch (% of Kiwifruit' 27.2 Matsumoto et al.

dried material) (1983)
Kiwifruit 0 Wills et al. (1986)
Kiwifruitb 29.1,29.8,30.5 Bowen et al. (1988)
Kiwifruit 40 Walton and de Jong
(1990)
Kiwifruit' 0-31.7 McMath et al. (1991)
Kiwifruit edible portion 2.7,3.5 Visser et al. (1990)
cone. during growth (1981)
season
Kiwifruit at harvestb 39.1,31.2 MacRae et al.
(1989a,b)
Kiwifruit at harvest 40 Given (1993)
Kiwifruit after 20 weeks 2 Given (1993)
storage
Amylose (% of Kiwifruit' 13.5 Matsumoto et al.

dry material) (1983)
Sucrose (% of Kiwifruit' 2.7 Matsumoto et al.

Staden (1984)
Kiwifruit' 6.6-31.5 Lintas et al. (1991)
(1991), Paterson
et al. (1991)
Hansmann (1992)
Kiwifruit 0.8-1.6 Walton and de Jong
(1990)
Kiwifruit at harvestb 1.4, 3.5 MacRae et al.
(1989a,b)
Fructose (% of Kiwifruit' 16.3 Matsumoto et al.

dry weight) (1983)
Staden (1984)
(1991), Paterson
et al. (1991)
Hansmann (1992)
l32 M. Kennedy et al.
Table 2. Continued
Kiwifruit 3-5 Walton and de Jong

(1990)
Kiwifruit at harvest 3.3 MacRae et al.
(1989a,b)
during growth (1981)
Glucose (% of Kiwifruit' 15.7 Matsumoto et al.

Staden (1984)
Kiwifruita 7.6-28.2 McMath et al.
(1991), Paterson
et al. (1991)
Hansmann (1992)
Kiwifruit 1.5-3 Walton and de Jong
(1990)
Kiwifruit at harvest' 2.3 MacRae et al.
(1989a,b)
Inositol (% of dry Kiwifruit' 0.4-0.8 Walton and de Jong

material) (1990)
(1991), Paterson
et al. (1991)
Thiamin vitamin Kiwifruit' 106 x 10-6 Beutel et al. (1976)

Bl (% dry Kiwifruitb 58.8 x 10-6 -117.6 X 10-6 Ferguson (1984)
material) Kiwifruit 59.5 x 10-6 , 56.5 X 10-6 Wills et al. (1986)
Kiwifruit edible portion a 82.4 x 10-6 Visser et al. (1990)
Riboflavin Kiwifruit edible portion 265 x 10-6 Beutel et al. (1976)

vitamin B2 (% Kiwifruit edible portion 178.5 x 10-6 , 28.2 X 10-6 Wills et al. (1986)
of dried Kiwifruit edible portion a 64.7 x 10-6,66.7 X 10-6 Visser et al. (1990)
material)
Pyridoxine Kiwifruit edible portion a 0.89 x 10-3, 1.05 X 10-3 Visser et al. (1990)
vitamin B6 (%
of dried
material)
Vitamin C (% of Kiwifruit' 0.50 Dawes (1972)

dry weight) Kiwifruit 1.26 Matsumoto et al.
(1983)
Kiwifruit' 0.18 Ferguson (1990)
Kiwifruit' 0.41-1.4 Lintas et al. (1991)
Hansmann (1992)
Table 2. Continued
Kiwifruit edible portion 0.56 Beutel et al. (1976)

Kiwifruit edible portion 0.41,0,42 Wills et al. (1986)
Kiwifruit edible portion' 0,47-0.80 Visser et al. (1990)
Kiwifruit at maturityb 0,47-1.76 Ferguson (1984)
Vitamin E (% of Kiwifruit edible portion' 0.01 Visser et al. (1990)

dried material)
Niacin - vitamin Kiwifruit edible portion 2.7 x 10-' Beutel et al. (1976)
(% of dried Kiwifruit edible portion 3.0 x 10-', 1.7 X 10-' Wills et al. (1986)
material)
a-Carotene (% of Kiwifruit edible portion 119 x 10--6, 113 X 10--6 Wills et al. (1986)
dried material)
~-Carotene (% of Kiwifruit edible portion 298 x 10--6, 395 X 10-6 Wills et al. (1986)
dried material)
Chlorophyll (% Kiwifruitb 0.015-0.017 Ferguson (1984)

of dry Kiwifruit 0.004-0.006 Robertson (1985)
material) Kiwifruitb 0.023 Lawes (1989)
Kiwifruit 0.014 Steele and Johnson
(1995)
Pheophytin (% of Kiwifruit 0.997, 0.002 Robertson (1985)

dried material) Kiwifruit 0.004 Steele and Johnson
(1995)
Ash (% of dry Kiwifruit 3.9 Dawes (1972)

weight) Kiwifruitb 4.1-5.9 Ferguson (1984)
Kiwifruit 5 Walton and de Jong
(1990)
Kiwifruit' 5.1-5,4 Fourie and
Hansmann (1992)
Kiwifruit edible portion 2.3 Beutel et al. (1976)
Kiwifruit edible portion 3,4-4.7 Wehmeyer and Von
Staden (1984)
Kiwifruit edible portion 3.6,4.0 Wills et al. (1986)
Kiwifruit edible portion' 3.6,4.7 Visser et al. (1990)
Kiwifruit pomace 6.2 Martin-Cabrejas et
al. (1995)
Kiwifruit puree 3.3 Castaldo et al.
(1992)
Calcium (% of Kiwifruit 0.17 Ferguson and

dry weight) Eiseman (1983)
Kiwifruit 0.19-0.20 Testoni et al. (1990)
Kiwifruit 0.205-0.257 Hopkirk et al.
(1990)
Kiwifruit 0.14-0.30 Prasad and Spiers
(1991)
Hansmann (1992)
l34 M. Kennedy et al.
Table 2. Continued
Kiwifruit 0.16 Smith et al. (1994)

Kiwifruit flesh 0.21 Clark and Smith
(1988)
Staden (1984)
Kiwifruit edible portion 0.143,0.l36 Wills et al. (1986)
Kiwifruit edible portion 0.20,0.15 Visser et al. (1990)
Kiwifruit at maturityb 0.147 -0.352 Ferguson (1984)
Kieldahl nitrogen Kiwifruit 1.12 Dawes (1972)

(% of dry Kiwifruit 0.89 Ferguson and
weight) Eiseman (1983)
Kiwifruit 0.87 Clark and Smith
(1988)
Kiwifruit 1.1 Walton and de Jong
(1990)
Kiwifruit 0.89,0.88 Smith et al. (1994)
Kiwifruit edible portion' 0.86,0.71 Visser et al. (1990)
Potassium (% of Kiwifruit 1.78 Ferguson and

dry weight) Eiseman (1983)
Hansmann (1992)
Kiwifruit 1.96, 1.84 Smith et al. (1994)
(1988)
Staden (1984)
Kiwifruit edible portion 1.55, 1.69 Wills et al. (1986)
Kiwifruit at maturity' 1.35-2.24 Ferguson (1984)
Phosphorus (% Kiwifruit 0.l3 Ferguson and

of dried Eiseman (1983)
material) Kiwifruit 0.12-0.14 Testoni et al. (1990)
Hansmann (1992)
(1988)
Staden (1984)
Kiwifruit edible portion' 0.l3,0.16 Visser et al. (1990)
Sulphur (% of Kiwifruit 0.12,0.11 Smith et al. (1994)

dried material) Kiwifruit flesh 0.09 Clark and Smith
(1988)
Table 2. Continued
Kiwifruit edible portion" 0.09,0.12 Visser et al. (1990)

Kiwifruit at maturity 0.14 Ferguson (1984)
Chlorine (% of Kiwifruit 0.24,0.22 Smith et al. (1994)

dried material) Kiwifruit edible portion" 0.23,0.28 Visser et aI. (1990)
Kiwifruit at maturitY' 0.15-0.29 Ferguson (1984)
Sodium (% of Kiwifruit 0.013-0.027 Testoni et al. (1990)

dried weight) Kiwifruit" 0.01l-0.014 Fourie and
Hansmann (1992)
Kiwifruit 0.03,0.02 Smith et al. (1994)
(1988)
Kiwifruit edible portion 7.11 X 10~3 -8.65 X 1O~3 Wehmeyer and Von
Staden (1984)
Kiwifruit edible portion 0.03,0.03 Wills et aI. (1986)
Kiwifruit edible 0.021, 0.016 Visser et al. (1990)
portion"
Magnesium (% of Kiwifruit" 0.160 Beutel et aI. (1976)

dried weight) Kiwifruit 0.08 Ferguson and
Eiseman (1983)
Kiwifruit" 0.171-0.224 Fourie and
Hansmann (1992)
(1988)
Staden (1984)
Kiwifruit edible portion" 0.083, 0.1l2 Wills et al. (1986)
Kiwifruit edible portion" 0.087, 0.059 Visser et aI. (1990)
Kiwifruit maturityb 0.082-0.159 Ferguson (1984)
Manganese (% of Kiwifruit" 0.5 x 1O~3 -0.7 X 1O~3 Fourie and

dry weight) Hansmann (1992)
Kiwifruit 0.65 x 1O~3, 0.70 X 1O~3 Smith et aI. (1994)
Kiwifruit flesh 0.8 x 1O~3 Clark and Smith
(1988)
Kiwifruit edible portion 004 x 1O~3, 0.7 X 1O~3 Visser et al. (1990)
Kiwifruit edible portion 0.29 x 1O~3 -0048 X 1O~3 Wehmeyer and Von
Staden (1984)
Boron (% of dry Kiwifruit 1.3 x 1O~3 -1.6 X 1O~3 Testoni et al. (1990)
weight) Kiwifruit 1.58 x 1O~3, 1.62 X 1O~3 Smith et aI. (1994)
Kiwifruit flesh 1.6 x 1O~3 Clark and Smith
(1988)
Iron (% of dry Kiwifruit 2.03 x 1O~3 Testoni et al. (1990)

weight) Kiwifruit" 1.4 x 1O~3 - 2.0 X 1O~3 Fourie and
Hansmann (1992)
Table 2. Continued
Kiwifruit 4.3 X 10-3 Smith et al. (1994)

Kiwifruit flesh 2.8 X 10-3 Clark and Smith
(1988)
Kiwifruit edible portion" 2.7 x 10-3 Beutel et al. (1976)
Kiwifruit edible portion l.67 X 10-3 - 2.63 X 10-3 Wehmeyer and Von
Staden (1984)
Kiwifruit edible portion" 2.4 X 10-3,2.8 X 10-3 Wills et al. (1986)
Kiwifruit edible portion" 1.4 x 10-3, l.7 X 10-3 Visser et al. (1990)
Zinc (% of dry Kiwifruit" 0.9 x 10-3 _1.1 x 10-3 Fourie and

weight) Hansmann (1992)
Kiwifruit 0.55 x 10-3,0.54 X 10-3 Smith et al. (1994)
Kiwifruit flesh 0.8 x 10-3 Clark and Smith
(1988)
Kiwifruit edible portion 0.76 x 10-3 -0.91 x 10-3 Wehmeyer and Von
Staden (1984)
Kiwifruit edible portion l.2 X 10-3, l.1 X 10-3 Wills et al. (1986)
Kiwifruit edible portion" 0.5 x 10-3,0.7 X 10-3 Visser et al. (1990)
Copper (% of dry Kiwifruit" l.7 x 10-3 -2.5 X 10-3 Fourie and

weight) Hansmann (1992)
Kiwifruit 0.6 x 10-3, 0.8 X 10-3 Smith et al. (1994)
Kiwifruit flesh 0.9 x 10-3 Clark and Smith
(1988)
Kiwifruit edible portion 0.81 x 10-3 -1.12 x 10-3 Wehmeyer and Von
Staden (1984)
Kiwifruit edible portion" 0.3 x 10-3, 0.5 X 10-3 Visser et al. (1990)
"Calculated back to 0% moisture from wet basis data using a given dry matter data.
bCalculated back to 0% moisture from wet basis data assuming 17% dry matter in kiwifruit.
Table 3. Summary of the elemental analysis of kiwifruit data from

the average of data in Table 2
Element Dried material (%)
Potassium l.70
Nitrogen 0.97
Chlorine 0.24
Calcium 0.19
Phosphorus 0.16
Sulphur 0.11
Magnesium 0.11
Sodium 0.03
Iron 2.32 x 10-3
Boron l.54 X 10-3
Copper l.03 X 10-3
Zinc 0.82 X 10-3
Manganese 0.58 X 10-3
Others e.g. Carbon, Oxygen, Hydrogen 96.5
Total 100
Kiwifruit water 82.9%
1
Solid (dry ratter) 17.1%
-- I
Ash Phenols Chlorophyll Simple Complex Acids Vitamins Lipid Protein
4.3% 0.013% Carbohydrate Carbohydrates 3.2% 5.2%
I
Tannin
0.29%
Starch Amylose Pectin Lignin Cellulose Bl B6 B2 C Niacin E

23.7% 13.5% 3.7% 3.2% 6.8% 80.1 x 10 6 % 0.97 x 10'% 121 x 10'% 0.69% 2.5 x 10)% 0.01%
Sucrose Frudose Glucose Quinic Malic Glucuronic Citric

6.2% 18.0% 16.9% 3.4% 2.2% 0.52% 6.0%
Fig. 4. Summary of kiwifruit composition data (all data dry weight basis except water and solid matter which are on a wet weight basis), compiled from data in Table
2. The total comes to 117.8%! The most likely source of error is starch, sucrose, fructose, and glucose contents changing during ripening. Another potential source
of error is confusion related to reporting data on "as dried" basis (17% water) or a "bone dry" basis (0% water). The data above is, however, a useful proximate
summary of Kiwifruit composition.
kiwifruit croLP_ _ _ _....:.7..::.3.:..;;o/~ ww direct consumption
27%ww
kiwifruit for 81/" ww kiwifruit juice

juice extraction-------+
19'}'0 ww
wet kiwifruit pomace
Fig. 5. Mass flows of components from the kiwifruit crop based on data from Table 2
4.1 Dry Material
The moisture content or dry material content is one of the most often measured
and most fundamental of variables used when dealing with fruit wastes or rejects.
The traditional method of obtaining it is oven drying overnight at 70C. Other
variations such as partial drying in an air oven prior to employing the official
AOAC (Association of Official Analytical Chemists) method which uses vacuum
oven drying, have also been tried (Scott et al. 1986). These methods are involved
and lengthy, resulting in unwanted delays, especially when used for obtaining data
important for predicting flavour quality and harvest maturity.
This prompted the development of rapid drying techniques such as micro-
wave oven drying (Spraggon 1988; Ragozza and Colelli 1990). Using the microwave
oven technique, the drying time required to obtain dry weight data was reduced to
approximately 30 min, but the method still had several disadvantages. The main
ones being the possibility of over drying or charring samples due to localised
drying effects and the variability of drying time and power settings, depending on
the sample type and size.
An innovation is the use of infrared drying, which overcomes the problems
of long drying times and localised heating effects associated with the other
methods (Kennedy and Phillips 1996). The infrared drier heats the sample
by transmission of electromagnetic radiation which penetrates and heats the
sample from the bottom, giving uniform drying. Sample size and temperature
can be easily varied, and drying progress monitored to obtain rapid drying
times of approximately 30 min, without compromising the accuracy of the
determination.
.
35 , - - - - ------------ - - -- - - -------------------------------- -- .
.... . I
I
Predic ted % dry wI
30
'.. ~

Oven % dry wI
Infrared % dry wI
25
."'E"
..
"0
;!.
20 1.
' .....
":;.,
15
.,'"'" .~ .
E
10 ! .. ......... .. .. ... .. .
.. .. . ... . .. . . .
". " .
5 ... .. . ~
0
-50 0 50 100 150 200 250 300 350 400
% dilution of kiwifruit pomace
Fig. 6. A comparison of the oven and infrared drying techniques fo r estimating the water content
of kiwifruit pomace. (Data from Fenton and Kennedy 1998)
A study by Fenton and Kennedy (1998) comparing the traditional oven drying
technique with the infrared drying technique, using kiwifruit pomace, showed
that the infrared drier could be used to accurately measure the dry weight of
samples with a wide range of moisture contents. Also, there was shown to
be excellent agreement between the two methods for the dry weight data obtained,
indicating that the much quicker infrared method could produce reliable data
(see Fig. 6).
4.2 Enzyme Activity Measurement
Conditions for the extraction of enzymes from kiwifruit can be critical for the
accurate determination of the required enzyme activity. Crude methods involve
homogenisation of fruit pulp 1: 1 with a (pH 6.5) buffered solution, followed by
centrifugation and testing of the supernatant for enzyme activity (Pn!stamo 1995;
Ogawa et al. 1990a). However, the most common kiwifruit enzyme extraction
method uses polyvinylpolypyrrolidone (PVPP; McLeod and Poole 1994; Boyes et
al. 1992) and sodium tetrathionate (Park and Luh 1985; Boland and Hardman
1972) in the extraction buffer in order to complex phenolic compounds which can
inhibit some enzyme activities. Samples of tissue, frozen with liquid nitrogen, are
crushed with a mortar and pestle. The homogenised material is extracted 1: 2 with
ice cold 0.1M phosphate buffer, pH 6.0, containing 1-2% (w/w kiwifruit sample)
PVPP, 10 mM sodium tetrathionate and 1 mM EDT A. The extraction mixture is
stirred for 30 min and then centrifuged at 20000 g for 20 min. The supernatant is
then tested for enzyme activity.
The list of kiwifruit enzymes and the methods used to determine their activity,
cited below, is not exhaustive but it does reflect either the presence of enzymes
unique to kiwifruit, or enzymes commonly monitored as markers of fruit develop-
ment and ripening.
4.2.1 Protease
Actinidin thiol protease (EC 3.4.22.14) is by far the most abundant enzyme
in kiwifruit, making up 60% of the total soluble protein in the fruit pulp
(Praekelt et al. 1988). There are a number of ways to determine the actinidin
activity.
Digestion of a Protein Substrate. A 2% solution of casein (Tello-Solis et al. 1995) or

azo-casein (Paul et al. 1995; Sarath et al. 1994) buffered with 50mM phosphate, pH
6.0, is prepared. A sample of extract is incubated with the substrate in the ratio
0.75: 1 at a fixed temperature, usually either 25 or 40C, for 10 min. The reaction is
stopped by acid precipitation with a 2x reaction volume of 5% trichloroacetic acid
(TCA) or 12% perchloric acid. The precipitate is removed by filtration (Whatman
No. 40) or centrifugation (10000 g, 5 min) and the absorbance of the supernatant is
read at 280 or 340 nm for casein or azocasein substrates respectively. The activity
of 1 unit of protease is defined as the amount of enzyme that produces a 1.0 unit
change in absorbance per min.
Hydrolysis of a Synthetic Substrate. Actinidin has been characterised by the

hydrolysis of N-CBZ-Iysine p-nitrophenyl ester (Boland and Hardman 1973).
This reaction is monitored colorimetrically (A348 ) by the release of p-nitrophenol.
The substrate is prepared as a 12 mM stock solution in acetonitrile: water
(19: 1 v/v) and added (50f.l1) to 2.98ml 70mM phosphate buffer, pH 6.0
(containing 0.2mM EDTA and 1.6% acetonitrile). Spontaneous hydrolysis of
the substrate is monitored for 20 s (A 348 ). Enzyme extract (50 f.ll) is added
and the change in A348 (dA/dt) recorded for 260s. The non-enzymatic substrate
hydrolysis is subtracted from the enzymatic dA/dt. One unit of activity is
the amount of enzyme which produces 1 f.lmol of p-nitrophenol per min
at pH 6.0, 25C where the extinction coefficient of p-nitrophenol (348) is
5400M- 1 cm- l
Active Site Titration. The total number of actinidin active sites can be deter-
mined spectrophotometrically using 2,2' -dipyridyl disulphide (Salih et al.
1987) which reacts stoichiometrically with the actinidin active site to pro-
duce an absorbance change at 343 nm. The number of active species can be
calculated from the extinction coefficient of the resulting pyridine 2-thione
(343 = 8080M- 1 cm- l ).
4.2.2 Carbohydrate Modifying Enzymes
Glycosidase. The predominant glycosidase activities in Kiwifruit are a and ~

galacto- and mannosidases (Ogawa et al. 1990a,b; Ross et al. 1993). Glycosidase
activity is measured by the hydrolysis of the appropriate p-nitrophenyl glycoside.
Enzyme extract (10111) is added to 0.25 ml, 2 mM substrate in 50 mM citrate buffer,
pH 4.5, and reacted at 37C for between 5 and 30 min. The reaction is stopped with
1.75ml 0.2M borate, pH 9.8, and nitro phenol release determined at 400nm. One
unit of enzyme is the amount of enzyme required to release 11lmol nitro phenol!
min where the extinction coefficient of nitro phenol 400 is 17700 M- 1cm-I.
Lysozyme. Lysozyme from Kiwifruit has been characterised (Lynn 1989).

