Novel Uses of Lignin and Hemicellulosic Sugars From Acidhydrolysed

NOVEL USES OF LIGNIN AND HEMICELLULOSIC SUGARS FROM ACIDHYDROLYSED LIGNOCELLULOSIC MATERIALS
by
Mojtaba Zahedifar
(B.Sc., Animal Husbandry - University of Tehran)

(M.Sc., Animal Nutrition - University of Gdll)
Thesis submitted for the degree of Doctor of Philosophy

in the University of Aberdeen
September 1996
DECLARATION
I hereby declare that this thesis has been composed by myself and has not been presented
or accepted in any previous application for a degree. The work has been done by myself
and all help given has been acknowledged and sources of information has been specially
acknowledged by means of references.
Mojtaba Zahedifar
September 1996
DEDICATION
To my wife
ABBREVIATIONS
ADF
BSA
CD
CrI
DM
EA
EAEP
ETOH
FSG
GLC
H
HMF
HPLC
HT
LM
MW
N
NDF
PEG
PVP
RT
SUG
TEP
TT
VFA
WIP
WSE
acid detergent fibre

bovine serum albumin
carpinus duinensis
crystallinity index
dry matter
ethyl acetate
ethyl acetate extractable phenolics
ethanol
functional specific gravity
gas liquid chromatography
hydrogen
hydroxymethyl furfural
high performance liquid chromatography
heating up time
lignocellulosic materials
molecular weight
nitrogen
neutral detergent fibre
polyethylene glycol
polyvinylpolypyrrolidone
reaction time
sugar
total extractable phenolics
total tannins
volatile fatty acid
water insoluble phenolics
water soluble extract
ACKNOWLEDGMENT
I am extremely grateful to my supervisor Dr. E.R. rskov at The Rowett

Research Institute for his continuous encouragement, comments and advice during my
studies at the Rowett. I should also thank my university supervisor Dr. H. Galbraith for
his valuable comments during the course of this study.
I would like to express my gratitude to Dr. F.B. Castro for his invaluable
comments on my studies and on the manuscript of this thesis.
I am grateful to Dr. R.N.B. Kay for his useful comments on the manuscript of this
thesis.
I would like to thank the Director of The Rowett Research Institute, Professor
W.P.T. James for the provision of the facilities at the Rowett and through him all the
Rowett Research Institute who have helped me in one way or another.
My very appreciation to Drs R. Begbie, G.J. Proven, J.A. Lomax and T.M. Wood
for their valuable comments and discussion during the course of this work.
I am grateful to all the staff from the International Feed Resources Unit with
special reference to Mr D.J. Kyle, Mr W.J. Shand and Mrs A. Marsden, who have
always been helpful and kind. I must also thank Dr X.B. Chen for his valuable
comments throughout this study.
Many thanks to the members of the Analytical Department of The Rowett
Research Institute specially Mr P.J.S. Dewey, Mr D.S. Brown and Miss M.G. Annand
who have helped me in the chemical analysis.
My great appreciation to the Ministry of Jihad Sazandegi and the Ministry of

Culture and Higher Education of The Islamic Republic of Iran for their financial support
throughout this study.
Finally, I wish to express my sincere gratitude to my wife and my children
Mohsen, Hamed and Sara for their constant encouragement and love.
SUMMARY
Lignocellulosic materials (LM) are an ever present renewable and available

energy source. The energy stored by photosynthesis in the form of vegetation is about
ten times more than world's annual energy consumption (Zsuffa, 1982). This source is
the only alternative for chemical production after fossil fuels.
Formation of organic acids (mainly acetic acid) from hemicellulose during steam
treatment of LM leads to acid hydrolysis of cell wall components. Solubilization of
hemicellulose and depolymerization of lignin are the most important changes that occur
during the process.
During hydrolysis of LM appreciable amounts of sugar degradation products,
organic acids and phenolics are produced. Inhibitory effects of the compounds on yeast
during alcoholic fermentation have been reported and several methods have been
proposed to overcome the problem. Among the new compounds phenolics derived from
lignin depolymerization have received most attention. Another problem during enzymic
saccharification of cellulose is partial inactivation of cell free enzymes.
The above mentioned constraints were investigated in this study.
Dry matter (DM) loss especially under harsh treatment conditions may be
regarded as a form of nutrient loss. Results of this study showed that greater nutrient loss
could occur through the formation of indigestible soluble and insoluble browning
reaction compounds. In vitro digestion of water soluble extract showed that only
carbohydrates in this fraction were utilized by rumen microbes. The water soluble
extract from steam-treated LM has been used for some biological purposes due to being
rich in soluble carbohydrates. This fraction can also be used as animal feed for both
ruminants and those monogastric animals which can ferment them in the hind gut to
produce volatile fatty acids (VFA). The highest sugar content (57%) of water soluble
fraction was obtained at 19 bar pressure and 0 min reaction time. At this treatment
condition only about 8% of soluble carbohydrates were in the form of monomeric
pentoses. Increase in harshness (pressure, reaction time and/or exogenous acid) of
treatment conditions negatively affected carbohydrate content of water extract.
Concentration of browning compounds in steam-treated LM is rather high if steam
treatment exceeds optimum conditions. The nitrogen present in steam-treated LM is in
the form of Maillard products. These compounds should be considered as nutrient loss
as they are also indigestible.
The antinutritive materials produced during steam treatment of LM are classified
mainly as lignin-based phenolics and browning reaction compounds.
The study of the inhibitory effect of browning compounds showed that these
compounds were not toxic to rumen microbes. Furfural and hydroxymethyl furfural with
known toxic effect on yeasts, did not affect rumen microbes. Production of these
compounds should be considered only as nutrient loss.
The detailed study of the effect of lignin-based phenolics on rumen microbes under different conditions showed that neither low molecular weight nor high molecular
weight phenolic derived from lignin depolymerization affected ruminal fermentation.
The concentrations of tannin-like compounds in steam-treated wheat straw were too low
(maximum 0.97% as tannic acid equivalent) to affect rumen microbes. Measurement of

the total monomeric phenolic compounds with a known negative effect on rumen
microbes also showed that their concentrations were far too low to affect microbial
activity. Alkali-extractable lignin did not show any effect on rumen microbes either. In
general it was shown in this study that no material toxic to rumen microbes is produced
during steam treatment of wheat straw.
Steam treatment has been used as an approach to increase enzymic hydrolysis of
cell wall polysaccharides. The advantages of steam treatment in this respect arise from
physical (cell wall swelling and reduction in particle size) and chemical (hemicellulose
and lignin depolymerization) changes which increase the availability of cell wall
polysaccharides to enzymic attack. Reduction in enzymic activity of cell-free enzymes
during saccharification of steam-treated LM has been considered as a constraint as more
enzymes have to be used. Measurement of the enzymic activity of xylanase and cellulase
mixed with lignin extracted from steam-treated wheat straw showed that lignin was
responsible for inactivation of enzymes. Both tannin and lignin were more potent in
precipitating protein (bovine serum albumin - BSA) than in deactivating enzymes.
A study of the lignins extracted from different LM sources (wheat straw, barley
straw, rice straw and sugar cane bagasse) showed that both treatment conditions
(pressure, reaction time and exogenous acid) and LM source affected the proteinprecipitating capacity of lignin. Very harsh treatment conditions (using high pressure
and a long reaction time in presence of exogenous acid) did not improve lignin quality.
Autohydrolysis at 19 bar pressure and 0 min reaction time was found to be more suitable
for lignin production.

In vitro degradation of protein mixed with lignin showed that lignin, like tannin,
was able to protect the protein from ruminal fermentation. It was also shown that the
protein adsorbed to lignin was released at low pH ( 2.2). The similarities between
tannin and lignin suggest the presence of hydrogen bonds and hydrophobic interactions
between lignin and protein since both types of the reactions are responsible for
precipitation of proteins by tannins. The results suggest that, like tannins, lignin could be
used as a protein protective material. In this respect lignin may also be used for the
chemical protection of proteins. An advantage of lignin is that it does not affect normal
ruminal fermentation. However, its low activity compared to tannins is a drawback.
More research is needed to increase the protein adsorbing capacity of lignin in order to
decrease lignin:protein ratio for agricultural and industrial purposes.
TABLE OF CONTENTS:
Page No.
CHAPTER 1 - LITERATURE REVIEW

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Plant cell wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3 Cell wall components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3.1 polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3.1.1 cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3.1.2 hemicellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3.1.3 pectic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.3.2 proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.3.3 phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.3.3.1 lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.3.3.2 tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1.3.3.3 phenolic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1.4 Acidolysis reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.4.1 cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.4.3 hemicellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.4.4 lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
1.5 Protein precipitating capacity of phenolic compounds . . . . . . . . . . . . . . . 27
1.6 The effect of steam treatment-associated toxic compounds on
rumen microbial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
1.6.1 furfural and 5-hydroxymethyl furfural . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
CHAPTER 2 - CHARACTERIZATION OF WATER-SOLUBLE EXTRACT

FROM STEAM-TREATED WHEAT STRAW
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Page No.
2.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

2.2.2 steam treatment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.2.3 extraction of water soluble extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.2.4 statistica analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38
2.2.5 total neutral sugar analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.2.6 dry matter loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.2.7 ash content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.2.8 nitrogen (N) content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.2.9 total extractable phenolics analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.2.10 in vitro gas production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
2.2.11 browning reaction products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.3.1 steam treatment procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.3.2 soluble carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2.3.3 nutritive value of water soluble extract . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
CHAPTER 3 - IDENTIFICATION OF INHIBITORY COMPOUNDS

PRODUCED DURING STEAM TREATMENT OF LIGNOCELLULOSICS
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.2 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.2.1 substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.2.3 extraction of water soluble extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.2.4 effect of addition of PVP on microbial fermentation . . . . . . . . . . . . . . . . . 63
3.2.5 extraction of phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.2.5.1 ethyl acetate extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.2.5.2 extraction of water insoluble phenolics . . . . . . . . . . . . . . . . . . . . . . . . 65
3.2.6 effect of extraction procedure by ethyl acetate on

nutritive value of feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.2.7 HPLC analysis of phenolic compounds of steam-treated wheat
straw samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Page No.
3.2.8 effect of ethyl acetate extractable phenolics and water
insoluble phenolics on rumen microbes . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.2.9 extraction of phenolic compounds using ethanol solutions . . . . . . . . . . . . 67
3.2.10 effect of EAEP and WIP at high substrate loading on
microbial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.2.11 statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.2.12 optimum level of substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.2.13 effect of furfural and HMF on rumen microbes . . . . . . . . . . . . . . . . . . . . . 69
3.2.14 extractable phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.2.15 total tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.3.1 effect of PVP on microbial fermentation . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.3.2 effect of EAEP and WIP on rumen microbes . . . . . . . . . . . . . . . . . . . . . . 74
3.3.3 effect of phenolic compounds from steam-treated wheat
straw on rumen microbes at high substrate loading . . . . . . . . . . . . . . . . . . 81
3.3.4 effect of ethanol extract from steam-treated
wheat straw on rumen microbial activity . . . . . . . . . . . . . . . . . . . . . . . . 83
3.3.5 effect of furfural and HMF on ruminal fermentation . . . . . . . . . . . . . . . . . . 84
3.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
CHAPTER 4 - PROTEIN PRECIPITATING CAPACITY OF LIGNIN

EXTRACTED FROM STEAM TREATED LIGNOCELLULOSICS
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
4.2.1 substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4.2.3 lignin extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4.2.4 enzyme assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
4.2.4.1 effect of lignin and tannin on xylanase inactivation . . . . . . . . . . . . . . . . . 94
4.2.4.2 enzymic inhibition by steam-treated lignin using

gas production technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4.2.5 effect of lignin extracted from steam-treated
wheat starw on rumen microbial activity . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.2.6 statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Page No.
4.2.7 protein precipitating capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.2.6 protein analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
4.2.7 reducing sugar analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.3.1 inhibition of enzymes by lignin extracted from
steam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
CHAPTER 5 - EFFECT OF TREATMENT CONDITIONS AND SOURCE OF