Lysozyme activity is determined by monitoring the turbidity change (L'1440 nm) of
a 0.01 % w/v suspension of dried Micrococcus lysodeikticus cells in 0.1 M acetate,
pH 4.6.
Xyloglucan Endotransglycosylase. Xyloglucan endotransglycosylase (XET) is in-

volved in the ripening and softening of kiwifruit (Redgwell and Fry 1993; Percy et
al. 1996). Its activity measurement involves the transfer ofaxyloglucan donor to
a tritium labelled heptasaccharide. Enzyme extract (20 Ill) is incubated for 1 h
at 25C with 20 III substrate (0.25% xyloglucan, 0.05 M acetate, pH 5.8, and
283kBqmr i eH] heptasaccharide). The reaction is stopped with 5011140% formic
acid. The unreacted [3Hl heptasaccaride is removed and the enzyme activity deter-
mined by [3Hl scintillation counting. One unit of activity is defined as 1 Bq of 3H
xyloglucan polymer formed kBq-1 h- I
4.2.3 Oxido-Reductases
Peroxidase. A number of substrates can be used to determine peroxidase activity.

The peroxidase in kiwifruit has been characterised (Soda et al. 1991; McLeod and
Poole 1994) by measuring the A470 increase after the addition of 0.1 ml kiwifruit
extract to 2.9 ml of 5 mM guaiacol and 1.5 mM hydrogen peroxide in 0.1 M potas-
sium phosphate, pH 6.5. One unit of peroxidase activity is defined as the amount
of enzyme required to give a 1.0 OD 470 change per min.
Lipoxygenase. Lipoxygenase contributes to the "grassy" aroma associated with

kiwifruit juice (Boyes et al. 1992). Lipoxygenase activity is determined spectropho-
tometrically at 234nm. Linoleic acid is prepared as a 6.6mM stock solution (0.1 ml
linoleic acid, 0.15ml Tween 20, 10ml 0.1 M NaOH in 50ml total volume). The
linoleic acid substrate (0.3 ml) is added to 2.6 ml 0.2 M phosphate, pH 6.0. The
increase in A234 is monitored after the addition of 0.1 ml enzyme sample.
PolyphenolOxidase. Polyphenol oxidases (PPO) cause enzymatic browning in all

fruits and vegetables. The PPO level can be determined using a variety of phenyl
substrates, however, Park and Luh (1985) used the optical density change of cat-
echol at 420nm to measure kiwifruit PPO activity. Enzyme extract (0.2ml) was
added to 2.8 ml 0.01 M catechol in 0.1 M citrate-0.2 M phosphate, pH 7.3 at 30 C.
One unit of PPO activity causes a 0.001 OD 42o increase per minute.
4.3 Polyphenol Components
The polyphenol fraction is predominantly concentrated in the skin of kiwifruit.

While collectively the polyphenols amount to only a small fraction of each pomace,
they nevertheless are of interest as a potential source of nutriceuticals because they
possess free radical scavenging and antioxidant activities. They consist of different
classes of compounds, and in kiwifruit pomace the major polyphenols consist of
chlorogenic acid, epicatechin and their oligomers, quercetin glycosides and p-
coumaric acid (Lu and Foo, personal communication, 1997). For analysis, these
compounds are best extracted with an aqueous organic solvent mixture, the most
common being 70% aqueous acetone. Good resolution of these compounds can be
obtained by HPLC using a LiChroCART 125-4, Lichrospher 100 RV -18(5 /-lm)
column set at 30 C. Solvents are (A) acetic acid/water (2/98) and (B) acetic acid/
acetonitrile/water (2/20/78) with solvent programming starting with 80% A and
20% B to give 40% A and 60% B at 40 min and 100% B at 60 min with flow rate of
0.5 mllmin. Detection is by UV absorption at 280 nm for all phenols and 350 nm for
flavonoids. The HPLC chromatogram of a 70% aqueous acetone extract of kiwi-
fruit pomace is reproduced in Fig. 7 which shows good resolution of the flavans
and their oligomers as well as the various quercetin glycosides.
Where the identity of individual compounds is not required, the total
polyphenol content can be determined by a variety of assays. The preferred assay
DADl A. 5'0=280.8 Ret::;590.10

mAU
kiwifruit pomace. HP20 purified
3Q
25
tl 5
20 II
15
I I " I
'~I~~~vJ vJ~
10
10 20 30 40 50
Fig. 7. HPLC chromatogram of HP20 purified kiwifruit pomace extract

today is the Folin-Ciocalteu method which is based on the reduction of a

phosphotungistic-phosphomolybolic reagent in a slightly alkaline medium. This
colorimetric assay has been evaluated by Scalbert (1992) to be the most ap-
propriate for the determination of total phenol in plant extracts. However, several
substances, including ascorbic acid and excess sugars which are present in the
pomace, cause some interference with the analysis and this can be avoided by
passing the extracts through a column of hydrophobic resin such as XAD-2 or
HP20, washing the resin with water to remove the ascorbic acid and sugars before
eluting the polyphenols with methanol. After removal of the methanol, the residue
is then taken up to volume in acidified methanol (0.3% HCl) and water (3:2v/v).
Extract aliquots of 100 III are dissolved in 2.0 ml of 2% Na2 C0 3 solution and after
2min 100111 of a 50% Folin-Ciocalteu reagent is added and left to stand at room
temperature for 30 min. The absorbance of the solution is measured at 750 nm
against a blank consisting of all the reagents and solvents minus the test sample.
The standards commonly used are catechin or gallic acid and they are prepared at
concentrations from 0.05 to 0.5 mg/ml, and the results are expressed as catechin or
gallic acid equivalent.
4.4 Antioxidant Analysis
To assess the antioxidant activity of the pomace extracts (or purified compounds
derived from them) the relative free radical scavenging activity (compared with
ascorbic acid or a-tocopherol) is measured using 2,2-diphenyl-1-picrylhydrazyl
(DPPH) as the radical species. This is carried out by adding 0.1 ml of a polyphenol
solution (0.1 mg/ml in 95% ethanol) to 2.0ml of a solution DPPH (0.1 mM in 95%
ethanol). The mixture is kept at room temperature for 60 min and at the end of this
period the absorbance is measured at 517 nm. The radical scavenging activity
(RSA) is calculated from the formula:
RSA = (Ao - As)/ Ao x 100,
where Ao is the absorbance of the blank, and As is the absorbance of test sample.
The relative free radical scavenging activity is the ratio obtained by dividing the
RSA of sample by the RSA of ascorbic acid or a-tocopherol.
Another method which is popularly used is the superoxide dismutase
(SOD) method. This assay is based on the generation of O~ by the xanthine
oxidase system which then reduces nitro blue tetrazolium (NBT). Essentially, a
0.1 ml solution of various concentrations (0 to 100 units/ml) of SOD (Sigma,
S-2515) or test samples dissolved in DMSO (0.1 mg/ml) are added to a 1.0ml
mixture consisting of 0.4 mM xanthine and 0.24 mM NBT in 0.1 M phosphate
buffer (pH 8.0). Xanthin oxidase (0.049 unit in 1.0ml of 0.1 M phosphate buffer)
is added to the samples, and the mixtures incubated at 37C for 20 min. After
this period, 2.0 ml of 69 mM sodium dodecylsulfate is added to terminate
the reaction and the absorbance of the solutions is measured at 560 nm. The
SOD activity is calculated from the standard curve as SOD equivalent per mg
of extract.
4.5 NMR Analysis
Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for

characterising pomaces. One of the advantages ofNMR is that it allows us to study
the pomace and other plant material in the solid state, without requiring selective
extraction or other chemical pretreatment. A brief outline of the technique is
provided elsewhere (Kennedy et al., this Vol.).
Applications of NMR are illustrated (Figs. 8 to 10) by spectra obtained on a
Varian Inova-200 spectrometer operating at a carbon-13 NMR frequency of
50.3 MHz with magic angle spinning (MAS) at 4 kHz. The sample of kiwifruit
pomace was freeze-dried, but the moisture was later allowed to equilibrate with
room air. In the case of the cross-polarization (CP) NMR spectrum (Fig. 8), each
5!ls proton preparation pulse was followed by a 2 ms CP contact time, 30 ms of data
acquisition, and a recovery delay of 3 s.
Signals were averaged over a period of 8 h. The pulse sequence was then
modified by gating off the decoupler output during a delay of 42!ls between the
contact time and data acquisition in order to produce an "interrupted decoupling"
(ID) spectrum (Fig. 9). Signal averaging was extended to 24h. In the case of the
"single pulse excitation" (SPE) spectrum (Fig. 10), each 5!ls carbon-13 preparation
pulse was followed by 30 ms of data acquisition and a 2 s delay. Signals were
averaged over 12 h. Each of these three spectra will be discussed in turn.
The CP NMR pulse sequence is the standard sequence for obtaining solid-state
carbon-13 NMR spectra of solid organic matter. The CP NMR spectrum of
the kiwifruit pomace (Fig. 8) is dominated by signals assigned to carbohydrates,
fructose
cellulose C j2
C-l ~ +
200 180 160 140 120 100 80 60 40 20 o

Chemical shi ft (ppm)
Fig.8. Carbon-13 NMR spectrum of kiwifruit pomace, obtained with the CP (cross-polarization)
sequence, showing signals from relatively solid components
fructose
C-2
-COzH
and
-CO( NH)-
1 tann ins
fit / , ~
200 180 160 140 120 100 80 60 40 20 o
Fig. 9. Carbon-13 NMR spectrum of kiwifruit pomace, obtained with the ID (inter-
rupted decoupling) sequence in order to suppress signals from CH and CH, carbon in rigid
environments
-C(H)=C(H)-
n
200 180 160 140 120 100 80 60 40 20 o

Fig. 10. Carbon-13 NMR spectrum of kiwifruit pomace, obtained with the SPE (single pulse
excitation sequence), showing signals from relatively liquid-like components. Labels refer to
carbon atoms in seed oils
including cellulose and fructose. The peak at 105ppm (assigned to C-l) is a par-
ticularly useful indicator for cellulose. The assignment of the peak at 99 ppm to
fructose is discussed below. Minor components of the pomace identified by NMR
include tannin and protein. The condensed tannins can be identified by character-
istic signals at 155 and 145 ppm, assigned to oxygen -substituted aromatic carbon
in the A and B rings, respectively (Newman and Porter 1992). Protein cannot be so
positively identified by characteristic signals, but a band between 18 and 35 ppm is
consistent with side chain carbon in protein. Signals from secondary amide carbon
(173 ppm) and C-2 of each amino acid (typically 55 ppm) coincide with other
signals in pomace, e.g., signals from C-6 of pectin structural units at 170 to
180ppm (Jarvis and Apperley 1990).
The signals at 155 and 145 ppm in Fig. 8 correspond to just five carbon atoms
out of 15 in each structural unit of a condensed tannin (Newman and Porter 1992).
After allowance for the other carbon atoms in the structure of a condensed tannin,
it becomes clear that the tannin content of pomace amounts to a few percent by
weight. The lower value in Table 2 probably refer to water-soluble tannins.
The ID NMR experiment is used to identify carbon-13 nuclei lacking directly-
bonded hydrogen atoms, i.e., quaternary carbon in aliphatic or alicyclic structures,
or substituted carbon in aromatic structures (Opella and Frey 1979). Dipole-dipole
interactions between carbon-13 nuclei and protons suppress signals associated
with rigid CH and CH 2 groups.
The spectrum in Fig. 9 is dominated by a peak at 99 ppm assigned to fructosyl
C-2. Fructose differs from all other common carbohydrates in that it contains
non-protonated carbon, so ID NMR provides a useful method for distinguishing
fructosyl residues from other structural units in polymers or oligo saccharides. A
broader band underlying the fructose C-2 signal is assigned to condensed tannins
(Newman and Porter 1992). This ID NMR band provides a useful test for the
presence of condensed tannins and related degradation products. Lignin can con-
tribute a broad band in the same chemical-shift range, but the band is suppressed
byID NMR.
While CP and ID NMR spectra are used to investigate the most rigid compo-
nents of a sample, the SPE NMR spectrum (Fig. 10) is used to investigate relatively
liquid-like components. Sharp signals are assigned to seed oils. Schaefer and
Stejskal (1975) have shown that carbon-13 NMR provides a useful method for
characterising the major fatty acid components of seeds. In particular, a signal at
132 ppm is characteristic of linolenic acid (Gunstone et al. 1977). This signal ap-
pears on the extreme left of a group labelled -C(H)=C(H)- in Fig. 10. Some of
the carbohydrates appear to be contained in relatively liquid-like domains, con-
tributing signals across the chemical-shift range 60 to 100 ppm. Moistening the
sample (spectra not shown) resulted in enhancement and sharpening of some of
the signals assigned to carbohydrates, indicating selective rehydration of specific
components as the solid material became more liquid-like.
The distribution of pomace components between "solid-like" and "liquid-
like" domains hinders quantitative analysis by NMR. Any attempt at quantitation
based on CP NMR alone would yield estimates of contents as a percentage of
"solid-like" matter rather than as a percentage of total organic matter. Similarly,

SPE NMR alone yields an estimate of seed oil as a percentage of "liquid-like"
matter rather than as a percentage of total organic matter. One way forward might
be comparison of CP NMR signal strengths with those measured for a known
weight of a water-insoluble synthetic polymer mixed with the pomace. Meanwhile
it seems clear that solid-state NMR provides a useful overview of the contents of
kiwifruit pomace, at least in qualitative terms.
5 Discussion
While a large amount of work has been done on characterising kiwifruit, little has
been conducted on finding uses for or analysing kiwifruit pomace. Standard ana-
lytical techniques for analysing kiwifruit, e.g. enzyme assays, are well developed,
and applying more sophisticated analytical techniques, e.g. NMR analysis, will
drive further research and commercial activity in the future.
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Analysis of Tree Leaf Decomposition in Arid Soils
J.e. TARAFDAR
1 Introduction
Arid areas are characterized by low and erratic precipitation, high day tem-
peratures during the summer and low temperatures in winter, high erosive
winds, high evaporative demand by the atmosphere, low organic matter and
low moisture retention capacity of soils. These factors pose considerable difficul-
ties for the survival and growth of microorganisms involved in the decomposition
process. The problem is further accentuated since addition of organic matter to
arid soils is limited due to heavy demands on straw for fodder and cow dung for
fuel. As tree leaves are abundant and leaf fall is very common in desert regions,
there is the possibility of using tree leaves to build up soil organic matter in this
area.
Principal chemical constituents of tree leaves are: (1) cellulose, (2) hemi-
cellulose, (3) lignin, (4) water-soluble sugars, amino acids and aliphatic acids,
(5) ether and alcohol soluble constituents including fats, oils, waxes, resins
or pigments, and (6) proteins. The breakdown of these constituents is effected
as a sequence of specific reactions with the enzyme systems of specific organisms.
The major groups of leaf-decomposing organisms are bacteria, actinomycetes,
fungi, protozoa, nematodes, microarthropods, macro arthropods, enchytracid
worms, and lumbricid worms. The processes of leaf decay are largely controlled
by soil microorganisms and are therefore influenced by temperature, moisture,
pH and soil aeration (Jenkinson 1981). Plant material that is low in lignin and
other polyphenols and high in nitrogen and soluble carbohydrates, generally
decomposes relatively quickly (Tian et al. 1995). Thus the rate of initial break-
down varies between immature and mature tissue as well as between different
species.
The decomposition of tree leaves in arid soils is a complex process.
The substrate is a heterogenous mix of organic matter ranging from recently
introduced leaf material to humic material, and this conversion may often take
thousands of years (Jenkinson 1988). The soil microbial biomass, the agent
of decomposition, is also a varied mix of organisms having different popula-
tions where normally actinomycetes predominate. Despite this complexity,
considerable advances have been made in understanding the decomposition
process. Studies on CO 2 evolution during plant residue decomposition show
an initial rapid phase, in which about 70% of the C initially present in the

154 J.C. Tarafdar
residue is lost as CO 2 , followed by a slower phase (Jenkinson 1977; Sauerbeck

and Gonzalez 1977). The first phase is known as the labile (or decompos-
able) fraction and the second phase is known as the recalcitrant (or resistant)
fraction of the plant residue. A consideration of the chemical composition of
plant material suggests that residue decomposition is likely to exhibit two
phases characterised by different kinetics. The initial rapid phase corresponds
to the degradation of water-soluble free amino acids, amino sugars and car-
bohydrates, and cell cytoplasmic and membrane constituents. The slower
second phase corresponds to the decomposition of cell walls and structural
components, and the decomposition of secondary, more stable products of the
first phase (Sorensen 1975). In this chapter, the important tree leaves in arid
soils, their method of decomposition and analysis of decomposed materials is
discussed.
2 Important Trees in Arid Soils
Table 1 lists important trees in arid soils, along with their uses and potential.
3 Methodological Approaches for the Decomposition Process
Diverse efforts have been made over the years to quantify the decomposition
process. Among the approaches, the perfusion technique, the litterbag technique,
the tracer technique and the soil respiration technique have been widely adopted
in contemporary studies.
3.1 The Perfusion Method
Nyamai (1992) has developed a perfusion apparatus for study of organic matter
breakdown. The main components are a sample compartment, solution sampling
device, reservoir for collecting and mixing the perfusion solution before recircula-
tion, an air flow lift system for recirculation of the CaC12 solution, and a CO 2 trap,
all constructed out of glass with plastic tube connectors.
Lefroy et aI. (1995) developed an in vitro perfusion method to estimate break-
down rates ofleaflitter and crop residues. A simple, cheap and effective apparatus
was developed, utilizing a hospital drip bag and administration set. Results ob-
tained through this apparatus were comparable with those from a more elaborate
glass apparatus (Nyamai 1992). The technique allows a screening of a wide range
of leaf litters under a standarized set of conditions, without the complication of
differences in soil biota.
Analysis of Tree Leaf Decomposition in Arid Soils 155
Table 1. Important trees in arid soils
Botanical name Uses/Potential
Acacia nilotica Source of water-soluble gum, used for timber, tanning material,
fodder.
Acacia senegal Principal commercial source of gum; widely used in the food,
pharmaceutical, confectionary and paper industries. Seeds
relished as a vegetable in India; leaves are used as fodder.
Ailanthus excelsa Leaves are used as fodder.
Albizia lebbeck A potential firewood source; leaves are used as fodder.
Azadirachta indica Fruits yield an oil used in manufacture of soap. Bark, leaves and
fruit used in traditional Indian medicines and as insecticides.
Extracts of bark used in toothpaste.
Calotropis pro cera Seeds contain a semi-drying oil. Source of latex used for tanning
and dyeing. Leaves used to produce merissa, a native beer in
West Africa. Stems are used for making huts.
Colophospermum mopane Important firewood source.
Eucalyptus camaldulensis Used for timber, fodder etc.
Euphorbia tirucalli Latex, roots and branches used in indigenous medicine. Latex can
be used as rubber.
Ficus religiosa Used as fodder, firewood.
Leucaena leucocephala Utilised mainly for forage; various parts of plant used in
indigenous medicine, and as a source of dye. Leaves provide a
good green manure.
Maringa oleifera Lubricant, used in the preparation of cosmetics and perfumes. All
parts of the tree used in Indian indigenous medicine. Wood is
suitable for making paper pulp.
Osyris spp. Leaves used as source of sandie wood oil. Some species used as
timber. Seed oil may have industrial potential.
Pithecellobium dulce Used as fodder.
Prosopis cineraria Pods used in various foods. Gum, which exudes from cut stem, is
eaten and marketed. Wood gives high quality charcoal. Can be
used as furniture. The leaves are lopped for use as fodder and
are also composted.
Prosopis juliflora Its bark exudes a gum which is edible and is used in the
manufacture of mucilages. The bark is also used for tanning .
. The flowers yield nectar and honey. Leaf extracts used
medicinally locally. Wood is used as charcoal.
Salvadora pessica Used to make shelter belts. Shoots eaten as salad. The root
contains alkaloids. Fruits and the root bark are locally used
medicinally.
Simmondsia chinensis Oil from seeds can be used medicinally.
Tecomella undulata Used for making furniture.
Zizyphus nummularia Used as fodder, fuel wood etc.
3.1.1 Perfusion Apparatus of Lefroy et al. (1995) and Its Components
The perfusion apparatus used is shown in Fig. 1. The main components of the
apparatus are a sample compartment, a perfusion solution bag, a reservoir for
collecting and mixing the perfusion solution before recirculation, a solution sam-
pling tap, and a CO 2 -free air supply to carry CO 2 evolved during decomposition to
156 J.C. Tarafdar
Fig. 1. The UNE perfusion apparatus. A CaCl, Solution bag; B solu-

tion administration set; C sample compartment; D reservoir; E CO,
trap; F CO 2 Scrubber; G CaCl, sampling tap; H recycling tube
A
-~ =
,
~
C
G 0
.E.
'
:
I
~ B
J F 1
<QJ ~ I
= C2 '"
ii C3 OE
." C4
.", C5
H CJ C6
OC7
~C O o
Fig. 2. Components of the UNE perfusion apparatus
the CO 2 trap. Each part of the apparatus is shown in Figs. I and 2 and details
described below:
CaCl2 Solution Bag (Fig. IA). A 500-ml viaflex container with female luer adaptor,
manufactured by Baxter Healthcare Pty Ltd., UK.
Solution Administration Set (Fig. IB). A solution administration set with luer slip
adapter, manufactured by Baxter Healthcare Pty Ltd., England, was connected
between the CaCl2 bag and the sample compartment. The FLO-TROL clamp on
this set controls the flow of CaCl2 from the bag to the leaf material in the sample
compartment.
Sample Compartment Section (Figs. IC and 2). The two lids (CI and C2) are glued
together with the smaller one in the centre of the larger one. A 4-mm hole is drilled
through the centre of both lids for the CaCl2 inlet. A second 4-mm hole is drilled
through both lids about lO mm from the centre for the CO 2 free air inlet. A third
4-mm hole is drilled in lid CI, outside lid C2, for the air outlet.
The outer part of the sample compartment (C7) and the reservoir (D) are
made from 250-ml screw cap polycarbonate sample vials (100 x 65 mm size).
A 10 mm diameter hole is drilled at the bottom of the top vial (C7) to allow CaCl2
to pass to the lower reservoir (D). The top compartment (C7) is pushed into
the reservoir (D) and sealed with silicone sealant. A hole is drilled in the side
of the reservoir (D) as near to the base as possible, and a CaCl2 sampling tap
(G), a three-way luer lock stop-cock manufactured by Indoplas Pty Ltd., glued
in place. The other end of the tap is connected to the solution recirculation
tube (H), which is connected to the solution bag. This allows CaCl2 to be recircu-
lated from the reservoir to the solution bag by lowering the bag below the reservoir
once per day. The third outlet is used to remove samples of CaCl2 for nutrient
analyses.
The sample compartment (C6) is made from a 100 ml screw cap polycarbonate
sample vial (70 x 45mm size). Several 2.5mm diameter holes are drilled in the
bottom of this vial to allow CaCl2 solution and air to pass through. The leaf material
to be studied is placed between two layers of OA-mm nylon mesh (C4 and C5) in
the sample compartment with a stainless steel spring (C3) between the upper nylon
mesh and the lid. The nylon mesh is cut to a 50mm diameter to prevent the sample
from rising above the upper mesh.
CO2 Trap (Fig. IE). A 50 ml screw top plastic centrifuge tube is used as a CO 2 trap.
Two holes are drilled in the lid for the air outlet tube and for ventilation. Air is
passed through the system at the rate of I bubble S-I.
CO 2 Scrubber (Fig. IF). A fish tank air pump, or similar pump, is used as an
air supply. CO 2-free air is produced by pumping the air through a sodalime CO 2
trap. A tap is fitted to each air inlet tube to enable control of air flow into each
compartment.
158 J.C. Tarafdar
Plastic tube (4mm diameter) cut to the required length is used for the air inlet
and air outlet tubes and CaCl2 recirculation tube (H). All joints of the apparatus
(Fig. 1) are sealed with silicone sealant to ensure that there are no leaks. There is
some volume loss of CaCl2 as air is pumped through the system. The volume loss
must be measured when calculating nutrient release. This is done by measuring the
volume of CaCl2 remaining at the end of the experiment and correcting on the basis
of a linear decline. When a CaCl2 sample is removed for analysis from the sampling
tap (G), the volume removed should be replaced with fresh CaCI 2
3.1.2 Management of the Perfusion Apparatus
The UNE (University of New England) perfusion solution bag was filled with
200 ml of 0.005 M CaCl2 solution, a concentration similar to the ionic strength of
soil solutions. The 200 ml of CaCl2 solution flowed through the sample container
each day at a rate of approximately 1 drop per 10 s. The solution was collected in
the reservoir before being returned to the perfusion solution bag to begin the next
cycle. The recirculation was achieved by placing the CaCl2 bag below the level of the
reservoir and opening and closing the appropriate taps.
CO 2-free air was pumped through each apparatus to enable measurement of
the amounts of CO 2 produced by microbial respiration. The rate of flow was
standardized in each apparatus by counting the air bubbles in the CO 2 traps and by
checking daily for leaks, reduction of pressure or blockages.
The reservoir of the Nyamai perfusion apparatus was filled with 200 ml of
CaCl2 and this was continuously pumped by an air lift system to the plant material
in the sample compartment at a flow rate of approximately Smlh- 1 The CaCl2 was
then collected and mixed in the reservoir prior to subsampling for analysis.
3.1.3 CO 2 Measurement
The leaves/residues were cut into small pieces and oven -dried at 80C for approxi-
mately 24 h. An approximately 2-4 g sample was put in the sample compartment of
each apparatus. The experiment normally ran for 6-8 weeks at 28-30C. During
the first 14 days, the CO 2 trap (containing 35ml ofO.5M KOH) was replaced daily.
After this period, the rate of CO 2 evolution decreased and so the CO 2 trap was
reduced to 30 ml of 0.25 M KOH, which was replaced every 2-3 days. The trapped
CO 2 was measured by adding 15ml of 10% w/v of BaCL2 to the KOH, to precipitate
BaC03 The remaining KOH was then neutralised by back-titration with 0.5 M HCI,
to the phenolphthalein end-point. Finally, more HCI was added to the methyl
orange end-point to dissolve the BaC03 The amount of CO 2 was calculated by
using the formula:
(T2-Tl)
mg CO 2 evolved/day = x Mx22,
t
where T1 is the amount of HCI used to neutralise KOH, T2 is T1 + amount of HCI

used to dissolve precipitated BaC03 , M is the molarity ofHCI, 22 = 22 mg CO2 /1 ml
1 M HCI and t is the time in days.
The CO 2 in the control treatment was subtracted from the calculated value for
CO 2 release. The CO 2 evolution was also determined by measuring the conductivity
in the collection vials. A significant linear relationship of the form Y = 220.03 -
4.27 X (e = 0.98), where Y is the CO 2 measured by titration (mg/vial) and X is the
conductivity (mS) was used. This procedure is more convenient than titration.
3.1.4 Nutrient Analysis
The cation and P and S concentrations in the initial plant residues and the residue
remaining at the end of the experiment may be determined by ICAP-AAS after
digestion in the sealed chamber (Anderson and Henderson 1986). Analyses of the
digest for total P, Mg, Ca, Zn, Mn, Fe, Cu, and Al was accomplished using induc-
tively coupled argon plasma (ICAP) and K by atomic absorption (AA) spectro-
scopy. Nand C may be determined in an automatic nitrogen and carbon analyser,
with final determination by mass spectrometer (ANCA-MS). The CaCl2 solutions
may be sampled regularly for nutrient analyses.
3.2 Litterbag Technique
The litterbag technique given by Singh and Gupta (1977) has been widely adopted
for studying tree leaf decomposition under field conditions. Most studies have
shown that the litterbag method yields data which are pertinent for understanding
the role of different groups of soil invertebrates in the decomposition process.
Besides, the litterbag method minimizes loss due to fragmentation. This technique,
however, may underestimate the rate of decomposition because the litterbags
restrict the entry oflarge invertebrates, scavengers, and decomposers that could be
important detritivores. Further, the unavoidable disturbance of the vegetation
exposes the soil surface, resulting in an alteration of the microenvironment.
The steps involved in litterbag technique are as follows:
Leaf Collection and Preperation of Litterbag. Freshly fallen leaflitter was collected
separately from different regions of the study site in polythene bags, was brought
to the laboratory and was sorted out. All the litter samples were oven dried sepa-
rately at 80C. Litterbags of 20 x 15 em size, made from nylon wire netting cloth,
I-mm (2 mesh) size were prepared and filled with 20g dry weight of each type of
litter, and the bags were stitched closed.
Field Incubation of Litter Samples. Litterbags were placed in different regions. All
the bags were loosely covered with ground litter. It has been found that decompo-
sition was greatest in the order: foot of hill> hill top> slope (Joshi 1995).
160 J.C. Tarafdar
Sampling of Litterbags. At monthly intervals, three litterbags for each litter

type were recovered from each region and were brought to the laboratory
in polythene bags and the contents were washed under a fine jet of water over a
100-llm mesh screen to remove adhering soil, and were oven dried at 80C to
a constant weight.
The relationship between rate of weight loss oflitter samples and soil moisture
was investigated (Joshi 1995). Soil moisture is found to have more influence on
decomposition than soil temperature. The mean relative decomposition constant
R was calculated using the following formula:
LnWI-LnWo .
R= (Gupta and Smgh 1981),
t-t o
where R is the mean relative decomposition constant g/g/day, Wo is the original

weight of litter at time to and WI is the final weight oflitter at time t l Joshi (1995)
shows that the R value is at a maximum during the rainy season, and at a minimum
during the summer season. The higher rate of decomposition in the rainy season
could be attributed to suitable moisture and temperature conditions for the activ-
ity of decomposers (Rajvansi and Gupta 1980).
From the weight loss data, the decomposition constant K was calculated,
following the method of Olsen (1963):
LnX t
K=--
Xo '
t
where K is the decomposition constant, Xo is initial dry weight oflitter, and ~ is the
final dry weight, at time to. This model also follows calculation of the half-life
period (0.593/k) of litter, and the time required to reach 95% of loss (3/K). The
value of K is found to be higher at the foot of the hill and lower on the slope for
all the types of leaf litter tested. Higher values of K at the foot of the hill reflects
relatively better organic turnover due to efficient decomposer activity.
3.3 Tracer Technique
Tagging organic detritus with a radioisotope is a convenient way to measure the

loss of elements during decomposition. However, in most studies attempts have
been made to relate the fate of a specific element during decomposition (Witkamp
and Crossley 1966; Witkamp and Frank 1967; Thomas 1969), rather than to obtain
information on the process of decomposition as such. This technique has several
advantages including ease of analysis and allowance for contamination by el-
ements in rainfall and soil. Disadvantages include the difficulties of obtaining
isotopes with suitable radiological characteristics, and of obtaining detritus or
organisms tagged with isotopes incorporated naturally into tissues. However,
Murphy (1962) tried the radioisotope method for determining decomposition of
beech leaflitter. The leaves of young beech (Fagus sylvatica) were marked before
a few days of leaf fall with chemically inert radioactive material by using a large
surgical forcep. During the next 12 months they were sampled, photographed,
oven-dried, and weighed. He pointed out several advantages of this method, i.e.
litter of known age can be recovered from the ground and the distribution of leaf
material can be determined. Also, the leaves are maintained under conditions
closely resembling those occurring in the natural environment. This method is not
suitable for very small leaves because oflonger recovery periods vis-a-vis the decay
rate of radioactive substances.
3.4 Measurement of Soil Respiration
This approach provides a means of measuring the total soil respiration during
decomposition, mainly under laboratory conditions. Tarafdar and Rao (1992)
studied the decomposition of different tree leaves under arid soils. The soil was
powdered and sieved through a 20-mesh sieve before use. A hundred g of soil in
500-ml Erhlenmeyer flasks was used in the experiments. Two g of unground leaves
was mixed throughly with 100 g soil, moistened to 50% water holding capacity
before being placed in the flask. They found that 50% water holding capacity is best
for maximum CO 2 evolution. The soil was incubated at 30C in tightly stoppered
500-ml flasks containing a test tube of approximately 10ml1 N NaOH. The mois-
ture content was maintained by the gravimetric method. All the treatments were
replicated three times with the control (without leaf material). The trapped CO 2
was measured by adding 10ml of 10% w/v of BaCl2 to the NaOH to precipitate
BaC0 3 The remaining NaOH was then measured daily by back titration with
0.1 M HCI using phenolphthalein and bromo-phenol blue as indicators. The
amount of CO 2 absorbed was equivalent to the titre between the last pink colour of
the phenolphthalein and the first green colour of the bromo-phenol blue. It was
found that the rate of CO 2 evolution was at a maximum at 3 days and about 58-63%
CO 2 was evolved within the first week of incorporation under different moisture
levels.
The measurements of soil respiration under controlled laboratory conditions
ignore the effects of disturbance and of factors such as temperature, humidity and
CO 2 levels which have been shown to have considerable effect on CO 2 evolution
from the soil.
4 Analysis of Leaf Material Before and After Decomposition
4.1 Determination of Cell Wall Constituents in Leaf Samples
The cell wall constituents (CWC) or neutral detergent fibres (NDF) are a compo-
nent of the leaf which are not soluble in hot, neutral sodium lauryl sulphate
162 J.C. Tarafdar
solution. According to Van Soest and Moore (1965), the cell wall fraction of the
leaf is mainly composed of cellulose, hemicelluloses, insoluble nitrogen, lignified
nitrogen and alkali-soluble and insoluble lignin. The soluble fraction, called the
cell contents, includes all the water soluble organic and inorganic nutrients, lipids,
pectin, starch, soluble proteins and non-protein nitrogen.
The cell wall constituents of samples are now widely analysed adopting a
slightly modified procedure of Van Soest and Wine (1967). Currently, the addition
of sodium sulphite in refluxing the neutral detergent solution is omitted, as
sulphite reportedly removes the lignin fraction (Hartley 1972). Again, the use of
Decalin, an antifoaming agent, is also omitted as it allows poor filtration through
the sintered plates of the crucibles, thereby resulting in a high value for neutral
detergent fibres, especially when it is added prior to detergent solution.
4.1.1 Reagent and Apparatus
Neutral Detergent Solution (NDS). This was prepared by dissolving 30 g sodium

lauryl sulphate, 18.61 g dihydrogen ethylenediamine tetraacetic dihydrate,
6.81 g sodium borate decahydrate (Borax) and 4.56 g disodium hydrogen phos-
phate anhydrous or 5.71 g dis odium hydrogen phosphate dihydrate in ca. 800ml
distilled water. Ten mI2-ethoxy-ethanol was added and the pH of the solution was
brought to 7 by addition ofO.l N Hcl. The solution was made up to 11 with distilled
water.
Acetone. Pure grade.
Refluxing Apparatus. Bench heater, comprising four circular hot plates, each
heated by a 500W heating coil, regulated individually by Sun-Vic energy regula-
tors. The samples were refluxed in 500 ml tall Berzelius beakers, using 250-ml
round bottom flasks as condensers.
Sintered Glass Crucibles. 30 ml Gooch crucibles with 30-mm sintered glass discs of
coarse porosity grade 1 were selected for use. Crucibles with rims were found to be
better suited than those without rims.
4.1.2 Procedures
In the Berzelius beaker, 0.5-2 g ofleaf samples were placed together with 50 ml ND
solution. The beakers were rotated so as to mix all the particles of the samples with
the ND solution, and then kept on the hot plates. Condensers which were intercon-
nected by rubber tubing, were fixed on top of the beakers, with water continuously
passing through the condensers. The solution was allowed to boil, first vigorously
and then gently, with the help of the heat regulator on the hot plate. After 1h of
refluxing, the beakers were removed from the hot plates and their contents were
transferred to the tared sintered glass crucibles, fitted on a filtration apparatus.

The solution was allowed to percolate through the sintered plate; the vacuum was
increased slowly till all the wash was collected in the filtration flask. The contents
of the crucibles was washed with hot water (80-90C) several times until no more
foams were in the wash. Finally, the lumps in the crucible were broken up and the
crucible further filled with hot water, the suction was increased immediately and
the crucible was sucked dry. The residue was then washed three times with ac-
etone, each time with 20 ml of the solvent, and finally sucked dry. The crucibles
were dried overnight at 100C, in a hot oven and transferred to a desiccator, cooled
at room temperature and weighed. The weight of the residue indicated the amount
of NDF in the given samples. Calculations were then made to determine NDF per
g dry matter of the sample.
4.1.3 Filtration
Filtration is the most cumbersome process in the estimation of detergent fibres in

leaf samples. The use of asbestos or celite as a filtration aid tends to cause better
results. Bohra (1981) used Kieselguhr as a filtration aid and it was found that it
hastens the rate of filtration, and causes larger variation in the NDF values of the
same leaf. He concluded that it was better not to use any filtration aid.
The rate of filtration could be hastened by washing the NDF in the beaker,
insted of in the crucibles. The refluxed samples with the ND solution were allowed
to cool at room temperature and 200 ml boiling water was added to it just 5 min
before the initiation of the filtration. The contents of the beaker were allowed to
settle at the bottom and the supernatant was transferred to the sintered glass
crucibles. The contents of the beaker were washed several times, until no more
foam appeared in the wash. Finally, with the help of hot water, the contents of the
beaker were transferred to the crucible. The procedure was useful in reducing
the filtration time and in helping to loosen the NDF particles which had stuck to
the inner walls of the beaker while refluxing.
4.1.4 Cleaning of Crucibles
After the estimation ofNDF, the particles ofNDF were removed with the help of a
camel hair brush. The crucibles were then washed with water and kept in glass
beakers of appropriate capacity. Concentrated sulphuric acid was poured on the
crucibles to immerse them fully to remove NDF particles completely. After 1 h of
heating, ca. 5 ml of saturated sodium nitrate solution was added along the inner
wall of the beaker, drop by drop, into the hot acid solution, and the beaker and its
contents were further heated for 2 h. The beaker with its contents were allowed to
cool overnight. The next morning the crucibles were removed from the beaker and
kept in a shallow glass tray, washed by forcing water in reverse direction through
the sintered plates, followed by rinsing with distilled water. The crucibles were
164 J.e. Tarafdar
finally allowed to dry in a hot air oven, cooled in a desiccator, weighed and used for
further analysis.
4.2 Estimation of Crude Fibre (Acid Detergent Fibre ADF)
The acid detergent fibre of the leaf is composed of cellulose, lignified nitrogen, and
insoluble and alkali-soluble lignin. ADF was estimated with little modification of
the method of Van Soest (1963).
4.2.1 Reagent and Apparatus
Acid Detergent Solution (ADS). This was prepared by dissolving by continuous

shaking 20 g cetyltrimethyl ammonium bromide in II of previously standardized
N sulphuric acid.
Acetone, Refluxing Apparatus and Crucibles. As used in the NDF estimation.
4.2.2 Procedure
In a 500-ml Berzelius beaker, 0.5-2 g of leaf material was distilled by refluxing

with 50 ml ADS by boiling vigorously at first and then more gently. After 1 h
of reflux distillation, the contents of the beakers were transferred to tared cru-
cibles and the contents were allowed to percolate through the sintered glass
plates. The residue was repeatedly washed with boiling water until no more
foam appeared in the filtered solution. The residue was sucked dry and washed
three times, each time with 20 ml acetone and finally sucked dry. The crucibles
were kept overnight in a hot air oven at 100C and then cooled in a desiccator
and weighed. The residue which remained insoluble in the hot ADS was the
amount of ADF in given sample. The ADF content per g dry matter was calculated
on this basis.
4.3 Determination of Cellulose, Lignin and Insoluble Ash
The acid detergent fibre from leaf samples obtained as above was analysed for
cellulose, lignin and insoluble ash (Van Soest and Wine 1968)
4.3.1 Reagents
Saturated Potassium Permanganate. Prepared by dissolving 50 g potassium per-

manganate in II distilled water and stored in an amber coloured bottle.
Buffer Solution. Prepared by dissolving 6 g ferric nitrate nonhydrate and