LIGNOCELLULOSIC MATERIALS ON PROTEIN PRECIPITATING
CAPACITY OF LIGNIN
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
5.2.1 substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 5.2.2
steam treatment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
5.2.3 statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .111
5.2.4 effect of hemicellulose on lignin quality . . . . . . . . . . . . . . . . . . . . . . . . . 112
5.2.5 lignin extraction and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
5.2.7 protein analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
5.2.8 water soluble fraction of lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
5.2.9 total phenolic content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
5.2.10 ash content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
5.2.11 nitrogen content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
5.2.12 total sugar content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5.3.1 optimization of steam treatment conditions for lignin

production from wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
5.3.2 lignin quality from barley straw, rice straw
and sugar cane bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
5.3.3 lignin properties from different agricultural by-products . . . . . . . . . . . . . . . 120
5.3.4 effect of hemicellulose removal on lignin quality . . . . . . . . . . . . . . . . . . . 124
Page No.
5.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
CHAPTER 6 - THE USE OF LIGNIN TO PROTECT PROTEIN FROM

MICROBIAL DEGRADATION IN THE RUMEN
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
6.2.1 substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
6.2.3 extraction and purification of lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
6.2.4 In vitro protein and amino acid degradation . . . . . . . . . . . . . . . . . . . . . . . 129
6.2.5 NH3 analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
6.2.6 VFA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
6.2.7 statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .131
6.2.8 protein analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
6.2.9 adsorption of amino acids by lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
6.2.10 amino acid analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
6.2.11 release of adsorbed protein from
polyphenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
6.2.12 specific gravity of lignin-protein complex . . . . . . . . . . . . . . . . . . . . . . . . 133
6.3.1 protection of protein by lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
6.3.2 adsorption of amino acids by lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
6.3.3 effect of lignin on deamination activity . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
6.3.4 postruminal availability of protein adsorbed by lignin . . . . . . . . . . . . . . . . 138
6.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
CHAPTER 7 - GENERAL DISCUSSION AND CONCLUSIONS

7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143
7.2 Constraints of steam treatment of lignocellulosic materials . . . . . . . . . . . . 144
7.2.1 nutrient loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .144
7.2.2 identification of compounds toxic to rumen microbes in
steam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Page No.
7.3 Biological activity of lignin extracted from steam-treated

lignocellulosic materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
APPENDIXES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
LIST OF FIGURES
Figure No.
Title
Page No.
1.1.1
Model of arrangement of components in wood cell wall

(lignified) (Ker and Goring, 1975) . . . . . . . . . . . . . . . . . . . . . . 2
1.1.2
Relationship between lignin content and digestibility in rye

grass (data from Hartley, 1972) . . . . . . . . . . . . . . . . . . . . . . . . . .2
1.2.1
Illustration of primary and secondary cell walls

(Timell, 1964) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3.1
Distribution of cell wall polysaccharides (Worth, 1967) . . . . . . . . .6
1.3.1.1.1
Proposed arrangement of D-glucan chains within

cellulose microfibrills of primary cell walls of dicots
(Dey andBrinson, 1984) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
1.3.1.1.2
Diagrammatic representation of cellulose microfibrillar

structure (Hess et al., 1975) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.1.2.1
Hypothetical structure of plant xylan

(Puls and Poutanen, 1989). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3.3.1.1
Structure of three precursors of lignin

(Sarkanen and Ludwic, 1971) . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.3.1.2
Proposed lignin structure in softwoods (Nimz, 1974) . . . . . . . . . . 14
1.4.1.1
Production of furfural from D-xylose

(Tipson and Horton, 1988) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
1.4.1.2
Production of 5-hydroxymethyl-2-furaldehyde from

D-glucose (Tipson and Horton, 1988) . . . . . . . . . . . . . . . . . . . . . 21
1.4.4.1
The susceptible bounds (-aryl and -O-4) in lignin

molecule for cleavage under acidolysis reaction . . . . . . . . . . . . . . 24
1.4.4.2
Effect of reaction time on depolymerization/repolymerization

of lignin (Wayman and Chua, 1979) . . . . . . . . . . . . . . . . . . . . . .26
Page No.
2.2.2.1a
The High Pressure Steam Treatment Vessel in operation . . . . . . .36
2.2.2.1b
Components of the High Pressure Vessel . . . . . . . . . . . . . . . . . .36
2.2.2.1c
Putting the sample in the beaker (c) and transferring

into the vessel (d) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37
2.3.2.1
Effect of pressure and reaction time on soluble

carbohydrate (sCHO) content of the water soluble
extract from steam-treated wheat straw . . . . . . . . . . . . . . . . . . . 49
2.3.3.1
Nutritive value of the water soluble extract from

steam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.2.5.1
Extraction procedure of phenolic compounds (EAEP and WIP)

from steam-treated wheat straw and Carpinus duinensis
by ethyl acetate and acetone solution. . . . . . . . . . . . . . . . . . . . . 64
3.2.9.1
Extraction procedure by ethanol solution from

acid-hydrolysed (19 bar pressure and 10 min reaction time)
wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68
3.3.2.1
Efficiency of extraction by ethyl acetate of phenolic

compounds from water soluble extract of
steam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
3.3.2.2
Effect of ethyl acetate extractable phenolics and

and water insoluble phenolics extracted from C.
duinensis on rumen microbial activity . . . . . . . . . . . . . . . . . . . .78
3.3.3.1
Effect of substrate (rye grass) loading on pH . . . . . . . . . . . . . . . 82
4.2.3
Extraction procedure of lignin from steam-treated

(19 bar pressure and 0 min reaction time) wheat straw . . . . . . . . .93
4.3.1.1
lignin effect on cellulase activity . . . . . . . . . . . . . . . . . . . . . . . 101
4.3.2.1Effect of lignin on the Bradford method for determining
protein adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103