0.15g silver nitrate in 100mi distilled water, well mixed with 500ml of 1% (w/v)
potassium acetate in glacial acetic acid, and finally adding 400ml of tert-butyl
alcohol. All the reagents were mixed thoroughly and stored in an amber coloured
bottle.
Buffered Permanaganate Solution. This was prepared by mixing two parts of

saturated potassium permanganate and one part buffer solution (v/v) as prepared
above. This was prepared just before use.
Demineralizing Solution. Prepared by dissolving 50 g of oxalic acid dihydrate in

700ml of95% ethanol and mixing with 50ml12 N Hel and 250ml of distilled water.
Ethanol 80%. Prepared by mixing 800 ml of absolute alcohol with 200 ml distilled
water.
Acetone. Pure grade.
4.3.2 Procedure
ADF obtained as described above was used for the estimation of cellulose, lignin
and insoluble ash in the leaf samples. Horizontal staining jars, which can
accomodate two 30ml sintered glass crucibles, containing ADF, were found
most suitable for this analysis. Water was raised in the jars but not in the crucibles,
until the incoming water in the crucibles entering from the bottom through the
sintered plates, just started to moisten the contents of the crucibles. Immediately,
20 ml of freshly prepared buffered permanganate solution was stirred in with a
10 x 0.5 cm (L x aD) glass rod. With occasional shaking, the contents of the
crucibles were allowed to react with the buffered permanganate solution for
30 min. The crucibles were then transferred to the filtration system and vaccum
was applied to remove the brown hue formed as a result of the reaction between
lignin and the buffered permanganate solution. The crucibles were then kept in
clean staining jars and the level of water in the jars was adjusted. Twenty ml of the
buffered permanganate solution was poured into each crucible. With occasional
shaking, the contents of the crucibles were allowed to react for 15min and then
sucked to dryness.
The crucibles then were placed in another set of clean jars and filled with 20 ml
demineralizing (DM) solution with continuous stirring with a glass rod. After
5 min, the solution was sucked to dryness. The crucibles were further filled with
20ml ofDM solution. This time the contents of the crucibles were allowed to react
with the DM solution for 15min and then sucked to dryness. This was repeated
once more. Usually three treatments, each with 20 ml DM solution, were found to
be sufficient to remove the oxidized lignin, formed as a result of reaction with the
buffered permanganate solution.
166 J.C. Tarafdar
Lignin could not be removed until this time, and appeared as a yellow colour
in the contents of the larger fibre particles. It was removed by further treatments of
the DM solution until all the fibres became white. The crucibles were then trans-
ferred to the filtration apparatus, sucked to dryness with the water suction pump
and washed three times, each time with 20 ml 80% ethanol, followed by two
washings with acetone, finally sucked to dryness and transferred to a hot air oven,
dried overnight at 100C, cooled in a desiccator and then weighed. The loss in
weight during these treatments represented the lignin content of the sample.
Further calculations were made to determine the lignin content per g dry matter
of leaf.
The residue remaining in the crucibles was used for the estimation of cellulose
content by firing the contents in a muffle furnace at 500-550C for 3 h. The cru-
cibles were then cooled to 100C in the furnace and transferred to a desiccator for
further cooling. The crucibles were weighed, and the loss in weight due to firing
represented the cellulose content of the sample.
The insoluble ash content of the same sample was calculated by the difference
between the weight of the empty crucible and that of the crucible with its contents
after firing.
4.4 Determination of Cell Contents and Hemicellulose
Cell contents and hemicelluloses were determined by substracting the value of

NDF (%) from that of dry matter (100) and the value of ADF (%) from that ofNDF
(%) respectively.
4.5 Direct Estimation of Cellulose, Hemicellulose and Lignin

(Moubasher et al. 1982)
Figure 3 shows the procedure for estimation of cellulose, hemicellulose and lignin.
4.6 Fibre Degrading Enzymes
4.6.1 Carboxymethyl Cellulase (Endo-1, 4-~-Glucanase, EC 3.2.1.4)
Principle. The enzyme catalyses the hydrolysis of cellulose releasing glucose

by breaking the ~-1,4 linkages and is of microbial origin. The amount of
glucose released is measured colorimetrically to estimate the enzyme
activity.
The enzyme is active against cellodextrins and phosphoric acid swollen
cellulose. Carboxymethyl cellulose is the most common substrate used for the
estimation of enzyme activity. Thus, the enzyme is also termed carboxymethyl
cellulase.
2 g of leaf residue
t
Residue boiled for 15 min with ethanol (4 times)
t
Resultant residue kept in oven at 40C overnight
t
Material obtained treated with 30 ml 1% diastase enzyme for 30 min
t
Washed thoroughly with distilled water
t
Kept in oven for dry weight at 40C
t
Divided into two parts
J 1
First part residue Second part residue
t t
Kept at 80C for taking dry weight (A fraction) Treated with 24% KOH for 4 h at 25C
t
Residue washed thoroughly with distilled water
Kept in oven at 80C overnight and dry weight taken

(8 fraction)
8 fraction treated with 72% H2 S04 for 3 h to

hydrolyze cellulose
Reflux with 5% H2 S0 4 again for 2 h

t
H2 S04 removed by washing residue with distilled
water
Kept in oven for dry weight at 80C (C fraction)

Calculation:
Cellulose = B-C
Hemicellulose = A-8
Lignin = C itself
Fig. 3. Flow chart for estimation of cellulose, hemicellulose and lignin
168 J.e. Tarafdar
Reagents
l. 0.1 M phosphate buffer, pH 7.
2. Carboxymethyl cellulose (1%): dissolve 1 g of carboxymethyl cellulose in dis-
tilled water and dilute to 100 mI.
3. Dinitrosalicylic acid (DNS) solution: dissolve 109 of sodium hydroxide pellets
in 500ml distilled water. Add 109 DNS and 2g phenol and dilute to 11 with
distilled water. Sodium sulphite (0.05%) is added just before use.
4. Rochelle salt solution (40%): dissolve 40g of Rochelle salt (sodium-potassium
tartarate) in distilled water and make volume up to 100mI.
5. Standard solution of glucose (0.1%): dissolve 10mg glucose in lOml distilled
water.
Procedure
l. Test: take 1-ml phosphate buffer, 0.5 ml carboxymethyl cellulose solution and
0.5 g sample in test tube and mix well.
2. Control: mix 1 ml phosphate buffer, 0.5 g sample and 0.5 ml carboxymethyl
cellulose solution.
3. Blank: 2 ml distilled water is put in a test tube.
4. Standard: prepare the standard with glucose solution to plot a calibration curve
of 0, 100, 200, 300,400, 500, 600, 700 and 800 ~g of glucose concentration.
Incubate test (1) for 1 h at 39 DC. Add 3 ml DNS reagent to all the tubes. Keep
the tubes in a boiling water bath for 10 min. Add 1 ml Rochelle salt solution to each
tube and then cool them to room temperature. Make up the volume to 20 ml with
distilled water. Read absorbance (A) at 575 nm against blank. Prepare calibration
curve by plotting A against glucose concentrations.
Calculations
Change in absorbance A = A'es' - Aeon,rol

Read A on the calibration curve to get the ~g glucose released.
Enzyme activity = ~g glucose/h/g = ~g glucose/T x S,
where T is the incubation time (1 h), and S is the amount of sample (0.5 g). Note:
DNS solution can be stored at room temperature for a month without adding
sodium sulphite. Sodium sulphite should be added at the time of use.
4.6.2 a-Amylase (l,4-a-D-Glucanohydrolase, EC 3.2.1.1.)
Principle. The enzyme attacks a-1,4-glucan linkages of starch and glucogen, re-
leasing maltose, isomaltose, larger oligo saccharides and glucose. The a-amylase
activity is determined by measuring the rate of release of reducing sugars during
the incubation of the enzyme with the substrate.
Procedure. The procedure for estimation of a-amylase activity is similar to that of

Endo-1-4-I3-glucanase with the following differences:
1. Starch solution (1 %): dissolve 1 g starch in 90 ml of distilled water by heating at
39 DC. Make up the volume to 100 ml.
2. Assay mixture: it contains 0.5 ml phosphate buffer, 0.25 ml starch solution and
0.25 g sample.
3. Incubation time: 30min.
4. Enzyme activity is expressed as f..Lg reducing sugar released per min per g.
4.6.3 Xylanase (1,4-I3-Xylan Xylano Hydrolase; Endo-1,4-I3-Xylanase; EC 3.2.1.8)
Principle. The xylanase catalyses hydrolysis of xylan and releases its structural
unit D-xylose by breaking 13-1,4-linkages. The enzyme is of microbial origin. The
activity of the enzyme is determined by estimating colorimetrically the amount of
D-xylose released during incubation of enzyme with substrate.
Reagents
1. 0.1 M phosphate buffer, pH 7.
2. 0.25% xylan solution: dissolve 250mg xylan in 100ml distilled water.
3. Dinitrosalicylic acid (DNS) reagent.
4. Rochelle salt solution (40%).
5. Standard solution of D-xylose (0.1 %): dissolve 100mg of xylose in 100ml
distilled water.
Procedure
1. Test: mix 1 ml phosphate buffer, 0.5 ml xylan and 0.5 g sample in a test tube.
2. Control: mix 1 ml phosphate buffer, 0.5 ml xylan and 0.5 g sample in another test
tube.
3. Blank: 2 ml distilled water in a test tube.
4. Standard: prepare tube in duplicate in graded concentration of xylose for
plotting a calibration curve of 0, 300, 600, 900, 1200 and 1500f..Lg in distilled
water.
Incubate test (I) for 15min at 39 DC. Proceed for colour development by DNS
method. Enzyme activity is expressed as f..Lg xylose/min/g.
4.6.4 I3-Glucosidase (I3-D-Glucoside Glucohydrolase, EC 3.2.1.21)
Principle. The enzyme catalyses the hydrolysis of cellubiose and short chain
oligo saccharides to glucose. l3-glucosidase is active towards salicin, phos-
phoric acid swollen cellulose, cellubiose, p-nitrophenol and I3-D-glucopyrano-
side (PNPG). With PNPG as a substrate, the enzyme activity is determined by
170 J.e. Tarafdar
measuring the amount of p-nitrophenol released during incubation with the

enzyme.
Reagents
1. 0.1 M phosphate buffer pH 7.
2. PNPG solution (0.1 %): dissolve 100 mg of PNPG in 100 ml phosphate buffer.
3. p-Nitrophenol solution (0.01%): dissolve 10 mg of p-nitrophenol in 100 ml
distilled water.
4. Sodium carbonate solution (2%): dissolve 2g of sodium carbonate in 100mi
distilled water.
Procedure
1. Test: mix 0.1 g material and 0.9 ml PNPG solution.
2. Control: mix 0.1 g material and 0.9ml PNPG solution.
3. Blank: 1 ml distilled water.
4. Standard: prepare tubes of graded concentration of p-nitrophenol in duplicate
to plot calibration curve as 0, 2.5, 5.0, 7.5, 10.0, 15.0,20.0 and 25.0 ~g in distilled
water.
Incubate test (I) for 10 min at 39C. Add 1 ml sodium carbonate solution to all
the tubes. Read absorbance A at 400 nm against blank. Prepare a calibration curve
by plotting A against standard p-nitrophenol concentrations.
Calculations
Change in absorbance A for sample = AteS! - Acontrol'
Read A on calibration curve to get the amount (~g) of p-nitrophenol released.
Enzyme activity = ~g p-nitrophenollmin/g = ~g p-nitrophenollT x S,
where T is 10 min and S is 0.1 g.
4.6.5 a-Glucosidase (EC 3.2.1.21)
Principle. a-Glucosidase is an enzyme which breaks linkages of disaccharides,

releasing their structural units. The enzyme group includes sucrose (sucrose
D-glucohydrolase, EC 2.2.1.20) and isomaltase (dextrin D-glucanohydrolase,
EC 3.2.1.10). The enzyme activity is determined by measuring the amount of
p-nitrophenol released during the incubation of the enzyme with the substrate
p-nitrophenyl a-D-glucopyanoside.
Procedure. Procedure for a-glucosidase estimation is the same as for B-

glucosidase with the only difference being that here the substrate is 0.1%
p-nitrophenyl-a-D glucopyranoside.
4.6.6 ~-Xylosidase (1,4-~-D Xylan Xylohydrolase: Exo-1,4-~-D Xylosidase,

EC 3.2.1.37)
Principle. Hemicellulose is predominantly the xyloglucan polymers in plant

tissue which, on hydrolysis, release small-chain xylose polymers and xylose. ~
Xylosidase further hydrolyzes these small polymers to release xylose.
Activity. Using p-nitrophenyl ~-D xylopyranoside as a substrate, the activity

of the enzyme is determined colorimetrically by measuring the amount of p-
nitrophenol released during incubation of the enzyme with the substrate.
Procedure. The procedure for ~-xylosidase estimation is similar to that for ~

glucosidase, the only difference being that the substrate is 0.1 % p-nitrophenyl-~
D-xylopyranoside.
4.7 Protein-Degrading Enzymes
4.7.1 Urease (Urea Amidohydrolase, EC 3.5.1.5)
Principle. Urease enzyme hydrolyses urea to generate ammonia. The enzyme ac-
tivity is determined by measuring the amount of ammonia during the incubation
of the enzyme with urea.
Reagents
1. 100mM phosphate buffer, pH 7.
2. 10 mM urea solution: dissolve 15 mg urea and 8 mg EDT A disodium salt in 25 ml
phosphate buffer.
3. Solution A: dissolve 1 g phenol and 5 mg sodium nitroprusside in 100 ml of
distilled water.
4. Solution B: dissolve O.5g NaOH and 0.84ml sodium hypochlorite in 100mi
distilled water. Solutions A and B are to be stored in amber coloured bottles in
a refrigerator.
5. Standard solution of ammonium sulphate: dissolve 0.048 g ammonium sulphate
in 100mi distilled water to get a final concentration of 10mg of ammonia
nitrogen per 100ml of solution.
Procedure
1. Test: 1 ml of assay mixture contains 0.25 g sample, 0.25 ml urea solution and
0.5 ml buffer.
2. Control: 0.25 g sample, 0.25 ml urea solution and 0.5 ml buffer.
3. Blank: 1 ml distilled water.
4. Standard ammonium sulphate solution: prepared tubes of graded con-
centration of ammonium nitrogen in duplicate for plotting the calibra-
172 J.C. Tarafdar
tion curve, containing 0, 0.5, 1, 2, 3, 4, 5, 6, and 71lg of ammonia

nitrogen.
Incubate test (1) for 15min at 39C. Add 5ml solution A to all the tubes and
mix well. Add 5 ml solution B to all the tubes and mix vigorously. Incubate all
the tubes for 15 min at 39C for colour development. Record absorbance (A) at
625 nm against blank. Prepare calibration curve by plotting A against standard
ammonium nitrogen concentrations.
Calculation. The enzyme activity is defined as Ilg ammonia nitrogen released per
min per g sample.
4.7.2 Proteases
Principle. Leaf proteins are hydrolyzed successfully into pep tides and amino acids
by the action of proteolytic enzymes. The enzyme proteases are present both in
plant tissues and in microorganisms.
The activity of these enzymes is determined by measuring the amount
of hydrolysed protein produced during incubation of the substrate with the
enzyme.
Reagents
1. 100mM phosphate buffer, pH 7.

2. Casein solution (1%): dissolve 1 g casein in 1 N NaOH. Adjust to pH 7.5
by adding 1 N HCI and make the final volume 100ml with phosphate
buffer.
3. Standard solution of bovine serum albumin (0.1% BSA): it is prepared in
distilled water to contain 1 mg BSA/ml.
4. Trichloroacetic acid (TCA), 10% solution: dissolve 10 g TCA in distilled water to
make up the volume 100 ml.
5. Solution A: dissolve 2g sodium carbonate in 100ml of 0.1 N NaOH solution.
6. Solution B: dissolve 1 g sodium-potassium tartrate in 100 ml distilled water.
Add to it 0.5 g copper sulphate. Keep it overnight and then filter to remove the
precipitate, if any. Solutions A and B can be stored at room temperature.
7. Solution C: mix 50 ml solution A and 1 ml solution B just before use.
8. Solution D: mix 1 ml Folin and Ciocalten's phenol reagent and 2ml distilled
water just before use.
Procedure
1. Test: take 1.5ml buffer, 0.25ml casein solution and 0.25g sample in a test tube
and incubate for 2 h at 39C.
2. Control: mix 1.5 ml buffer, 0.25 ml casein solution and 0.25 g sample.
3. Blank: 0.5 ml distilled water.
4. Standard curve: prepare tubes of 0.1 % BSA in duplicate so that BSA concentra-
tions are 0, 30, 60, 90,120,150, 180,210,240,270 and 300~g.
Stop reaction in (1) by adding 2ml TCA solution and keep it overnight. Next
day, centrifuge at 2500rpm for 10min and collect the supernatant. Take O.5ml of
the supernatant in a test tube for colour development. Add 5 ml of solution C to all
the tubes and leave for 10 min at room temperature. Add 0.5 ml solution D and
immediately mix it vigorously. After 10min, record absorbance (A) against blank
at 600 nm. Prepare calibration curve by plotting A against standard BSA solutions.
The enzyme activity is defined in ~g hydrolyzed proteins released per h per g
sample.
4.7.3 Transaminases
Two enzymes are considered here; aspartate aminotransaminase: Glutamic oxalo-

acetate transaminase; GOT (EC 2.6.1.1) and alanine aminotransaminase: Glutamic
pyruvic transaminase: GPT (EC 2.6 1.2). These enzymes catalyze the following
reactions:
glutamic acid + oxaloacetic acid ~ a-keto-glutaric GOT acid + aspartic
acid, and ,.-----
glutamic acid + pyruvic acid ~ a-keto-glutaric acid + alanine.
The reactions are reversible. Colorimetric estimation of the enzyme activity is

done using the backward reaction. GPT activity is determined by measuring the
amount of pyruvic acid released during the incubation of the substrate with the
enzyme. Pyruvic acid gives the brown colour of hydrazone, formed by its reaction
with 2,4-dinitrophenyl hydrazine (DNPH). In GOT, oxaloacetic acid is released
during incubation of the substrate and the enzyme converts it spontaneously into
pyruvic acid by decarboxylation. Thus pyruvic acid is estimated for measuring
GOT activity.
Reagents
1. 100mM phosphate buffer, pH 7.4.
2. GOT substrate: dissolve 2.6 g DL-aspartic acid in a minimum volume of 1 N
NaOH. Adjust to pH 7.4 and add 0.02g a-ketoglutaric acid. Dissolve it by
adding a little more 1 N NaOH. Adjust pH to 7.4 and make it up to 100mi with
phosphate buffer. Store it frozen in small aliquots of lOmI.
3. GPT substrate: dissolve igalanine in water and adjust pH to 7.4 by adding 1 N
NaOH. Add 0.028g a-ketoglutaric acid and dissolve it by adding a little more
1 N NaOH. Adjust pH to 7.4 and make it up to 100mi with phosphate buffer.
Store it frozen in small aliquots of 10 mI.
4. 4 mM pyruvate standard: dissolve 11 mg sodium pyruvate in 25 ml phosphate
buffer. Store frozen in 1 ml aliquots.
174 J.C. Tarafdar
5. 1 mM 2,4-dinitrophenyl hydrazine (DNPH): dissolve 18.8mg DNPH in 10ml of

concentrated HCI and make upto 100 ml with distilled water.
6. DAN sodium hydroxide: dissolve 16g NaOH in distilled water and make up
to 11.
Procedure (GOT)
1. Test: take 0.1 g sample and O.Sml substrate in a tube and incubate for 60 min at
39C.
2. Control: take 0.1 g sample and 0.5 ml substrate. No incubation is required.
3. Pyruvate standard: take OAml substrate, 0.1 ml pyruvate solution and 0.1 ml
distilled water. No incubation is required.
4. Blank: 0.5 ml substrate and 0.1 ml distilled water.
Add 0.5 ml DNPH immediately and leave the tube for 20 min at room tempera-
ture. Add Sml of DAN NaOH. Record absorbance (A) at SlOnm against reagent
blank.
Calculations. Enzyme activity is expressed as /lmol pyruvate produced per kg

sample per min.
T-C OAx1 1000 T-C I

- - x - - x-- = - - x 67/lmo
S-B 60 0.1 S-B '
where T is the test; C is the control; S is the standard; and B is the blank.
Procedure (GPT). The procedure for GPT estimation is same as for GOT with the
following differences:
1. Use GPT substrate.
2. Incubation time for (1) test is 30 min.
3. Enzyme activity
4. The calculation equation used is:
T-C OAx1 1000 T-C

- - =- - x - - =- - x 133/lmoI
S- B 30 0.1 S- B
4.704 Glutamate Dehydrogenase (GDH) (L - Glutamate:

NADP + Oxidoreductase EC 1.4.1.4)
Microorganisms utilize the GDH pathway for ammonia assimilation to convert

a-keto-glutaric acid into glutamic acid. The enzyme catalyses the reaction
NH: +a-ketoglutarate + NADHP GDH) glutamate + NADP+ + H 20
GDH activity is determined by measuring the rate of increase in absorbance at

340nm due to oxidation ofNADPH to NADP.
Reagents
1. Tris-HCl buffer: dissolve 0.454g Tris in distilled water. Adjust pH to 7.8 with
HCl and make volume up to 100ml.
2. 0.1 mM mercaptoethanol: dissolve 0.039 ml mercaptoethanol in 5 ml distilled
water.
3. 0.15M a-ketoglutarate: dissolve 0.105g a-ketoglutarate in 5ml distilled water.
4. 0.005 M ethylene-diamine-tetraacetate disodium salt: 0.008 g per 5 ml tris
buffer.
5. 0.01 M reduced nicatinamide-adenine-dinucleotide phosphate (NADPH): dis-
solve 0.008 g NADPH in 10 ml distilled water.
6. 1.2 M ammonium chloride: dissolve 0.32 g NH 4 Cl in 5 ml buffer.
Procedure
1. Prepare blank and test for each sample direct in the cuvette.
2. To both the tubes add 0.5 ml buffer, 0.1 ml mercaptoethanol, 0.1 ml NH 4 CI,
0.03 ml EDTA, 0.1 g sample and 0.5 ml NADPH. Add 0.62 ml distilled water in
blank and 0.57 ml in test.
3. Incubate the tubes at room temperature for 20 min.
4. Read the optical density (El) of blank. Take another reading after 10min (E2).
5. To the tube being tested, add 0.05 ml a-ketoglutarate and read the absorbance
(E3), take another reading after lOmin (E4).
Calculations
E1- E2 = A,
E3 - E4 = B,
AA/10min = B - A, and
MxV
1U= X10 6 ,
exdxvxt
where E is the molar extinction coeffcient of NADPH at 340nm (6.22 x 103 l1moi
x cm), d is the diameter of the cuvette in cm (1 cm), V is the total volume, v is
the sample volume, and t is the time. Multiplication by 106 converts mo1!l into
Mmolll.
1U = AA/10 x 241.12llm/g of sample.
4.8 Determination of Gross Energy (GE)
4.8.1 Principle
The quantitative measurement of heat generated by complete oxidation or com-

bustion of 1 unit of organic matter is called heat of combustion or gross energy
176 J.C. Tarafdar
(GE). The heat thus produced is measured with the help of a bomb calorimeter.
Here, a quantity of organic matter is ignited under the pressure of oxygen for
complete oxidation. In general, an adiabatic oxygen bomb calorimeter is widely
used.
4.8.2 Things Required
The following items are needed:

1. An adiabatic bomb calorimeter
2. Oxygen cylinder fitted with pressure guage
3. A small pelleting machine
4. Cotton thread of uniform thickness
5. Fuse wire etc.
4.8.3 Chemicals and Reagents
The following chemicals and reagents are required: benzoic acid (GE = 6.318k
cal per g); distilled water; 0.1 N Ba(OH)2; 0.1 N Na2C0 3 ; 0.1 N HCI; methyl red
indicator.
4.8.4 Procedure
Preparation of Sample. Weigh about 0.5 to 1 g of dry sample and press it into
the form of a pellet. Now record its accurate weight. Take another sample
from the same material for dry matter determination. If pelleting is not
possible, it may be wrapped in a piece of tissue paper of known weight and GE
content.
Loading the Bomb

1. Place a weighed sample in a crucible.
2. Hang the crucible with sample between the parallel bars of the bomb calorim-
eter top.
3. Connect a lO-cm-Iong piece of fuse wire with the two electrodes through the
sample in the crucible. A lO-cm-Iong cotton thread of uniform thickness and
weight is used to connect the liquid sample to the fuse wire.
4. Put 2 ml distilled water in the body of the bomb.
5. Fix the top of the bomb onto the body.
6. Fill the bomb with oxygen to a pressure of 20 to 2Slb.
Adjustment of Water Temperature. Put 2000ml distilled water in the bucket.

Submerge the bomb in water. Now put the bucket in an outer jacket. Adjust the
temperature in the outer and inner jackets by adding cold or hot water to the outer
jacket.
Ignition or Charging. Connect the bomb points with the mains of the swich box.
Place the thermometer. Switch on the stirrer for mixing the water. Record initial
temperature after 5 min. Charge the bomb. Record final temperature when it is
constant.
Removal of Bomb After Ignition. Open the top of the calorimeter after removing
the thermometer. Disconnect the bomb from the mains. Slowly release the gas in
the bomb. Open the bomb in a vertical position. Wash the interior of bomb and
crucible with a jet of distilled water. Collect washings in a 250ml glass beaker for
the estimation of sulphuric acid and nitric acid.
Acid Correction. Boil the washings in the beaker, cool and titrate against
0.1 N, Ba(OH)2 using phenolpthalein as the indicator. After titration, add 5ml of
0.1 N Na2C0 3 solution and boil slowly. Filter while hot through a Whatman
filter paper No.1, giving a few washings to beaker and filter paper with hot dis-
tilled water. Titrate against 0.1 N HCI, using methyl red indicator. The following
factors are used for the calculation of the caloric values of sulphuric acid and nitric
acid.
1 ml 0.1 N Ba(OH)2 = 3.60 Cal.
1 ml 0.1 N Na2C0 3 = l.43 Cal.
Determination of Water Equivalent. The GE value of dry benzoic acid is

6318 Cal.!g. Ignite exactly 1 g benzoic acid in the bomb calorimeter as described
earlier and calculate water equivalent of the calorimeter with the help of the
following equation:
HxM+Cl+C2+C3
W=--------
t
where W is the water equivalent of the calorimeter (CaUOF), H is the GE of standard

benzoic acid (Callg = 6318 Cal), M is the weight of dry benzoic acid, Cl is the GE of
sulphuric acid (Cal), C2 is the GE of nitric acid (Cal), C3 is the GE of fuse wire (Cal),
C4 is the GE of cotton thread (Cal), if used and C5 is the GE of paper (Cal), if used.
GE value of tissue paper is 3234 Callg, GE value of cotton thread is 3962 Callg and
GE value offuse wire is 1400Callg for platinum wire.
Calculation of GE in samples:
TxW -(Cl+C2+C3+C4)
GE= ,
m
where GE is the gross energy of the sample (Callg), T is the temperature rise due to
ignition, W is the water equivalent of the bomb calorimeter, Cl is the sulphuric
178 J.C. Tarafdar
acid correction (Cal), C2 is the nitric acid correction (Cal), C3 is the fuse wire
correction (Cal), C4 is the paper correction (Cal), and m is the quantity of
sample in g.
5 Conclusion
Evaluation of leaf decomposition rates becomes difficult because of one sort or

another limitations of each of the available methodological approaches. The prin-
cipal challenge is the quantification of the process under natural field conditions,
which is fulfilled only to a certain degree by the litter bag technique. The most
predominantly used litter bag technique for studying decomposition needs further
standardization in respect of surface area, mesh size, thickness and amount of
litter enclosed, and ventilation. Each habitat may require litter bags of different
specifications and this should perhaps be established first by trail runs. The in vitro
UNE perfusion method seems to be a powerful technique.
Similarly, the measurement of soil respiration by alkali absorption suffers
from certain limitations, notwithstanding its simplicity of handling, lower cost and
ease with which observations can be replicated. Its limitations arise from the lack
of standardisation of the absorption surface, concentration and volume of alkali
and the specific area enclosed. Increasing use of radioisotopes is being made, but
restrictions in the use of isotopic material in the natural environment may limit
their use even though the results might be more precise. A rewarding result may
possibly be obtained if the use of a combination of more than one method is
adopted.
There seems to be a gap in our knowledge regarding the individual contribu-
tions of different decomposers, including the variabilities which arise from differ-
ent leaf material.
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Measurement of Leaf Litter Decomposition
S.R. GUPTA and V. MALIK
1 Introduction
Decomposition is a complex and multistep process of litter breakdown through

leaching, mechanical and invertebrate fragmentation, and transformation through
the activity of soil microorganisms (Swift et al. 1979). The rate of litter decompo-
sition varies with chemical composition, abiotic factors and biotic factors (Singh
and Gupta 1977). The linkages between plant quality, soil biota, physico-chemical
environment and the decomposition processes are important (Swift et al. 1979).
During the decomposition of organic matter, a large proportion of carbon is lost
via respiration of decomposer organisms, and nutrients are released during
mineralization. A knowledge oflitter decomposition rates and biological processes
could be useful for the management of soil fertility (Swift 1986), ecological resto-
ration of environmentally fragile sites, detoxification of soil contaminants and
reducing the emission of trace gases to the atmosphere (Heal et al. 1997).
Litter quality refers to the intrinsic characteristics of litter that influence its
utilization by heterotrophs and mineralization by decomposer organisms. The phy-
sical, chemical and inhibitory components oflitter regulate decomposition rates in
different types of ecosystems (Swift et al. 1979; Cadisch and Giller 1997). Lignocellu-
lose is the predominant component oflitter which consists of cellulose, hemicellu-
lose and lignin. It is of interest to study lignocellulose transformation processes
under various environments for making use oflignocellulose components in indus-
trial processes and biotechnological approaches (Deobald and Crawford 1997).
Analysis oflitter decomposition in terrestrial ecosystems has mainly focussed
on energy flow, nutrient cycling, soil biology and modelling of soil organic matter.
The decomposition constant, as defined by Jenny et al. (1949) and Olson (1963) on
the basis of mathematical models, has provided the conceptual framework for
describing the decomposition process. The litter bag technique introduced by
Bocock and Gilbert (1957) has been widely used in decomposition studies in
different ecosystems for comparing litter species, soil conditions and experimental
manipulations. The use of gas chromatography, mass spectrometry, nuclear mag-
netic resonance and stable isotopes have proved to be useful for analyzing litter
quality and understanding organic matter transformations (Norden and Berg
1990; Thompson 1996; Palm and Rowland 1997; Hopkins and Chudek 1997).
In this chapter, we review the methodological approaches for the analysis of
resource quality and decomposition rates of plant litter.

182 S.R. Gupta and V. Malik
2 Plant Litter Sampling and Preparation
Plant litter sampling and preparation for determining its quality involves the
collection of a representative sample of plant litter depending upon age of the
plant, the season, the association with twigs and stems or manures mixed with crop
residues (Palm and Rowland 1997). In forest ecosystems, freshly senescent,
undecomposed litter samples are collected from the ground or collected on dry
cloth or plastic sheets after shaking the plant gently. Leaves significantly attacked
by herbivores are discarded. For herbaceous plants in a grassland ecosystem, the
shoots are harvested just after senescence (Gupta and Singh 1981). Petioles are
considered as part of the leaf, and the initial petiole dry weight as percentage of
total dry weight has been assessed (Arun Lekha and Gupta 1989; Cornelissen 1996).
In the case of compound leaves, laminae and rachis may be treated separately,
depending upon the objective of the experiment. Samples of cover crops are
collected just before slashing and air dried (Luna-Orea et al. 1996) and straw is
collected at the time of crop harvest (Andren and Paustian 1987). To account
for natural field variability, composite samples of litter could be useful (Tanner
1981).
The method of drying of plant material, i.e. freeze drying, air drying, sun
drying, or oven drying has effects on lignin, polyphenol and tannins (Van Soest
1982). For soluble carbohydrates and polyphenols, the drying of plant material
contained in paper bags/ventilated bags in a forced air oven at 30-40C has been
found optimal (Allen 1989; Marstorp 1996). For the analysis of structural compo-
nents (lignin, cellulose and hemicellulose) a drying temperature of 60-80 C has
been used. All the results of chemical analysis are represented on an oven dry
weight basis (100C).
For analyzing the chemical composition of litter, the residues need to be
characterized as a function of their age, plant part composition, particle size and
fineness of grinding (Vanlauwe et al. 1997), nutrient conditions, ambient CO 2
concentration (Cotrufo et al. 1994), residue treatment and drying regime
(Mafongoya et al. 1997). A homogeneous, throughly mixed and adequate replica-
tion of subsamples are taken for chemical analysis.
3 Characterization of Resource Quality of Litter
3.1 Physical Properties of Leaves
Toughness, hardness and particle size affect decomposition rates and nutrient
release from the litter. Toughness and hardness determine the texture of leaves
and are often related to sclerophylly (Loveless 1961). The degree of sclerophylly
has been estimated from crude fibre to crude protein content ratio (Loveless 1961;
Cromack and Monk 1975). From values of acid detergent fibre and nitrogen
Measurement of Leaf Litter Decomposition 183
content, the sclerophylly index was calculated as (total acid detergent fibre x 0.64}1
(nitrogen (%) x 6.25) (Ellis et al. 1946; Loveless 1961). The sclerophylly index gives
information about the carbon quality for decomposition in terms of cellulose,
lignin and crude fibre content (Cromack and Monk 1975). Litter decomposition
rates of some forest tree species were significantly correlated with leaf carbon to
nitrogen ratio and sclerophylly index (Cromack and Monk 1975).
The particle size of the material with a high sclerophylly index is important
(Palm and Rowland 1997). The combined particle area and its weight, known
as specific leaf area (g em-2) could be a simple means of estimating toughness
(Table I). The toughness ofleaves can be measured as the pentometer resistance
(Choong et al. 1992). Woods and Raison (1983) measured the total area of the
leaves enclosed in litter bags before and after the decomposition period using an
electronic leaf area planimeter.
The arrangement of different components within plant tissues (architecture)
and physical properties of cell walls influence the digestibility of forages and
possibly litter decomposition in soil (Chesson 1997). The surface area of the leaf
litter is an important consideration in the degradation process, and this can be
measured by a gas adsorption method. The principle of the method is that nitrogen
Table 1. Parameters and methods used to characterise plant litter quality for decomposition
Parameter Method used Reference
Sc1erophylly index Crude fibre dry weight! Loveless (1961)

crude protein content
Specific leaf area Weight per area Choong et al. (1992)
Toughness Pentometer Choong et al. (1992)
Total surface area Gas adsorption Chesson (1997)
Wax content Dichloromethanelether Ryan et aI. (1990), Schlesinger
extraction and Hasey (1981)
Cellulose + Acid-soluble fraction Effland (1977)
hemicellulose method
Lignin ADF(H,S04) ADF-residue Van Soest (1963), Rowland
and Roberts (1994)
Cellulose ADF-residue Rowland and Roberts (1994)
Soluble carbon Hot-water extraction TAPPI (1988)
Simple sugars Hot-water extraction Dubois et al. (1956)
Phenol-sulfuric acid
reaction
Soluble phenolics Aqueous methanol (50%) Constantinides and Fownes
extraction (1994)
Condensed Acid butanol Waterman and Mole (1994)
tannins Vanillin assay Waterman and Mole (1994)
Protein binding capacity
Total nitrogen Kjeldahl Anderson and Ingram (1993)
Total phosphorus Acid digestion Anderson and Ingram (1993)
Total carbon Digestion method Kalembasa and Jenkinson
(1973)
Nelson -Sommers Nelson and Sommers (1982)
Ash-free dry weight Ash 3-5 h, 500C
gas used for gas adsorption/desorption measurements freely enters the intact cells
through the micro- and meso-pores. The volume of the gas adsorbed is a measure
of the total surface area.
Pubescence of leaves affects fungal growth and feeding by soil animals. The
wax content ofleaves has been determined by using dichloromethane (Ryan et al.
1990) or ether (Schlesinger and Hasey 1981). A method for studying the influence
of fragmentation and bioturbation on the decomposition of 14C-Iabelled beach
leaf litter has been described by Scheu and Wolters (1991), using perpex chambers
containing soil along with intact, mechanically fragmented litter, and faeces of
millipedes deposited after feeding on 14C-Iabelled beech leaf litter.
3.2 Chemical Composition of Litter
The rate of litter decomposition is markedly affected by the amount of water

soluble components, readily available carbohydrates, structural carbohydrates
(cellulose, hemicellulose and lignin) and various refractory components (polyphe-
nols). Nitrogen, phosphorus and sulfur are the essential nutrients required by the
decomposer organisms. In the soil system, mineralization of N, P and S is asso-
ciated with decomposition of litter and soil organic matter. The residues of high
quality (high in nutrients and low in lignin) decompose rapidly, whereas the
residues oflow quality (high in lignin and polyphenols) decompose slowly (Swift
1986). Some polyphenols such as condensed tannins could inhibit exozyme activ-
ity and have been found to reduce decomposition rates (Palm (lnd Sanchez 1990;
Tian et al. 1995). The lignin content of litter exerts a control over the rate of
decomposition in a forest ecosystem (Aber and Melillo 1982; Upadhyay and Singh
1989). Initial lignin concentration or the lignin/nitrogen ratio has proved to be an
effective index of decomposition rates and nitrogen release from the litter (Melillo
et al. 1982). Nitrogen release by legumes with high polyphenol content was slower
(Palm 1995). In order to understand nitrogen release and uptake by rice, the
interaction among chemical constituents of plant residues has been analyzed by
Clement et al. (1995). The analysis of the chemical composition of plant residues
could be useful for selecting appropriate green manure species in rice cropping
(Clement et al. 1995; Malik 1997). A brief description of the various methods for
the analysis of the chemical composition of litter is given in the following sections
(Table 1).
3.2.1 Soluble Carbohydrates and Amino Acids
Dubois et al. (1956) has given a colorimetric method for the determination of
sugars. Simple sugars, oligo saccharides and polysaccharides give an orange -
yellow colour by treating with phenol and conc. sulphuric acid. The reaction is
sensitive and the colour remains stable. The phenol sulphuric acid reaction is a
sensitive method to determine submicro amounts of sugars. In combination with
paper partition chromatography, the method is also useful for the determination
of the composition of the polysaccharides (Dubois et al. 1956).
Total non-structural carbohydrates (TNC) can be estimated by acid hydrolysis
of the plant material followed by Shaeffer-Somogyi copper iodometric titration
(Smith 1969). The details of methods for the analysis of soluble carbohydrates
and amino acids in Lalium multiflarum shoots have been adopted from Marstorp
(1996). Soluble carbohydrates (glucose, fructose, sucrose and fructans) were
extracted with a sodium acetate buffer (pH 5) at 60C. In extracts, glucose and
fructose were determined enzymatically, and the contents of sucrose and fructans
were determined after acid hydrolysis (Steen and Larsson 1986). Free amino acids
were determined using 500mg samples, extracted with 10ml of 80% ethanol for
18h with norleucine as an internal standard. The amino acid analyses were done
on an amino acid analyzer (Biotronic LC5001). Total amino acids were analyzed
using hydrolysates produced from a 50-mg sample, hydrolysed for 24h at 110C
with 6M HCI containing 2mg phenol mr l on an Alpha plus amino acid analyzer
(LKB Model 4151; Marstorp 1996).
3.2.2 Analysis of Cellulose, Hemicellulose and Lignin
Lignocellulose is the predominant component of leaf litter and consists of cellu-