4.3.2.3
Protein (BSA) adsorbing capacity of 1 g of the three

phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Page No.
5.3.1.1
Effect of steam treatment conditions on protein

precipitating capacity of lignin extracted from
auto-hydrolysed wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5.3.1.2
Effect of steam treatment conditions on protein

precipitating capacity of lignin extracted from
acid-hydrolysed wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . 116
6.3.1.1
Effect of levels of lignin extracted from steam-treated

wheat straw on degradation of casein by rumen microbes
as indicated by production of NH3. . . . . . . . . . . . . . . . . . . . . . .135
6.3.3.1
Effect of lignin extracted from steam-treated

(19 bar pressure and 5 min reaction time) wheat straw
on deamination activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .139
6.3.4.1
Effect of pH on releasing of protein from

tannin (tannic acid) and lignin . . . . . . . . . . . . . . . . . . . . . . . . . 140
7.2.1
Physical and chemical changes of LM during steam treatment . . .145
7.3.1
Method for reducing protein degradation in the rumen . . . . . . . . .155
LIST OF TABLES
Table No.
Title
Page No.
2.2.7.1
Composition of macro-mineral, micro-mineral and buffer

solutions used for preparing artificial saliva . . . . . . . . . . . . . . . . 42
2.3.1.1
Effect of sample DM content on chemical composition of

steam-treated wheat straw at 19 bar and 0 min reaction time . . . . 45
2.3.1.2
Sugar, phenolics contents and DM loss of wheat straw by

steam treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.3.2.1
Neutral sugar composition of total and monomeric

carbohydrates in water soluble extract from steam-treated
wheat straw at different conditions . . . . . . . . . . . . . . . . . . . . . . .48
2.3.3.1
Chemical analysis of water soluble extract from

steam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
3.2.2.1
Steam treatment conditions applied to wheat straw . . . . . . . . . . . .62
3.3.1.1
Effect of steam treatment and addition of PVP on

the in vitro gas production after 12 and 24 h incubation . . . . . . . . 72
3.3.1.2
Total extractable phenolic and total tannin content from

auto-hydrolysed and acid-hydrolysed wheat straw . . . . . . . . . . . . .73
3.3.2.1
Effect of ethyl acetate extraction procedure on

sugar fermentation by rumen microbes . . . . . . . . . . . . . . . . . . . . 75
3.3.2.2
Extractable phenolic, tannin and soluble carbohydrates

data of the ethyl acetate extractable phenolics and water
insoluble phenolics from steam-treated wheat straw and
Carpinus duinensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
3.3.2.3
Effect of ethyl acetate extractable phenolics and water

insoluble phenolics from steam-treated wheat straw and
Carpinus duinensis on in vitro gas production . . . . . . . . . . . . . . . 79
3.3.2.4
Concentration of monomeric phenolic compounds in

steam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Page No.
3.3.3.1
Effect of ethyl acetate extractable phenolics at high

substrate loading on rumen microbes . . . . . . . . . . . . . . . . . . . . . . . 82
3.3.4.1
Effect of supernatants and precipitates obtained by ethanol

solutions on in vitro gas production from rye grass . . . . . . . . . . . . .85
3.3.4.2.
Amount of dry matter extracted (supernatants and precipitates)

from 380 g acid-hydrolysed wheat straw by ethanol extraction . . . . .85
3.3.5.1
Production of furfural and hydroxymethyl furfural from

steam-treated wheat straw under different treatment conditions . . . . . 86
3.3.5.2
Effects of furfural and hydroxymethyl furfural on

gas production in vitro from rye grass . . . . . . . . . . . . . . . . . . . . . .87
4.3.1.1
Effect of lignin and tannin on xylanase activity . . . . . . . . . . . . . . .100
4.3.1.2
Effect of different levels of lignin on rumen

microbial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .101
5.3.2.1
Protein precipitating capacity of lignin extracted from

auto-hydrolysed wheat straw, barley straw, rice straw and
sugar canebagasse at different treatment conditions . . . . . . . . . . . . . 119
5.3.3.1
Characteristics of lignin extracted from auto-hydrolysed

and acid-hydrolysed wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . 121
5.3.3.2
Characteristics of lignin samples from barley straw,

rice straw and sugar cane bagasse . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.3.4.1
Protein precipitating capacity of lignin and quantity of

lignin extraction from acid detergent fibre of wheat straw . . . . . . . . 125
6.3.1.1
Effect of levels of lignin extracted from steam-treated

wheat straw on iso-acid production. . . . . . . . . . . . . . . . . . . . . . . . 135
6.3.1.2
Functional specific gravity of lignin-protein complexes . . . . . . . . . . 137
7.3.1
Lignosulphonate sale in Europe, 1986

(Wayman and Parekh, 1992) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
CHAPTER 1
LITERATURE REVIEW
1.1 Introduction
Cellulose, hemicellulose and lignin are the main organic compounds which make
up the biomass of trees and agricultural by-products (lignocellulosic materials - LM).
LM are the most important renewable resources of the terrestrial ecosystem and have
been used for many biological and industrial purposes. From the nutritional point of
view, agricultural by-products such as cereal straws can provide only a fraction of the
daily requirement of energy by ruminant animals. The main constraint for using LM as
an animal food is its low nutritive value. Cell wall barriers observed in such materials
negatively affect the bio-degradation of both cellulose and hemicellulose.
In the mature plant cell wall polysaccharides are encrusted with lignin (Fig 1.1.1)
thereby limiting the access of enzymes (Baker, 1973) and rumen microbes (Hartley,
1972) (Fig 1.1.2) into the cell wall matrix and limiting its degradation. Another
important factor is the presence of lignin-carbohydrate complexes in the cell wall which
inhibit enzymic activity (Morrison, 1979; Neilson and Richards, 1982). Unavailability of
carbohydrates in lignin-carbohydrate complexes to rumen microorganisms has also been
reported (Wallace et al., 1991).
A possible solution to overcome such barriers is to subject the LM to specific
treatments, e.g. chemical (Chandra and Jackson, 1971), physical (Morrison, 1983) or
Fig 1.1.1 Model of arrangement of components in wood cell wall (lignified) (Ker and
Goring, 1975).
Fig 1.1.2 Relationship between lignin content and digestibility in rye grass (data from
Hartley, 1972).
biological (Agosin et al., 1986; Akin et al., 1993).