lose, hemicellulose and phenolic polymers of lignin. Cellulose is a linear polymer
of glucose linked through ~-1,4 linkages. Hemicellulose is a matrix of polysac-
charides of plant cell walls excluding pectin and cellulose (Whistler and Richards
1970). Hemicelluloses are generally heteroglycans containing two to four or more
different monosaccharides. Cellulose and hemicellulose account for 25 to 50% of
plant material (Rowland and Roberts 1994). Lignin is a phenolic polymer found in
plant cell walls and the middle lamella surrounding the cellulose microfibrils. It
provides rigidity to cell walls and makes plants resistant to plant pathogens and
mechanical stresses.
Methods for measuring cellulose and hemicellulose have been described by
Allen (1989). The acid soluble fraction method of Effland (1977) has been used
for the analysis of cellulose and hemicellulose (Kemp et al. 1994). Cellulose can
be estimated accurately using the acid detergent fibre method (Van Soest 1963;
Rowland and Roberts 1994). The flow charts for the routine analysis of chemical
fractions oflitter have been given by Palm and Rowland (1997).
The various methods for determining lignin have been adopted from Rowland
and Roberts (1994) and Palm and Rowland (1997). The forest products technique
also known as Klason lignin involves treating of plant materials with 72% H2 S0 4
followed by boiling in 3% H2S0 4 (Effland 1977). The forage fibre method involves
digestion with an acid detergent solution (CTAB = cetyltrimethyl ammonium
bromide) followed by treatment with 72% H2S0 4 (Van Soest 1963). The treatment
of residue with potassium permanganate is a modification given by Van Soest and
Wine (1968). For the litter and leaves with acid detergent fibre (ADF) greater than
35%, the permanganate modification may not be essential (Rowland and Roberts
1994). The lignin and cellulose determined by the forage fibre technique is simpler
and more precise than the Klason lignin (Ryan et al. 1990; Rowland and Roberts
1994).
The near infrared reflectance spectra and proximate analysis data from
various plant litters have been used to develop multiple regression equations for
estimating nitrogen, lignin and cellulose (McLellan et al. 1991). This method is
applicable to both fresh and decomposed materials, and can be easily standard-
ized. Being cost effective, this method could be useful for repeated measurements
on material during decomposition.
3.2.3 Polyphenols
Phenolics are compounds that have a hydroxyl group bonded to an aromatic ring.
They include a range of compounds differing in size, complexity and reactivity.
Caffeic acid, coumarins, flavonoids and tannins are all phenolic compounds
(Waterman and Mole 1994; Harborne 1997). The condensed and hydrolysable
tannins are most important in terms of decomposition and nutrient dynamics
(Palm and Rowland 1997).
Several methods are available for the extraction and analysis of phenolics
(Waterman and Mole 1994). Aqueous acetone, aqueous methanol and hot
water are the most frequently used extractants. A procedure given by King
and Heath (1967) recommends 37.5mg of plant material per ml of extractant.
A concentration of 2mgmtl has been found to be more effective for the
extraction of soluble phenolics in tropical leaves (Constantinides and Fownes
1994). Kuiters and Sarink (1986) have analyzed leaching rates of water-soluble
phenolics from leaf and needle litter of deciduous and coniferous trees. Whole
leaf material was used for the extraction of water-soluble phenolics. A sample
of 20g dry weight was shaken for 20h in 1000mi distilled water and after filtra-
tion (Whatman paper No. 1), a subsample was used for the analysis of soluble
polyphenols.
Total soluble phenolics in the extract are most commonly measured by Folin-
Denis, or more recently the Folin-Ciocalteu assay (Waterman and Mole 1994).
Total soluble phenolics determined with Folin-Ciocalteu reagents were higher and
showed less precipitation than with the Folin-Denis method (Constantinides and
Fownes 1994). The phenolics extracted with the Folin-Denis assay show significant
correlation with net nitrogen mineralization (Palm 1995).
The condensed tannins are the major groups of phenolics that bind proteins.
The various assays for the analysis of condensed tannins include acid butanol and
vanillin assay (Waterman and Mole 1994) and protein binding capacity of the
phenolics and the bovine serum albumin precipitation assay (Dawra et al. 1988;
Waterman and Mole 1994). For the extraction of polyphenols in green manure
species, Clement et al. (1995) have used a solution of 1% HCI in methanol and
determined the soluble polyphenols using the Folin-Ciocalteu reagent with tannic
acid as standard (Singleton and Rossi 1965). The same extract was used to analyze
condensed tannins using the vanillin assay with +catechin as standard

(Broadhurst and Jones 1978).
3.2.4 Plant Nutrient Analysis
Methods for the analysis of plant nutrients have been given by Allen (1989),
and Anderson and Ingram (1993). Nitrogen is analyzed by the micro-Kjeldahl
procedure. Carbon is analyzed by the Nelson Sommers method (Nelson and
Sommers 1982) and acid digestion method (Kalembasa and Jenkinson 1973). The
automatic CNH analyzers are now widely used for analyzing total C and N in plant
materials.
The ash free dry weight of plant material is determined in order to make any
necessary corrections due to contamination by mineral soil in the decomposing
material. Samples are ashed at 450-500C for 3 h.
4 Lignocellulose Transformation
Enzymes involved in the breakdown of cellulose have been studied in a wide

variety of microorganisms. ~-1,4 endo- and exoglucanases are the extracellular
enzymes responsible for the hydrolysis of cellulose chains, whereas ~-glucosidase
converts disaccharide cellobiose to glucose. Different types of enzymes take part in
complete hydrolysis of hemicelluloses because of the complexity of sugars and
linkages in hemicelluloses. Methods for studying cellulose and hemicellulose
degradation make use of the reducing capacity of sugars (Miller 1959; Somogyi
1952) and measurement of hydrolytic activity on the basis of the release of soluble
dye from insoluble, dyed polysaccharide (McCleary 1988). The adsorption of
Trichoderma viride'cellulase components by several types ofleaflitter were mea-
sured by Sinsabaugh and Linkins (1988). Exocellulases are adsorbed preferentially
to holocellulose while endocellulases had greater affinity for lignin (Ooshima et al.
1983). The endocellulase to exocellulase activity ratio increased during the course
of decomposition of litter.
For analyzing lignin biodegradation, Deobald and Crawford (1997) have
described a liquid culture system, solid state fermentation methods and model
compounds for measuring enzyme activities for lignin depolymerization. The
dehydrogenative polymerizate of coniferyl alcohol or DHP is the most commonly
used lignin model. 14C-Iabelled synthetic lignin or DHP is also used as a substrate.
The standard assays for detecting ligninolytic enzymes i.e. lignin peroxidases,
manganese peroxidases, laccases, and peroxide producing enzymes have been
compiled (see Hammel 1997; Deobald and Crawford 1997). For the fungi growing
on litter/in solid state cultures, the enzymes can be detected using indirect
methods (Kawai et al. 1995).
The chemical changes in the lignin components during microbial degradation

can be studied using spectrophotometric methods, UV -visible spectroscopy,
infrared spectroscopy and l3C nuclear magnetic resonance spectrometry (Deobald
and Crawford 1997).
5 Methods for in Situ Litter Decomposition Rates
The decomposition rates of litter have been measured by using containers of wire
screening, by placing the material in layers of open wire bags, by using the tethered
leaf technique, by using litterbags or by using the litter basket method. The utility
and drawbacks of the various techniques have been discussed (Singh and Gupta
1977; Blair et al. 1991; Anderson and Ingram 1993).
5.1 The Litterbag Technique
The litterbag technique as introduced by Bocock and Gilbert (1957) is the most
widely used method for examining litter decomposition rates assuming that
litterbags reflect characteristic trends of unconfined decomposing litter (Singh and
Gupta 1977; Wieder and Lang 1982). This method has proved useful for comparing
species, sites and experimental manipulations. Decomposition of one species on
several sites has been compared (Gupta 1986; Hunt et al. 1988; Upadhyay et al.
1989), several species have been compared on one site (Tanner 1981; Melillo et al.
1982; Woods and Raison 1983; Upadhyay et al. 1989; Gupta and Rout 1992), one
species has been used to compare the effects of mesh sizes (Crossley and Hoglund
1962; Gupta and Singh 1977; Cornelissen 1996). The comparative approach using
litter of both standard and variable quality has been used in several long-term
studies and intersite comparisons in Europe, North America, Canada and the
tropics (Anderson et al. 1992; TSBF 1992; Trofymow et al. 1995).
Cromack and Monk (1975) studied the integrated decomposition rate for
mixed hardwood leaves using litterbags of 0.125 m 2 containing 40 g of material. The
upper half of the large bag was made of 2.54-cm mesh nylon netting, while the
lower half was 2-mm mesh nylon. This facilitated the free entry of animals and
inhibited loss of small leaf fragments from the bags. In a montane rain forest,
Tanner (1981) studied the seasonality in the decomposition of leaf litter using
composite leaflitter samples from four species placed in 15 x 15 cm bags of 2-mm
nylon mesh. Luna-Orea et al. (1996) determined the rate of decomposition and
nutrient (N, P, K, Ca and Mg) release patterns of managed fallows, using the air-
dried plant materials of Pueraria phaseoloides and Desmodium adscendens placed
in I-mm nylon mesh bags.
The decomposing leaf material from litterbags is retrieved at different time
intervals or after long exposure time in the field, and analyzed for dry mass,
moisture content, ash content and nutrient composition. The decomposing litter
materials often show contamination by soil particles, which needs to be corrected
for by calculating percent litter mass remaining on an ash-free dry weight basis.
During rinsing of litterbags with deionized water, some loss of water-soluble ma-
terials occurs which could be measured by determining the decrease in litter
weight due to loss of water solubles under laboratory conditions (Andren and
Paustian 1987).
In agricultural systems, the decomposition of crop residues and green
manures can be studied using mesh cylinders to maintain contact between
the material and the soil (Anderson and Ingram 1993). The mesh bags are opened
to form mesh tubes (20 cm diameter x 30 cm deep) and positioned with
half of their length in the soil. The mesh cylinders are located randomly in the
field and a known weight of litter is added to the mesh tube. To facilitate
handling of material, a metal sleeve can be fitted inside the bags to hold the
mesh tubes rigid during their placement in the soil. The mesh tubes along with
the litter (3 to 5 replicates) are collected on a random basis at regular intervals.
The litter bags are collected from the soil surface at periodic intervals. In
the laboratory, the bagged material is sorted by hand and sifted through a coarse
sieve to separate litter fragments, stones, soil aggregates, and soil fauna. The
residual litter along with the soil can also be separated by a floatation method
(Anderson and Ingram 1993) or by using a commercially available root washing
apparatus.
Cornelissen (1996) has used tube shaped litter bags made up of 0.3 mm mesh
and a double layer of 5-mm mesh nylon net. The double layer of 5-mm mesh was
found to be effective in avoiding loss of leaf fragments as well as allowing free
movement of macrofauna. For needle shaped species and herbaceous materials,
O.3-mm mesh bags were used. After retrieval of the litter bags from the field, the
samples were stored at -14C. After defrosting, adhering soil, soil fauna and other
materials was removed by brushing or rinsing with water.
Decomposition rates oflitter have been studied using the litter bag technique
in various types of ecosystems in India (Gupta and Singh 1981; Arun Lekha et al.
1989; Upadhyay and Singh 1989; Upadhyay et al. 1989; Gupta and Rout 1992;
Aggarwal 1997).
Studies on grassland, forest and cropland ecosystems at Kurukshetra showed
that litter bag studies provided useful information on decomposition rates in
relation to climatic seasonality, resource quality and the role of leaching and soil
fauna in litter decomposition. The decomposition rates of leaf litter, straw and
green manure as determined by using the mesh bag technique are summarized in
Table 2.
In central Himalayan forest ecosystems, Upadhyay et al. (1989) found that
lignin was the best indicator of the annual loss rates. The climate and litter quality
models showed that lignin concentration with actual evapotranspiration or lignin
and annual mean temperature predicted weight loss as well as variations from
habitat to habitat (Upadhyay et al. 1989).
Table 2. Decomposition rates for various plant materials in grass-

land, forest and agricultural systems at Kurukshetra, India
Ecosystem/plant material Decomposition rate (percent day-i)
Grassland
Chenopodium album' 0,248
Desmostachya bipinnata' 0,221-0,243
Dichanthium annulatum' 0.338-0.345
Mixed grass a 0.238-0.242
Sesbania bispinosa' 0.315-0,356
Dry deciduous forest

Dalbergia siSSOOb,d 0,254-0,238
Acacia nilotica d 0,176
Breynia rhamnoides d 0.251
Butea monosperma d 0,254
Capparis sepiaria d 0,257
Carrisa spinarum d 0.216
Cordia dichotoma d 0,159
Diospyros cordifolia d 0,255
Mixed -herbaceous b 0,234
Cropland
Vigna unguiculata' 0.307
Sesbania aculeata' 1.043-0.708
Agroforestry system
Populus deltoides' 0.319
Leucaena leucocephala' 0.329
Rice straw' 0.274
Sorghum straw' 0.288
a Gupta and Singh (1981).

b Gupta and Rajvanshi (1991).
C Arun Lekha and Gupta (1989).
d Aggarwal (1997).
'Malik (1997).
5.2 Litter Basket Technique
A litter basket technique for studying the interaction of fauna, microbes and litter
quality in decomposing litter has been described by Blair et al. (1991). The litter
baskets (10 x 10 x 10 cm) were made up of wire hardware cloth with 6-mm mesh
and a plastic window screen (mesh 1.5 x 1.8 mm) separating the forest floor profile,
(L-Iayer, F-Iayer and the soil). The test material dogwood (Comus florida) leaves
were placed between the F-Iayer and the L-Iayer. The litter baskets were collected
over time to determine litter decay rates and patterns of nutrient immobilization/
mineralization. Microarthropods were extracted using Tullgren Funnels and by
high gradient extraction. Subsamples were used for enumeration of bacteria, fungi
and nematodes. Microbial populations were estimated using a direct count analy-
sis, whereas nematodes were extracted on Baermann funnels.
According to Blair et al. (1991), the litter basket technique has several advan-
tages i.e., reduced micro climatic effects, use of stable isotope tracers, analysis of
nutrient content changes, easy extraction of invertebrates and quantification of
microbial populations and ability to quantify the movement of radioactive or
stable tracers from the litter.
5.3 13C Nuclear Magnetic Resonance (NMR)
Considerable progress has been made in soil organic chemistry due to the avail-
ability of advanced methods of analysis such as 13C nuclear magnetic resonance
C3C NMR), pyrolysis and mass spectrometry (Golchin et al. 1994). Solid state 13C
NMR spectroscopy offers the possibility of direct characteriztation of organic
materials in intact soil or separated fractions. The technique of cross-polarization
and magic angle spinning (CP/MAS) was developed by Barron and Wilson (1981)
to study various carbon compounds in whole soil. The general principles of 13C
NMR spectroscopy have been widely discussed (Simpson 1986). Using 13C CP/MAS
NMR for the whole soil and its density fractions, Barron and Wilson (1981) and
Baldock et al. (1991,1992) obtained well-resolved spectra of organic compounds of
soil organic matter. The chemical shift ranges of organic compounds as reported
by Baldock et al. (1991) are reproduced in Table 3. The resonance at 10-45ppm
(methyl and alkyl-C) was mainly from aliphatic lipids, fatty acids, waxes and
hemicelluloses. The small signals in the aromatic region (110-160ppm) were at-
tributed to aromatic C in lignin, and the lignins at 60-90 ppm (O-alkyl-C) resulted
from oxygenated C in carbohydrates.
Norden and Berg (1990) used high resolution 13C NMR (CP/MAS) spectros-
copy for analyzing the decomposition of Scots pine (Pinus sylvestris) litter. This
method is briefly described here. The litter material (1.5 g air-dried needles) was
enclosed in litterbags (8 x 8cm2 , l-mm mesh terylene net). The litterbags were
placed on the surface of the litter layer (ADD) in subplots (1 x 1 m 2 ) within each of
25 main measurement plots according to the randomized block design. The
litterbags were recovered three times in a year (25 replicates), processed for re-
moving extraneous materials and dried at 82C. A pooled sample was prepared,
ground in a laboratory mill (l-mm mesh screen) and analyzed for chemical
composition. The subsamples were analyzed for l3C CP/MAS NMR spectra (Bruker
MSL-IOO, 25.178MH z, 7.5-mm rotors made up of Al 20 3 and equipped with
Kel-F caps). The chemical shift scale was determined with reference to external
adamantane whose methylene resonance was set at 38.3 ppm.
In the initial, undecomposed material, l3C signals from the carbohydrate
region were evident (60-100ppm). The NMR signals showed a decrease in carbo-
hydrate content with the progress of litter decomposition (Fig. 1). By using a
multivariate data analysis method (partial least square), and NMR data, lignin
content was also determined in the decomposing litter (Norden and Berg 1990).
The transformation of 13C-enriched grass material was studied by analyzing
the redistribution of 13C and identifying various functional groups on the basis of
Table 3. Chemical shift ranges, chemical assignments and classes of compounds. (Baldock et al.
1991)
Shift range (ppm) Type of C Class of compounds
10-45 Methyl-and alkyl-C Lipids, waxes and aliphatic hydrocarbons