Amongst the treatments that have been studied over the years steam treatment has
shown promise method for upgrading LM bio-availability. The need for specific
equipments and the high cost of setting up the processing unit have been important
constraints for its application on a farm scale. Nevertheless, steam treatment has been
widely used in sugar refineries in Brazil (Wayman and Parekh, 1990) as a method to
upgrade sugar cane bagasse for animal feed.
Steam treatment affects cell wall structure as a result of an acid-hydrolysis type of
reaction (Baugh and McCarty, 1988). Production of organic acids from acetyl and
formyl groups present in hemicellulose leads to acidolysis of cell wall components.
Solubilization of hemicellulose and depolymerization of lignin are the main results of
this reaction. The water soluble fraction which contains easily fermentable carbohydrates
(hemicellulosic sugars) could be used for many biological purposes such as alcohol and
single cell protein production or as an animal feed. Production of chemicals such as
furfural and 5-hydroxymethyl furfural is an alternative use for steam treatment (Sproul et
al., 1985). The solid fraction, rich in cellulose, can be used as a raw material for the pulp
industry, for alcohol production or as a ruminant feed.
Although steam treatment is an effective method for upgrading LM, production
of inhibitory compounds are still considered to be an important constraint. Such
compounds restrict enzymic hydrolysis as well as fermentative batch processes (MesHartree and Saddler, 1983; Sutcliffe and Saddler, 1986). Inhibition of rumen microbial
activity has also been reported in steam-treated lignocellulosics (Britton, 1978; Castro et
al., 1995). There is no detailed report of the inhibitory compounds formed during steam
treatment of LM.
Depression of enzymic activity during hydrolysis of steam-treated LM with cell
free enzymes have also been reported (Sinitsyn et al., 1982). One possible way in which
free enzymes are inactivated is through binding to phenolic compounds. It should be
mentioned that extractable phenolic compounds detected in steam-treated LM are
formed during lignin depolymerization. It is worth noting that natural phenolic
compounds, e.g. tannins are capable of complexing with proteins and protecting them
from rumen microbial degradation.
All the effects related to inhibitory compounds formed during steam treatment
have been described as negative from the point of view of enzymic hydrolysis and
fermentation processes. Such properties can however be used to control certain
biological processes; e.g., extent of rumen protein degradation. To do so, it is essential to
identify such compounds and measure their activities in specific processes.
In the present study two insight were obtained concerning the effects of steam
treatment upon the properties of both lignin and hemicellulosic sugars. First a negative
effect which normally is detrimental to the utilization of food and second a beneficial
effect in which such products of steam treatment can be used to manipulate fermentation
and protein degradation.
1.2 Plant cell wall

Plant cell walls consist of primary and secondary cell walls (Fig 1.2.1). Primary
walls are deposited at early stages of growth while the cells are expanding (Fry, 1987).
The middle lamella which forms a common boundary layer between adjacent cells
occupies the site of the cell plate (Timell, 1964; Fry, 1987). Contiguous cells are bound
together by deposition of lignin in the middle lamella (Ornston et al., 1987).
Secondary cell walls are formed at later stages either lignified or unlignified.
Unlignified walls have a high tensile strength and stiffness mainly due to the mechanical
properties of the cellulose microfibrils (Northcote, 1972). Lignification results in a rigid
cell wall with high compressive strength and low porosity. The highest concentration of
lignin is observed in the middle lamella and the primary wall (Saka and Goering, 1985).
The secondary wall contains most of the cell lignin as it forms the greatest volume of
cell wall (Harris, 1990).
1.3 Cell wall components

Plant cell wall is a layer of structural material involving the protoplast which can
be 0.1-10 m thick and composed of polysaccharides (cellulose, hemicellulose and
pectin) and lignin (Fry, 1987). The presence of polysaccharides in different parts of the
cell wall is illustrated in Fig 1.3.1. Cell wall contains a small amount of glycoproteins
(Cassab and Varner, 1988).
Fig 1.2.1 Illustration of primary and secondary cell walls (Timell, 1964)
Fig 1.3.1 Distribution of cell wall polysaccharides (Worth, 1967)
1.3.1 polysaccharides
Classically, cell wall polysaccharides have been grouped into three fractions. a)
Cellulose: the most resistant to chemical disruption. b) Hemicellulose: extracted by
relatively strong alkali solution or mild acid hydrolysis; and c) Pectic polysaccharides:
extracted by hot water, ammonium oxalate solution, weak acids or chelating agents:
(Dey and Brinson, 1984; Chesson and Forsberg, 1988).
1.3.1.1 cellulose. Cellulose is the most abundant cell wall polysaccharide in nature and
consists of long chains of -1,4 linked glucose residues. The chains are held together by
hydrogen bonds between oxygen of alternating glycosidic bond in one glucan chain and
the primary hydroxyl groups at position 6 of glycosyl residues in another chain
(Wolfgang et al., 1973) to form thin, flattened, rod-like structures that are referred to as
microfibrils (Fig 1.3.1.1.1) (Dey and Brinson, 1984).
The cellulose microfibrils are bound to each other and to hemicellulose polymers
by hydrogen bonding (Valnet and Albersheim, 1974) but there is no evidence of
covalent linkage between cellulose and other cell wall constituents (Morrison, 1979).
Cellulose microfibrils contains regions with highly oriented molecules or less
oriented microfibrils called crystalline and amorphous regions respectively (Cowling and
Kirk, 1976) (Fig 1.3.1.1.2). The crystallinity index of cellulose, i.e. degree of
microfibrils orientation, is highly variable and depends on the source and age of the
tissue (Harris, 1990). Because of its structure, cellulose is insoluble in most
Fig 1.3.1.1.1 Proposed arrangement of D-glucan chains within cellulose microfibrills of

primary cell walls of dicots (Dey and Brinson, 1984).
10
Fig 1.3.1.1.2 Diagrammatic representation of cellulose microfibrillar structure (Hess et

al., 1975).
11
solvents and has a low accessibility to aqueous acids and cell-free enzymes (Tipson and
Horton, 1988).
Several properties of cellulose can influence its susceptibility to enzymic
degradation (Cowling and Kirk, 1976), i.e capillary structure in relation to the size of
cellulases, crystallinity index, dimension of crystalline portions of the microfibrils and
the nature of substances (lignin) with which the cellulose is associated (Kirk 1983,
Shambe and Kennedy, 1984). Among different characteristics of cellulose, crystallinity
index has been reported to be the most important factor affecting cellulose digestibility
(Fan et al., 1981; Gharpuray et al., 1983).
1.3.1.2 hemicellulose. The term hemicellulose is applied to cell wall polysaccharides

which occur in close association with cellulose, especially in lignified tissues. It is often
restricted to substances extracted with alkaline reagents (Aspinal, 1959).
Hemicelluloses are built up of a relatively limited number of sugar residues (100200 sugar units), eg. D-xylose, D-mannose, D-glucose, D-galactose, L-arabinose, 4-Omethyl-D-glucuronic acid, D-galacturonic acid, D-glucuronic acid and to a lesser extent,
L-rhamnose, L-fucose and various O-methylated neutral sugars (Timell, 1964).
Xylans are quantitatively the most important hemicellulose of graminaceous cell
walls (Fig 1.3.1.2.1) (Dey and Brinson, 1984). Xylan is composed of chains of 1-4
linked -D-xylopyranose residues. Different plants may contain the same basic xylan
structure but different arrangements with other sugar residues, especially L-arabinose,
12
D-glucuronic acid and 4-methyl ether, may occur (Wilkie, 1979).
13
14
Fig 1.3.1.2.1 Hypothetical structure of plant xylan (Puls and Poutanen, 1989).
15
Xylans from cereals and grasses are generally characterized by the presence of Larabinofuranose residues linked to the backbone as single-unit side-chains, usually to
position 3 of xylose. Similarly, D-glucuronic acid and/or 4-O-methyl-D-glucoronic acid
residues are also present in a similar proportion. The hemicellulose in wheat straw is
characterized by the xylan backbone, carrying through position 3 of D-xylose residues,
side-chains terminated by non reducing L-arabinofuranosyl-(1-3)-O--D-xylopyranosyl(1-4)-D-xylopyranose (Aspinal, 1959).
There is evidence of the presence of xyloglucans in primary cell walls of mono
and dicotyledonous plants which account for approximately 2 and 19% of the cell wall,
respectively (Dey and Brinson, 1984). In monocotyledons, xyloglucan polymers are
hydrogen bonded to cellulose fibrils forming a non-covalent link in the network of
polymers which cross-link the cellulose fibres (Burke et al., 1974; Valnet and
Albersheim, 1974).
All xylans contain backbones composed of -(1-4) linked xylosyl residues. There
is a wide variety in the nature of the side chains attached to this xylan backbone. The
most common side chains encountered are single L-arabinofuranosyl groups attached to
O-3 of some of the backbone xylosyl residues or single D-glucosyluronic or 4-O-methylD-glucosyluronic groups attached to O-2 of some of the backbone xylose units.
However, oligomeric side chains containing other glycosyl residues are also found
(Wilkie, 1979).
The composition of hemicellulose in softwoods and hardwoods is different from
16
that in grasses. Galactoglucomannans are the major constituents (20%) of softwood

hemicelluloses. Hardwood hemicellulose contains glucoxylan (15-30%) polymer
backbone similar to that of the softwoods (Timell, 1965).
Hemicellulose is linked to lignin through D-galactose, L-arabinose and D-xylose
by glycosidic linkages (Sarkanen and Ludwic, 1971). Isolation of lignin-carbohydrate
complexes from rumen liquor of animals fed roughage confirms the presence of bonds
between lignin and carbohydrates (Jung, 1988; Wallace et al., 1991). According to
Chesson et al. (1983), ester linkage by the O-5 position of arabinose to core lignin seems
to be a major bond between lignin and hemicellulose in forages.
1.3.1.3 pectic compounds. Pectic polysaccharides make up approximately 35% of the

primary cell walls of dicotyledonous plants, the main components being galactosyluronic
residues (Worth, 1967). The middle lamella is particularly rich in pectic polysaccharides.
Other major polysaccharide component found in pectic polysaccharides are rhamnose,
arabinose, and galactose. Monocotyledons appear to contain minor proportion of such
polysaccharides (Dey and Brinson, 1984). Pectic substances are hydrophillic and
therefore have certain adhesive properties which may also be a means of translocation of
water (Worth, 1967).
1.3.2 proteins
Proteins are a minor component of the plant cell wall which may be covalently
17
cross-linked
with
lignin
and
polysaccharides
(Lamport,
1965).
Extensins
(hydroxyproline-rich glycoprotein) are the most abundant protein in the plant cell wall
(Cassab and Varner, 1988). Primary cell walls of dicotyledons contain between 5 and
10% extensin which is rich (20%) in hydroxy-L-proline (Dey and Brinson, 1984).
1.3.3 phenolic compounds
1.3.3.1 lignin. Lignin is the most abundant natural non-carbohydrate organic compound
in fibrous materials. The importance of lignin in plants should be considered from
different aspects, i.e. its role in plant development, contribution to mechanical strength
and protection from degradation (Walker, 1975). From the nutritional point of view,
lignin has always been blamed as an important barrier to polysaccharide utilization. (Van
Soest, 1994).
Lignin is made up of three primary precursors, ie. trans-coniferyl, trans-sinapyl
and trans-p-coumaryl alcohols (Fig 1.3.3.1.1 and Fig 1.3.3.1.2) (Sarkanen and Ludwic,
1971). Lack of enzymic control during lignin polymerization (formation) results in an
almost random series of bonding and a very complex structure (Jung and Fahey, 1983).
The existence of strong carbon-carbon (C-C) and ether (C-O-C) linkages in the lignin
affects its susceptibility to chemical disruption (Harkin, 1973).
Lignins are always associated with hemicellulose, not only in intimate physical
mixture, but also anchored to the latter by actual covalent bonds (Sarkanen and Ludwic,
18
1971). Soluble lignin-carbohydrate complexes have been isolated from LM (Morrison,