45-60 Methoxyl-and alkyl-amino-C Lignin substituents, amino acids and
amino sugars
60-90 O-alkyl-C Carbohydrates
90-110 Acetal-and ketal-C Carbohydrates
110-160 Aromatic-C Phenyl-propylene sub-units of lignin
160-200 Carbonyl-C Organic acids and amino acids/peptides
Fig. 1. A J3C-NMR spectrum of original sample; B a sample

after decomposition for 553 days (45.9% mass loss); and C a
sample after decomposition for 1438 days (73.2% mass loss).
PPM (Norden and Berg 1990)
NMR signals (Hopkins and Chudek 1997). I3C-enriched Lolium perenne leaf mate-
rial (12 atom% I3C; C: N ratio = 20, cellulose = 39%, hemicellulose = 29%, lignin =
6%) was amended in a sandy loam soil (pH = 7, Carbon = 3.4%) at the rate of 40 mg
plant material g-l soil. The CO 2 evolution rates from amended and unamended
soils were measured by gas chromatography and I3C remaining in the soil was
determined at 0, 14,28, 56, 112 and 224 days. I3C NMR spectra were recorded on
dry, ground soil (0.4 g of particle size <100 Ilm) using NMR spectrophotometry
(Bruker AM 300 MHz/WB FT). During the early stages of decomposition, there
were clear NMR signals at 10-45,45-60,60-90,90-110 and 160-200 ppm. The rate
of decline of carbohydrate carbon was found to be highest when compared with
other forms of 13C during the course of decomposition.
The advantage ofNMR is that it is a nondestructive method that does not alter
the chemical composition of the plant material. This approach is useful in studying
the component processes of decomposition against the unlabelled soil organic
matter (Hopkins and Chudek 1997). The rates of decomposition of soil organic
matter in particle size fractions (>2000/-lm and >63/-lm) isolated from two grass-
land stagnogley soils, compared with their chemical composition, have also been
analyzed by solid-state 13C NMR (Hopkins and Chudek 1997). Analysis by NMR is
expensive, but it is a valuable approach for understanding the continuum between
plant decomposition and soil organic matter.
5.4 Tracer Techniques
5.4.1 Tagging Technique
Tagging organic detritus with a radioisotope is a convenient way to measure loss of

litter and nutrient release during decomposition. Murphy (1962) tried the radio-
isotope method for determining decomposition of beech leaf litter. The leaves of
young beech (Fagus sylvatica) were marked before a few days of leaf fall with a
chemically inert radioactive material by using a large surgical forceps. During the
next 12 months, the leaves were sampled, photographed, oven-dried and weighed.
The advantage of the method is that litter of known age can be recovered from the
ground, the distribution of leaf material can be determined and the leaves are
maintained in natural environmental conditions. However, the method is not
suitable for small leaves like pine needles and the study period is longer vis-a-vis
the decay rate of the radioactive substance.
5.4.2 14C Technique
The radioisotope 14C has been used to label plant material for studying its rate of
decomposition and analysis of residual carbon addition (Jenkinson 1977; Ladd
et al. 1985; Amato et al. 1987). The studies showed that '4C-labelled green plant
material added to the soil showed an initial rapid phase of decomposition for 1-2
months followed by a slow phase of decay. Amato et al. (1987) obtained recoveries
of -65 and 40% at 4 weeks and 12 months following incorporation of '4C-labelled
green Medicago littoralis shoots to the soil. The decomposition of 14C_ and 15N_
labelled medic materials was studied to analyze the effect of soil type on decompo-
sition rates (Ladd et al. 1985).
5.4.3 13C Stable Isotope Technique
Keeping in view the limitations of studying the decomposition oe 4C-labelled plant

materials under field conditions, the stable isotopes of carbon and nitrogen are
increasingly being used in decomposition studies. The decomposition of lSN_

labelled legume residues in soils have been studied (Ladd et al. 1981; Azam et al.
1985; Ladd and Amato 1986). Methods of analysis of plant and soil samples
labelled with l3C and lSN using mass spectrometry have been discussed in detail by
Mulvaney (1993).
The turnover of organic materials in soil can be studied by examining the
ratios of the stable isotopes C3C/ 12 C) of soil carbon (Balesdent et al. 1988; Golchin
et al. 1995; Ryan et al. 1995; Thompson 1996). The ratio of l3C/12C is generally
expressed as ol3C%o and given by the relationship:
(13C/12C) sample )
e
ol3C%o = ( 3 12
C/ C) reference
1 X 1000.
The reference is Pee Dee belemnite (PDB) which is CO 2 obtained from the carbon-
ate skeleton of a crustacean mollusc Belemnitella americana from the Pee Dee
formation in South Carolina (Lerman 1975; Golchin et al. 1995). Using the isotopic
dilution principle, the organic matter turnover in soil intensively cultivated with
C4 -type plants following forest clearance and long-term cultivation with C3-type
crops has been studied (Balesdent et al. 1987; Skjemstad et al. 1990; Golchin et al.
1995; Ryan et al. 1995).
A detailed methodological procedure for pulse labelling of Phacelia with l3C to
study its decomposition under field conditions has been given by Thompson
(1996) which is briefly described here. l3C-Iabelled shoot and root material were
applied at the field site within microplots (PVC cylinders 25 cm diameter, 60 cm
long). The cylinders were vertically pushed into the soil, so that the top of the
cylinder was -2cm above the soil surface. The soil from 0-15cm depth was re-
moved from the PVC cylinders and treatment material (2-cm segments) was
loosely mixed with the soil, followed by light repacking of the soil in the cylinders.
The sampling of cylinders (nine replicates) was done at 16 days and 179 days after
the addition of plant material. The subsamples were oven dried at 70C for 4 days
in a forced draft oven, and sieved (2-mm mesh sieve). All the material on the sieve
was collected and finely ground in a ball mill. The ground material was mixed with
the sieved soil and finely ground using stainless steel bars. The ground samples
were combined and thoroughly mixed.
The percentage recoveries of added excess l3C in soil total C were calculated
from the mass of excess l3C in the soil total C to the mass of excess l3C applied.
The background 13C values (atom % l3C) were: 1.0811 0.0005 Phacelia plant,
1.0864 0.0031 soil (0-15cm), 1.0854 0.0003 soil (15-30cm). According to
Thompson (1996), the mean total recoveries of added l3C in the soil (0-30 cm)
were 78 and 40%, respectively at 16 and 179 days (Table 4). The pulse labelling
with 13C could be a useful means for examining the decomposition of plant mate-
rials under field conditions provided a desired uniform labelled plant material
is available. The cost of using the 13C enrichment technique is high. Therefore, the
study has to be goal oriented depending upon the precision and applicability of
the results.
Table 4. Recovery of added excess 13C and total C contents of soil (0-15cm and 15-30cm)
following the addition of 13C-labelled Phacelia. (Thompson 1996)
Date Depth (cm) Soil total C Excess soil 13C % Recovery of

content (%) content (atom %) added 13C ( s.d.)
17 March 1994 0-15 0.75 1.0864

15-30 0.50 1.0854
5 April (after 0-15 0.87 0.1913 77.4 10.2
16 days) 15-30 0.55 0.0014 0.4 0.4
15 September 0-15 0.79 0.0985 39.9 2.4
(after 179 days) 15-30 0.51 0.0030 0.4 0.7
6 Analysis of Decomposition Data
The most commonly used statistical method for analyzing the decomposition data
is analysis of variance for the completely randomised block design, to compare the
effects of dates, sites, and litter type (Gupta and Singh 1981; Wieder and Lang
1982). The second general approach is the analysis of decomposition data using
mathematical models to estimate decomposition constants that describe mass loss
over time. The most frequently used model is the single exponential model pro-
posed by Jenny et al. (1949) and discussed in detail by Olson (1963). The single
exponential model assumes that the absolute decomposition rate decreases lin-
early as the amount of substrate remaining declines. The double exponential
model is particularly useful in experimental situations where the decomposition
of one litter type has been examined simultaneously in several sites (Wieder and
Lang 1982). The two models which describe the loss of mass over time are de-
scribed below.
The single exponential model (Jenny et al. 1949; Olson 1963):
Xt/X o = exp(-kt),
where Xo is the initial litter/nutrient content, ~ is the weight/nutrient remaining,

k is the decomposition constant (rate per day/year) and t is time.
The model assumes that plant tissue/nutrient will decompose or release at the
same rate over time. This exponential model also allows the calculation of the half-
life (O.693/k) or time required to reach 95% loss (3/k).
The double exponential model (Wieder and Lang 1982):
x = Ae -k,dt +(1- A)Ae -k,dt,

where X is the dry weight/nutrient remaining, A is the initial dry weight/nutrient
content, k and k2 are the decomposition or nutrient release rate constants and
j
t is time.
This model defines two phases of decomposition and nutrient release.
7 Laboratory Methods
7.1 Respirometric Techniques
Heterotrophic soil organisms degrade litter and the end products of the aerobic
degradation process are carbon dioxide and water. The overall metabolic activity
of soil organisms can be determined with respirometric techniques that monitor
CO 2 evolution or O2 consumption (Stotzky 1965; Anderson 1982; Gupta 1991).
CO 2 evolution can be used as a measure of microbial activity and the amount of
litter decomposed. The "master jar" system (Stotzky 1965) helps continuous col-
lection of respired CO 2 as well as facilitating other experimental manipulations
(transformation of substrate, species diversity and enzymic activities). The soils
are incubated at constant temperatures and maintained at 40-50% water holding
capacity. The amount of CO 2 absorbed in NaOH traps can be determined after
precipitation of the bicarbonate and carbonate ions with excess BaCl2 or by
automatic potentiometric titration with HCl. CO 2 evolution rates were measured
from straw amended soils under laboratory conditions by absorbing CO 2 in
1 M NaOH (Gupta 1991). CO 2 evolved from straw amended and unamended
soils was determined using automated pH titration (Jenkinson and Powlson 1976).
CO 2 evolution was calculated from the volume of 1 M HCI needed to bring the
pH of an aliquot of the 1 M NaOH absorbing solution from 8.30 to 3.70, using
a radiometer autotitrator (Shen et al. 1984). For details of various methods of
measuring CO 2 evolution, see Anderson (1982), Singh and Gupta (1977) and
Stotzky (1997).
In laboratory experiments, the decomposition of Lolium multiflorum shoots
(Marstorp 1996) and that of sugar beet leaves and rye grass shoots (Marstorp 1997)
was studied from automatic CO 2 measurement from the soil (40g d.wt.) amended
with plant material (40 to 50mg total C) contained in 250-ml respiration jars. The
moisture content of the soil was kept at 40% water holding capacity (WHC) and
jars were incubated at 20 DC. The shoots from which water has been extracted were
also used to differentiate respiration peaks from different kinetically defined com-
ponents. Respiration was measured in an automatic respirometer by absorbing
evolved CO 2 in KOH and taking hourly measurements of conductivity of the
alkaline solution. For details of the method and calculations of respiration rates,
see Marstorp (1996). On the basis of kinetically defined components of plant litter,
decomposition rates of most readily available litter components can be studied,
although the method may not be suitable for predicting the decomposition of
structural litter components (Marstorp 1997).
The effects of elevated CO 2 on decomposition of plant litter are receiving
increasing attention (Lambers 1993; Cotrufo et al. 1994). Elevated atmospheric CO 2
might increase litter C/N ratios, resulting in slower decomposition rates (Lambers
1993). For studying the effects of elevated CO 2, litter material was obtained from
solar domes where plants have been exposed to ambient (350ppm CO 2 ) and el-
evated levels of 600 ppm CO 2 (Cotrufo et al. 1994). The decomposition ofleaflitter
in a laboratory microcosm experiment showed that decomposition rates decreased

for the litters grown under elevated CO 2 (Cotrufo et al. 1994).
7.2 14C-C02 Evolution Rates
The decomposition of 14C-Iabelled plant materials on the basis of measurement of

CO 2evolution rates have been reported (Scheu and Wolters 1991; Van Gestel et al.
1993; Magid et al. 1996). Therefore, it is necessary to measure total C and its
radioactivity in soils and plant samples. 14C02 produced by wet oxidation or by
combustion of solid samples is trapped in alkali solution or in a CO 2 absorbing
cocktail for analyzing by liquid scintillation counting (Dalal 1979; Nelson and
Sommers 1982; Amato 1983).
Dalal (1979) developed a simple procedure for simultaneous measurement of
total C and its radioactivity in soil and plant materials. The soil or plant samples
were digested in a McCartney bottle containing a test tube with alkali for absorp-
tion of CO 2 in an autoclave at 121C. Amato (1983) described a modified wet
combustion method using a heating block and digestion tubes. This method allows
complete mixing of soil or plant material with the digestion mixture, and allows a
range of digestion temperatures. The CO 2 can be trapped in ethanolamine which
can be counted in total for more efficient scintillation counting of weakly labelled
materials. The wet combustion method is useful for the routine analysis of 12C and
14C in soil and plant samples. Trapping CO 2 in ethanolamine allows accurate
counting oflow radioactivity samples. Benoit et al. (1994) developed an automated
method for the rapid measurement of organic C as well as C and N elemental
composition.
Magid et al. (1996) have developed fractionation methods for studying the
decomposition of homogeneously l4C-Iabelled plant materials on the basis of
measuring CO 2 evolution rates and determining the recoveries of added 14C in
soil/quartz sand.
The decomposition of homogeneously 14C-labelled shoot material of
Lolium perenne grown at ambient CO 2 (350~1l-1) and elevated CO 2 (700~lrl)
in a phytotron were studied (Magid et al. 1996). The plant material (1.07mgg- 1dry
soil) was added to 150 g moist soil contained in 150 polyethylene beakers. The
soils were compressed to 100 ml to obtain bulk density of 1.3 g ml- 1on a dry weight
basis. Each beaker of soil was placed in a 11 jar containing a CO 2 trap (10 ml 1 N
NaOH). The soils were incubated in the dark at 14C for 200 days. The CO 2
evolution rates were measured periodically from the treatments with or without
soils.
The bicarbonate and carbonate ions in the NaOH solutions were precipitated
with excess BaCI2. Total CO 2was determined by titrating the remaining NaOH with
50 mM HCl. Total 14C in 0.5 ml of 1 N NaOH was determined by liquid scintillation
counting (Packard and Tricarb 4530) in 3 ml of Ultima Gold (Packard). Total C and
14C in the soil was determined by digesting 1 g soil in 5 ml of a solution of 2 mIl N
K2Cr20 7 in 25ml of conc. H2S04 and H3P0 4 (312 v/v) at 160C for 2h. Evolved
CO 2 was trapped in 5ml of 1 N NaOH and determined by acid titration, and 14C by
scintillation counting.
The effect of various density agents i.e. water, Ludox TM 40 (a colloidal silica
suspension) and sodium polytungstate on the decomposition rates of plant mate-
rial was analyzed by Magid et al. (1996). They took 2.5 g of coarsely ground 14C_
labelled material (grown at ambient CO 2 concentration) and leached this with
water. The structural plant material was soaked in water, Ludox (pl.4) or sodium
polytungstate (pl.4) for 15min followed by washing with water over a 100ilm
sieve. The treated plant material was mixed with acid-washed, quartz sand of
mixed grain size and incubated at 14C. The experimental set-up was similar to
that described for the soil.
On the basis of extensive experiments, Magid et al. (1996) found that mineral-
ization of carbon from previously leached plant material was enhanced by expo-
sure to Ludox and retarded by exposure to sodium polytungstate. Therefore, the
methods based on size separation and density fractionation with water seem to be
the most promising approach for studying the decomposition of isolated fractions
of soil organic matter.
7.3 Measurement of Enzymatic Activity
For an aquatic ecosystem, Sinsabaugh and Findlay (1995) proposed the enzymic
index of carbon quality as the ratio of cellulase activity to ligninase activity (the
ratio of hydrolytic to oxidative enzyme activity). The enzymic index of carbon
quality was found to be highly correlated with decomposition rates (Sinsabaugh
and Findlay 1995). The enzymatic activities are potentially more sensitive decom-
position indicators than less direct chemical measures (Sinsabaugh and Moorhead
1997). They have predicted mass loss as a function of extracellular enzyme activity
and developed a model for microbial allocation of resources among community
indicator enzymes (MARCIE). Using the MARCIE simulation model, Sinsabaugh
and Moorhead (1997) predicted litter mass loss, microbial biomass and ligno-
cellulose degrading enzymes for decomposing dogwood, maple and oak leaf
litter.
7.4 Nitrogen Mineralization from Decomposing Litter
Laboratory incubation methods have commonly been used for assessing nitrogen
mineralization under controlled conditions and various methods have been re-
viewed (Brown 1982; Hart and Binkley 1985). Several studies have analyzed the
effect of resource quality on nitrogen mineralization rates of plant residues in soils
using an aerobic incubation method (Frankenberger and Abdelmagid 1985; Palm
and Sanchez 1990; Clement et al. 1995; Malik 1997). The use of 15N pool dilution
techniques has become increasingly important for estimating N mineralization.
Microbial nitrogen immobilization-mineralization rates have been studied by
measuring microbial biomass 15N and mineral 15N after the additions of 15N either
as mineral nitrogen or labelled plant residue (Mary et al. 1996).
Using 15N labelled sugar beet tops, Gupta (1991, 1992) showed that microbial
biomass derived nitrogen preferentially from residue N than from fertilizer nitro-
gen (Fig. 2). The 15N-Iabelled sugar beet tops were obtained from the experimental
field at Woburn. A representative sample of the sugar beet tops was oven dried at
40C and ground through a mesh screen in a Wiley mill (40.69%C, 2.17% Nand
0.5990 atom percent excess 15N). Three treatments of the two soils (clay loam soil
and sandy loam soil) were prepared (soil alone, soil plus 15N-Iabelled sugar beet
tops, soil plus 15N-Iabelled sugar beet-tops plus KN0 3 ) by weighing a portion of
moist soil containing 50g soil oven dry mass (24h, 105C) in 200-ml jars. lsN_
labelled sugar beet tops (0.1875 g containing 75 f..lg N) with or without unlabelled
KN0 3 at 50f..lgN g-l soil were added. The soil was adjusted to 50% water holding
capacity. The jars containing the soils were placed individually in 11 glass bottles,
sealed and incubated for 5, 10, 20, or 40 days. The CO 2 evolved from the soils was
absorbed in 1 M NaOH and determined using a radiometer autotitrator. During
the total incubation period of 40 days, net decomposition rates (C0 2-C evolution
from sugar beet top amended soil- control soil/initial carbon in sugar beet tops)
varied from 54.84% to 61.91 %.
0- . -0 So il
t::r-----6 Soil + suga r beet tops
'0
CII -- - - Soil + N + sugar beet t ops
1 01
Z (a )
01
--- - -- -- --
::J
Tota l bi omass N
80
--
Z
CII
CII
o 60 /
E
:0
o '/
....
..
V
CI 40 - ' - 0 - ' _ . -t:>-- . - . - . - .-<>-- . _ ._ .- .- ._ ._ . - ' -0
o
t-
200~---S~---~~--------2~
0 ----------------~
40
(b)
B i om ass l~N
::: 20 -
CI
E
o
:0 10 20
I
Z
~
Tim e aft er sugar beet tops amend ment ( days )
Fig.2. Biomass nitrogen in unamended soil and soils amended with lsN-labelled sugarbeet tops
with and without inorganic in a clay-loam soil. (Gupta 1991)
Table 5. Labelled inorganic nitrogen (NH:-N + N0 3--N) in soils

amended with sugar beet tops (SBT) with or without inorganic
fertilizer nitrogen
Incubation time Soil + SBT Soil + SBT + inorganic N

(days)
Labelled inorganic N (J..lg 15N g-l soil)
Clay-loam soil
5 o 6.09 0.1
10 o 9.15 0.31
20 7.73 0.21 16.04 0.21
40 16.63 0.06 2l.88 0.33
Sandy-loam soil
5 o 3.86 0.03
10 o 7.55 0.24
20 o 14.01 0.41
40 1l.88 0.24 21.36 0.22
Soil microbial biomass nitrogen was measured using the fumigation extrac-
tion method (Brookes et a1. 1985; Odo and Brookes 1990). Total lsN after N0 3
reduction and digestion (Pruden et a1. 1985a,b; Odo et a1. 1991) was determined in
distillates trapped in 0.25M H2 S04 The distillates were evaporated on a hot water
bath and the concentrated samples (1-2 ml) were finally dried at 40C in an oven to
dryness. lsN enrichment was measured on dried samples using the mass spectro-
photometer (Europa isotope ratio mass spectrophotometer). Biomass lsN was
calculated as lsN = 2.22 eSN-labelled extractable nitrogen) assuming that labelled
and unlabelled biomass was formed in the same proportion during soil incuba-
tions (Brookes et a1. 1985).
Inorganic lsN (NH/ + N0 3--N) was determined in 25ml aliquots of soil
extracts (non fumigated soil) by reduction and steam distillation with
Devarda's alloy-MgO mixture (Bremner 1965). Atom % enrichment of distilled
NH/-N was determined as for total N in soil extracts. lsN content of samples was
calculated as lsN atom % excess. In both the soils, more labelled inorganic nitrogen
was in the soil to which unlabelled fertilizer was added along with sugar beet
tops (Table 5). The proportion of labelled inorganic lsN increased with time of
incubation.
Shen et a1. (1984) have described the procedure for determining isotope
ratio measurements of NH;-N and NO;-N in soil extracts to avoid cross con-
tamination between samples. An unlabelled NH;-N solution (containing 150~g
NH;-N) was distilled with MgO and the distillate discarded while processing
the actual K2 S04 soil extracts. The condenser and top of the distillation ap-
paratus were of stainless steel to minimize "memory" effects (Shen et a1. 1984).
Labelled N is calculated from the isotopic dilution formula of Hauck and
Bremner (1976), making a necessary correction for the atomic weight of the 14N _ls N
mixture.
8 Conclusions
Litter decomposition is an important soil biological process regulating nutrient

cycling and soil fertility. The most predominantly used litterbag technique needs
to be combined with analysis oflitter quality and various modern analytical tech-
niques such as gas chromatography, mass spectrometry and 13C Nuclear Magnetic
Resonance for the process level understanding of litter decomposition. The litter
bag technique or the litter basket technique could be modified with respect to
surface area, mesh size and amount of litter enclosed depending upon the ecosys-
tem type and experimental manipulations. For analyzing the chemical composi-
tion of litter, plant-part composition, method for drying and fineness of grinding
are important considerations. It is of interest to understand the process of
lignocellulose tranformation while studying litter decomposition. l3C nuclear
magnetic resonance and pulse labelling of plant material using 13C, though ex-
pensive, could be useful for the in situ analysis of decompostion. Laboratory
incubation methods based on respirometric measurements, decomposition of 14C_
labelled materials, measurement of enzymatic activity and nitrogen mineralization
rates can facilitate various experimental manipulations with respect to litter qual-
ity, soil conditions, species diversity and elevated CO 2,
Acknowledgements. Weare especially thankful to Dr. Bhupinder Kaur and Dr.