1974a and b; Nordkvist et al., 1989). Most lignins contain some aromatic carboxylic
acids (P-coumaric and ferulic acids) in ester bonds (Hartley, 1972).
19
P-Coumaryl alcohol Coniferyl alcohol
Sinapyl alcohol
Fig 1.3.3.1.1 Structure of three precursors of lignin (Sarkanen and Ludwic, 1971).
20
Fig 1.3.3.1.2 Proposed lignin structure in softwoods (Nimz, 1974).
21
Lignins differ mainly in the proportion of the three alcohol units. Softwood
lignins are made up of approximately 80% coniferyl, 14% p-coumaryl and 6% sinapyl
alcohols. In contrast, hardwood lignins are composed of 56% coniferyl, 4% p-coumaryl
and 40% sinapyl alcohols. Grass lignins are rich in p-coumaryl units (Jung and Fahey,
1983).
1
Sarkanen and Ludwic (1971) classified lignins into two groups, namely guaiacyl
and guaiacyl-syringyl lignin. Most of the gymnosperm lignins are typical guaiacyl
2
lignins, although they contain small amounts (<1.5%) of syringyl units and a rather
lower proportion of p-hydroxyphenyl units. Both dicotyledon and grass lignins are true
guaiacyl-syringyl lignins (Schwartz et al., 1989). The syringyl content of woody
angiosperm lignins varies between 20 and 60%. In herbaceous angiosperms
(dicotyledons) this range can be from 10 to 65%.
Lignin is poorly degraded under anaerobic conditions (Kirk and Farrell, 1987)
but extensively degraded by white-root fungi in presence of oxygen (Zadrazil, 1984;
Khazaal et al., 1993b).
Determination of lignin has not been an easy task, as lignin is always associated
with cell wall polysaccharides and also artifacts produced during cell wall preparation
can interfere with the determination of lignin (Hartley, 1978).
There are several methods for lignin determination. In the Klason method,
1
guiacyl is a collective term for the functional group common to coniferyl alcohol, ferulic acid and
vanillin.
2
syringyl is a collective term for the functional group common to synapyl alcohol, syringic acid and
syringaldehyde and.
22
hydrolysis of plant cell wall by sulphuric acid produces a residue containing lignin,
cutin, polyphenols, carbohydrate degradation products and nitrogenous material
(Theander et al., 1977). During H2SO4 hydrolysis some lignin may be lost due to partial
conversion to soluble phenolics (Hartley, 1981).
Goering and Van Soest (1970) proposed a method based on treatment with acid
detergent solution to render a residue free of hemicellulose and soluble compounds. The
cellulose-lignin residue is then hydrolysed with 72% sulphuric acid solution to remove
cellulose.
Determination of lignin by the permanganate method has also been described
(Van Soest and Wine, 1968). Important differences between the permanganate and the
acid detergent - sulphuric acid hydrolysis method arise from the fact that cutin is largely
retained in the lignin by the latter whereas it is removed in the former. Polyphenolic and
other unsaturated substances such as tannins, pigments and proteins that may not be
completely removed in the acid detergent fibre may react with permanganate and appear
as lignin.
The acetyl bromide method (Morrison, 1972) is another option for lignin
determination. It is based on the treatment of fibre in acetyl bromide solution to break
down the cross-linkages between structural units of lignin. Soluble phenolics are
measured by UV spectrophotometry. The limitations of this method are the lack of any
entirely satisfactory standard and the presence of non-lignin compounds, e.g. proteins,
phenolic acids and tannins, which interfere with the reading.
23
1.3.3.2 tannins
Tannins are known to occur in the vacuoles of plant cells (Forsyth, 1964; Jung
and Fahey, 1983). All tannins are polyphenolic compounds with a high molecular weight
(MW 500-3,000) (Haslam, 1981). Their presence in trees and woody shrubs produces a
bitter taste and astringency which may affect palatability and voluntary intake (Arnold et
al., 1980). Tannins can be divided into condensed and hydrolysable tannins. Condensed
tannins (proanthocyanidins) are the most widely distributed in vascular plants and are
made up by condensation of hydroxyflavans, leucoanthocyanidin (flavan-3,4-diol) and
catechin (flavan-3-ol) (Cope and Burns, 1974). Hydrolysable tannins are restricted to
angiosperm dicotyledons and usually contain glucose as a central core (Swain, 1979).
Tannins are known to affect the grazing behaviour and consequently depress the
food intake in sheep (Cope and Burns, 1974). Feed utilization appears to be negatively
correlated with tannin content due to depression in rumen microbial activity (Donnely
and Anthony, 1970; McLeod, 1974; Walker, 1975; Terrill et al., 1989).
1.3.3.3 phenolic acids

Phenolic acids are structural components of the lignin core in plant cell wall
(Shimada, et al., 1971). Their presence in food products has been associated with
24
astringency (Durkee and Thivierge, 1977), discoloration (Dorrell, 1976) and antioxidant
properties (Senter et al., 1980). The presence of carboxyl and phenolic groups in
phenolic acids enable such compounds to link to lignin and carbohydrates by ether
(Scalbert et al., 1985) or ester (Scalbert et al., 1986) bonds.
The concentration of phenolics in grass cell walls varies from 8 to 28 mg/g and is
less than 3 mg/g in legumes (Eraso and Hartley, 1990). Ester bonds are labile to alkali
treatment and as phenolic acids are ester-linked to both lignin and hemicellulose they
can be released by alkali treatment (Hartley and Jones, 1978; Hartley et al., 1985;
Scalbert et al., 1985).
The effect of phenolic acids on rumen microbial fermentation has been
extensively studied. Chesson et al. (1982) reported that p-coumaric and ferulic acids
were the most toxic phenolic acids to rumen cellulolytic bacteria. p-Coumaric acid has
an inhibitory effect on colonization of fibres by fungi (Akin and Rigsby, 1985) and
cellulolytic bacteria (Borneman et al., 1986; Varel and Jung, 1986; Akin et al., 1988).
1.4 Acidolysis reactions