C.B. Singh for their help and useful discussions.
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Subject Index
Acacia nilotica 155 literature 77-88

senegal 155 moisture content/density 104
acetogenic phase, degradation of high- mushroom compost 81
molecular substances 44 regional production 76
acetotrophe bacteria 45 tannin, condensed 112
acid detergent fibre (ADF) 164, 166 use 77-83
acidification phase, degradation of high- wine 79
molecular substances 44 xyloglucan extraction 80, 82
actinidin, meat tenderiser 123 seed oil 80, 81, 96
thiol protease, kiwifruit 140 vinegar 80
active site determination, actinidin 140 wax 80
adiabatic bomb calorimeter, loading 176 aroma compounds from apple pomace 82
Ailanthus excelsa 155 from kiwifruit 124
Albizia lebbeck 155 ash, % of dried material from apple
amino acid profile, apple pomace pro- pomace 90
tein 98 from kiwifruit 133, 137
acids determination in litter 184, 185 Azadirachta indica 155
ammonia determination in biogas 61-64
in sewage 49-51 Baermann funnels 190
amygdalin in apple seeds 83 Belemnitella americana 194
a-amylase, determination of activity 168, bio-accumulation, dioxins 26
169 bioassays PCDF/PCDD 37
anaerobic effluents treatment 42-45 bioavailable energy, analytical technique
prepurification 69 (apple pomace) 105, 106
brewery effluents 42 biofilters 69-71
purification, brewery sewage 41-73 biogas 45
reactors, sewage treatment 70 composition 71
analytical windows, mass spetrometry conversion 67-71
TCDD/PCDF 36 determination, sewage 59-67
animal feed, apple pomace 78 exploitation 71
anti -sapstain 22 extraction 43
antioxidant analysis 108, 109 from brewery effluents 42
kiwifruit pomace extracts 143 hydrocarbons 59-61
production from apple pomace 83 hydrogen sulphide determination 64-66
antioxidants, kiwifruit 125 mercaptane determination 67
apple juice extraction, mass flow chart 99 from waste treatment, apple pomace 79
manufacture 75-77 biomass nitrogen 199
press aids 85, 98, 10 1 bioreactor, brewery effluent
pips 97 purification 43
pomace 75-113 brewery effluent, composition 41
analysis, goal 83, 84 sewage 41-73
analytical requirements 84 Brix value 75
aroma compounds 82 Buchner pressing 97, 98
buffering capacity 105 bulk density, analytical technique
composition 84-101 (apple pomace) 103
disposal costs 81 butanol production via fermentation from
elemental analysis 101 apple pomace 80
210 Subject Index
14C-C02 evolution rates, decomposition of tree leaves 153-201

measurment 197 degradation of high-molecular sub-
13C NMR, direct characterization of organic stances 44, 45
material 191 denitrification, effluents 2
14C-technique to study the rate of decompo- density agents, effect on decomposi-
sition 193 tion 196
C/N analyzer, papermill sludge 24 Desmodium adscendens 188
calibration solution, composition of PCDDI Desulfomaculum 44
PCDF 31 Desulfurococcus 44
caloric value of biogas 71, 72 detection limits, TCDD/TCDF by mass spec-
Calotropis procera 155 trometry 36
carbohydrate modifying enzymes, kiwifruit dibenzofurans, chlorated, structure 26
analysis 141 dietary fibre, % of dry weight of kiwi-
carbon balance in sewage treatment 68 fruit 128
dissolved organic 1-16 digestion analysis of papermill sludge by
sources, regression relationships 11 ICP-AES 25
carboxymethyl cellulase 166-168 dioxin-containing samples, handling 30
cell wall constituents 161 dioxins, structure 26
in leaf samples, determina- substituted, toxicity 27
tion 161-178 disposal costs, apple pomace 81
cellulose, carbon-13 solid-state NMR dissolved organic carbon (see DOC)
spectrum 11 0, Ill, 113 matter (see DOM)
determination 164-166 DOC 1-17
% of dried material of apple po- acidified 10
mace 94 amounts 12
estimation, flow chart 167 effect of drying 14, 15
characterisation of plant litter 183 extraction, effect of pH 14-16
of pomaces, NMR analysis 144-147 fractionation 8-10
chemical oxygen demand (see COD) measurement 7, 8
shift ranges 192 molecular weight fractions 13, 14
Chlostridium 44 sources 6
chromatographic cleanup procedure PCDDI DOM I,ll
PCDF 32-34 DPPH (2,2-dipheny1-1-picry1hydrazy1) 108
citrus pectin 111 dry combustion 11, 12
cleanup columns 32, 33 matter apple pomace 85, 97
scheme for PCDD/PCDF in papermill kiwifruit 127, 137, 138
sludge 29, 32-34 dystrandept 4
CO 2 measurement, by perfusion appara-
tus 156, 157 Eidgenossisches Department des Inneren
COD 2,11 (EDI) 45,46, 48, 49, 51, 53, 54, 57
Colophospermum mopane 155 elemental analysis,% dried material of kiwi-
colour intensity, lake water 15 fruit 136
contaminants, apple pomace 83 elevated CO 2, decomposition 196
Comus florida 190 energy content, kiwifruit 127-136
CP techniques 191 environmental protection agency
"cross-polarisation" (CP), NMR (EPA) 25
method 109 enzymatic index of carbon quality 198
sequence, kiwifruit pomace 144, 146 enzyme activity measurement 139-142
crude fibre, apple pomace 86 inhibitors, kiwifruit pomace 125
estimation (ADF) 164 ethanol production via fermentation, apple
protein, apple pomace 87, 104 pomace 79
Eucalyptus camaldulensis 155
decomposition of crop residues 189 Euphorbia tirucalli 155
data, models 195 extraction scheme for papermill sludge 28
multistep process of litter break-
down 181 Fagus sylvatica 161
rate of plant material 190 fatty acid composition of apple seed oil 97
Subject Index 211
feed supplements, apple pomace 106 in vitro perfusion method, determination of

fibre degrading enzymes, determina- breakdown rates of leaf litter 154-159
tions 166-17l inductively coupled plasma atomic emission
Ficus religiosa 155 spectrometry (ICP-AES), digestion anal-
field incubation of litter samples 159, 160 ysis 25
filtration, papermill sludge 32 infrared drying, kiwifruit l36, l37
flavans, analysis of apple pomace 107 inorganic carbons (IC) 8
flocculation organic molecules, decrease of integrated decomposition rate for
extractibility 15 leaves 188
Folin-Ciocalteu method, analysis of total interfering substances, phosphorus determi-
polyphenol content 107, 108 nation 52
fuel use, apple pomace 79 international toxicity equivalency factors (I-
furans 26-37 TEF) 27
substituted, toxicity 27 interrupted decoupling, carbon-l3 NMR
furfural from apple pomace 80 spectrum of kiwifruit pomace 145, 146
ions, % of dried material of apple po-
gas analyser with flameionization detec- mace 90-92
tor 60
sampling sonde, hydrocarbons 60, 61 kiwifruit, actinidin thiol protease 140
fJ-glucosidase, activity determination 169, antioxidant analysis 143
170 antioxidants 125
glutamate dehydrogenase (GDH), determi- aroma compounds 124
nation of activity 174, 175 ash,% of dried material l33, l37
glutamic oxalacetate transaminase (GOT), composition 126-l37
measuring activity 173, 174 dietary fibre 128
pyruvic transaminase (GTP) 173, 174 dry matter 127, l37, l38
"grassy" aroma, kiwifruit 141 elemental analysis l36
green colour retention, kiwifruit 124 energy content 127-l36
gross energy determination 175-178 enzyme extraction l39, 140
inhibitors 125
haplothord 5 "grassy" aroma 141
hemicellulose determination 166 green colour retention 124
estimation, flow chart 167 infrared drying l36, l37
heterotrophic soil organisms, litter degrada- liqueur, destilled 124
tion 196 mineral content l33-l36
high protein feedstock from apple po- mushroom compost 125
mace 80 polyphenol component identifica-
resolution gas chromatography/high re- tion 142, 143
solution mass spectrometry (HRGD/ press aids 127
HRMS) 34 production 122
hydrocarbons, in biogas 59-61 reject 121, 123, 126
hydrogen sulphide determination, in bio- vinegar production 123
gas 64-66 vitamin content l32, l33, l37
hydrolysis phase, degradation of high-mo-
lecular substances 44 leaching of pomace 77
hydrophobicity, congeners dioxins 27 leaf collection 159
hydroxylated aromatic compounds decay 153
(HAC) 11 litter decomposition, measure-
ment 161-201
IC reactor 8 leaves, physical properties 182-184
identification PCDD/PCDF after HRGC/ Leco combustion 2, 8
HRMS 34-37 Leucaena laucocephala 155
immobilization/mineralization rates 198 lignin in apple pomace 112
Impinger sampling system for hydrogen sul- determination 164-166
phide determination 65, 66 estimation, flow chart 167
in situ litter decomposition rates, meth- lignocellulose, analysis in litter 185, 186
ods 188-195 transformation 187, 188
212 Subject Index
litter basket technique, decomposition stud- mineralization from decomposing lit-

ies 190, 191 ter 198-200
chemical composition 184 nuclear magnetic resonance (NMR) analy-
decomposition rate 184 sis, method of characterising apple po-
quality 181 mace 109-ll3
litterbag technique 159, 181, 188, 189
study of litter decomposition 159 odour emissions, avoidance in biogas pro-
Lolium multiflorum 185 duction 70
perenne 192, 197 organic nitrogen, determination in sew-
low sewage sludge residue 42 age 48,49
Osyris 155
macro nutrition, papermill sludge 24, 25 outgoing air, bioreactor treatment 43, 44
"magic number", Kjeldahl nitrogen determi- oxido-reductases, analysis of kiwifruit 141
nation 104, 105 oxygen concentration, aquifers 1
"magic-angle spinning" (MAS) NMR meth-
od 109 paper sludge 12, 21
maize strach 11 papermill sludge 22-37
Maringa oleifera 155 use as a soil amendment 38
MAS technique 191 waste water 21-38
mash, apple 75 Patua soil 6, 7, 9, 12, 15
enzymation 76 PCDD/PCDF equivalents, bioassays 37
mass balance in biogas 67-72 toxicity 26, 27, 37
brewery 41 peat water 3, 11
flow components, kiwifruit crop 138 pectin, carbon-l3 solid-state NMR spec-
"master jar" system 196 trum 1l0, lll, ll3
Medicago littoralis 193 extraction 77
medisaprists 4 Pee Dee belemnite, CO 2 reference 194
"memory effects" during destillation 200 Peptococcus 44
mercaptanes determination in biogas 67 perfusion method, study of organic matter
mesh cylinders, study of decomposi- breakdown 154-159
tion 189 solution bag 156
Methanobacterium 44 pesticide residues, groundwater 2
Methanobrevibacter 44 petroleum gas, composition 71
Methanococcus 44 Phacelia 194, 195
methanogene bacteria, anaerobic sewage phosphorus balance in sewage treat-
treatment 44 ment 69
methanogenic phase, degradation of high- determination in brewery
molecular substances 45 sewage 51-53
Methanothermus 44 Pithecellobium dulce 155
method performance tests, waste water resi- plant litter quality for decomposition, meth-
duals 29,30 ods for characterization 183
mineral content, kiwifruit l33-l36 sampling 182
moisture content/density, apple po- nutrient analysis, litter 187
mace 104 plenar halogenated hydrocarbons
molecular size fractions, DOC 14 (PHH) 37
Muencke washing flask sampling system, polychlorinated dibenzo-p-dioxin
ammonia determination 62 (PCDD) 22, 23, 26-37
mushroom compost 12 bibenzofurans (PCDF) 22, 23, 26-28,
from apple pomace 81 31
from kiwifruit 125 polyphenol components, apple
skin 106-108
"native" spike congeners, PCDD/PCDF 30 identification in kiwifruit 142, 143
neutral detergent fibres (NDF) 161, 163, polyphenols, HPLC method 107
166 in litter, methods for extraction and
solution (NDS) 162 analysis 186, 187
nitro blue tetrazolium (NBT) 109 pomace of apple 75-1l3
nitrogen balance in sewage treatment 68 liquefaction 76
Subject Index 213
postaeration tank 44 starch, carbon-l3 solid-state NMR spec-

press aids, apple juice 85, 98, 101 trum 111
kiwifruit 127 stream water 4, 6, 11, 12
proportion factor, organically bound carbon sugar content,% of wet material of apple po-
atoms in biogas 59, 60 mace 93
Prosopis cineraria 155 kiwifruit l30-l32, l37
juliflora 155 determination in litter 184, 185
proteases, determination of activities 172, sulphate/sulphite/sulphide determination in
173 sewage 53-59
protein, % of dried material in kiwi- sulphur balance in sewage treatment 69, 70
fruit 128, l37 superoxide dismutase (SOD), method for
protein-degrading enzymes, determina- antioxidant analysis 109
tions 17l-175 "surrogate" PCDD/PCDF congeners 30, 31
Pueraria phaseoloides 188
pulp, paper industry 21 tagging organic detritus 193
pulping process 22, 23 by tracer technique 160, 161
Pyrodictium 44 tamarind seed xyloglucan 82
tannin, condensed, in apple pomace 112
recovery standards, PCDD/PCDF calibration TCDD 22,26,27,32,34-36
solutions 31 equivalents 38
reject kiwifruit 121, 123, 126 isomer, separation 34, 35
resource quality of litter, characteriza- Tecomella undulata 155
tion 182-187 2,3,7,8-tetrachlorinated dibenzo-p-dioxin
respirometric techniques, decomposition (see TCDD)
analysis 196, 197 thermal exploitation biogas 7l
rotary vacuum filter, production of kiwifruit Thermococcus 44
pomace 122 Thermodiscus 44
Thermoproteus 44
safety, exposure to PCDD/PCDF conge- TOC 2, 8, 11, 47
ners 28 analyser 8
Salvadora persica 155 determination in brewery
sample preparation, process residues of pa- sewage 46-49
permill 30-32 Tokomaru soil 12
size, pulp mill effluent 32 total carbon (TC) 8
sampling, papermill sludge 23, 24 determination in sewage 46-49
Sarcina 44 organic carbon ( see TOC)
sclerophyll index 183 trace elements, papermill sludge 25
sclerophylly 182 technique, tagging organic detri-
secondary fibres 22 tus 160, 161
sewage prepurification plant, flow sheet 43 transaminases, activities 173-175
sampling 45, 46 tree leaf decomposition 153-201
sludge 3, 6, 9, 11, 15 trees in arid soils 155
analysis 72 trihalomethans 2
Simmondsia chinensis 155 Tullgren funnels 190
single cell protein (SCP) from apple po-
mace 81 UNE perfusion apparatus 155-158
pulse excitation sequence (SPE), carbon- urease activity 17l, 172
l3 NMR spectrum of kiwifruit po-
mace 145, 146 vinegar production, kiwifruit 123
sludge, sample size 32 vitamin content, kiwifruit l32, l33, l37
slurry 5 volcanic ash soils 3
soil microbial biomass nitrogen 200
respiration, measurement 161 waste, analysis 121-147
solubilization, soil organic matter wine 124
sorption chromatography, DOC 9 waste water, brewery 41
stable isotope technique (13 C, 15 N), study of water content, analytical methods of apple
decomposition 193-195 pomace 102
214 Subject Index
drying techniques 104 xeropsamment 4

equivalent, determiation 177 xylanase, enzyme determination 169
wax/cutin, identification by NMR III xyloglucan/cellulose interaction 113
wet mineralization, phosphorus in sew- extraction from apple pomace 80, 82
age 51, 52 p-xylosidase, activity determination 171
oxydation 7, 12, 13
wine from apple pomace 79 Ziziphus nummularia 155
kiwifruit 124

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Modern Methods of Plant Analysis

Springer-Verlag Berlin Heidelberg GmbH

P.J.S. Bain S. Baskaran N.S. Bolan M.S. Erich G. Fenton

Professor Dr. JOHN F. JACKSON

ISBN 978-3-642-08431-7 ISBN 978-3-662-03887-1 (eBook)

Springer-Verlag Berlin Heidelberg 1999

Typesetting: Best-set Typesetter Ud., Hong Kong

Modern Methods of Plant Analysis

1. the dependence of scientific progress in biology on the improvement of existing

Concept of the Series

Many methods described in the biochemical, biophysical and medical literature

Naturally, these three approaches to developments in analytical techniques for

Volume Twenty - Analysis of Plant Waste Materials

Adelaide and Nijmegen/Siena, Northern Spring 1999. H.F. LINSKENS

Methods of Measurement of Dissolved Organic Carbon of Plant Origin

Analysis of Papermill Waste Water Treatment Residuals

Analysis of Sewage from Anaerobic Purification of Effluent

Apple Pomace and Products Derived from Apple Pomace:

Kiwifruit Waste and Novel Products Made from Kiwifruit Waste:

1 Introduction .................................................. 121

Analysis of Tree Leaf Decomposition in Arid Soils

4.8 Determination of Gross Energy (GE) .......................... 175

Measurement of Leaf Litter Decomposition

Subject Index ................................................... 209

BASKARAN, S., Research Fellow, RC for Sustainable Cotton Production, Department

BOLAN, N.S., Massey University, Palmerston North, New Zealand

GUPTA, S.R., Department of Botany, Kurukshetra University, Kurukshetra 136119,

MALIK, V., Department of Botany, Kurukshetra University, Kurukshetra 136119,

Plant Analysis (1996) 27:2723-2737.

Modern Methods of Plant Analysis, Vol. 20

induces the reduction of nitrate (denitrification) in soils and effluents, resulting in

liquid/solid); back titration .,III

Peat soil (typic medisaprists) Extraction with By the Oceanography 6

straw, soil drainage centrifugation Analyser DC-80) ''"c"

2 Materials and Methods

2.1 Soils, Sludges and Stream Water

A number of environmental samples in which the dissolved organic carbon origi-

2.2 Extraction of DOC

Table 2. Characteristics of DOC sources

DOC sources pH Organic carbon CEC Total N Total P

Tokomaru soil 5.56 32 20 5.1 1.2

LSD (p = 0.05) 0.35 35 18 3.2 4.5

2.4 Effect of Drying

2.5 Measurement of DOC

2.5.1 Spectrophotometric Method

2.5.2 Wet Oxidation

2.5.3 Dry Combustion

Leco Combustion. Leco combustion was originally developed to analyse carbon

2.6 Fractionation of DOC

2.6.1 Molecular Weight Fractions

2.6.2 Sorption Chromatography

Amberlite +-- Hydrophobic acids

Duolite A-7 +-- Hydrophilic acids

3 Results and Discussion

3.1 Methods of Measurement of DOC

3.1.1 Spectrophotometric Method

Absorbance oflight by DOC decreased with increasing wavelengths. Absorbance at

DOC samples Regression equations' Wave Cell Reference

Lake water DOM = 1O.7A - 0.98 260 10 Banoub (1973)

bance, independent of DOC concentration (Moore 1987). The spectrophotometric

3.1.2 Dry Combustion

Water 0.lMK,S04 Leco TOC analyser

Tokomaru soil 0.56 0.42 1.02 0.98 55 3.18

LSD (p = 0.05) 0.25 0.38 0.39 0.21