Although steam treatment has been classified as a physical treatment, in fact it is
a chemical (thermo-chemical) treatment of acid hydrolysis nature. At high temperature
under acidic conditions hemicellulose is partly disrupted thereby releasing acetyl and
formyl groups of hemicellulose and pectin, e.g. 1-2% dry weight of grasses (Bacon et
al., 1975; Bacon and Gordon, 1980). The release of endogenous organic acids, e.g.
25
acetic and formic acids, will help to catalyze further reaction (Baugh and McCarty,
1988) as described below.
1.4.2 cellulose
Acid hydrolysis (saccharification) of cellulose produces a random cleavage of
glucosidic linkages containing hemiacetal and hydroxyl terminal groups (Harris, 1949).
+
Cellulose acid hydrolysis is dependent upon H concentration. A high cellulose

o
hydrolysis rate is observed even below 100 C when acid concentration is high. However,
under less acidic conditions higher temperature and/or longer reaction time are required
for achieving similar cellulose hydrolysis (Brownell et al., 1986).
The crystallinity index (CrI) of native cellulose has been suggested as a limiting
factor for enzymic hydrolysis (Fan et al., 1981). Interestingly, cellulose CrI is increased
during acid hydrolysis (Carrasco et al., 1994) whilst greater cellulose bio-availability is
achieved (Dekker and Wallis, 1983; Saddler et al., 1982; Wong et al., 1988; Toussaint et
al., 1991; Sawada et al., 1995). The reason for such an increase in cellulose availability
is the significant change observed in the chemical structure of lignin as well as in the
degree of polymerization of cellulose and accessible surface area (fibre swelling).
The degree of polymerization of cellulose can be significantly decreased during
acid hydrolysis (Knappert et al., 1980; Puri, 1984). The reduction of the degree of
polymerization of cellulose during steam treatment is also considered to be an important
26
factor affecting cellulose availability to enzymes (Cowling and Kirk, 1976; Wei and
Cheng 1985).
Fibre swelling also plays an important role in increasing cellulose bio-availability
from acid-hydrolysed lignocellulosics (Puri 1984; Morjanoff and Gray, 1987; Wong et
al., 1988).
1.4.3 hemicellulose
Hemicellulose is significantly more susceptible to hydrolysis than cellulose
(Kuznetsov et al., 1990; McDonald and Clark, 1992). Hemicellulose is very susceptible
to depolymerization at high temperature and under acidic conditions (Carrasco et al.,
1994) and the hemicellulosic sugars, e.g. xylose, arabinose, glucose, mannose and
galactose, released by hydrolysis of hemicellulose undergo the specific reactions
mentioned below.
o
At high temperatures (>100 C) and acidic condition all aldopentoses form 2furaldehyde (furfural) (Fig 1.4.1.1) in large quantity. Among the pentoses, D-xylose is
the most effective pentose in production of furfural (Tipson and Horton, 1988). Several
low molecular weight aldehydes (formaldehyde, acetaldehyde, and 2-butenal) have been
isolated from D-xylose after acid treatment.
Under acidic conditions hexoses produce mainly 5-(hydroxymethyl)-2furaldehyde (HMF) as a major end-product (Fig 1.4.1.2). The rate of formation of HMF
varies considerably among the hexoses. For instance, in sulphuric acid solution (2 M at
27
100 C) the order of reactivity is D-mannose > D-galactose > D-glucose (Tipson and
Horton 1988). Treatment of D-glucose, D-fructose, D-glucuronic acid and
28
Fig 1.4.1.1 Production of furfural from D-xylose (Tipson and Horton, 1988).
Fig 1.4.1.2 Production of 5-hydroxymethyl-2-furaldehyde from D-glucose (Tipson and

Horton, 1988).
29
D-galacturonic acid in acetate buffer at pH 3.5-4.0 at 96 C yields phenolic compounds at

a rate of 0.3% for hexoses and 7% for glucuronic acid (Popoff and Theander, 1976).
Similarly pectins can yield furfural under acid hydrolysis in aqueous solution at
high temperature as do pentoses.
Upgrading LM using steam treatment is normally associated with nutrient loss
via volatilization (Castro, 1994) and formation of undegradable browning reaction
products (Van Soest and Mason, 1991). Castro et al., (1993) reported an increase in acid
detergent fibre (ADF) after steam treatment of LM. The increase in ADF content was
attributed to formation of protein-carbohydrate complexes and polymerization of
hemicellulose breakdown products.
Three types of browning reactions have been described (Hodge, 1953). The
Maillard reaction (carbonylamino reaction) is the most common one and it includes the
reaction of aldehydes, ketones and reducing sugars with amines, amino acids, peptides
and proteins (Tipson and Horton, 1988). Heating of LM generates undegradable
Maillard products which are detected by an increase in the nitrogen content of the cell
wall. Maillard polymer is considered to be a lignin-like compound and poorly
degradable (Van Soest and Mason, 1991). There are many factors affecting the
formation of Maillard reaction products, e.g. temperature, reaction time, moisture
+
content, H concentration and type of substrate (Tipson and Horton, 1988).

A caramelization reaction is another type of browning reaction. It occurs when
polyhydroxycarbonyl compounds, e.g. sugar and polyhydroxycarboxylic acids are heated
30
to high temperature in the absence of amino compounds (Hodge, 1953).

A third type is known as oxidative reactions. Conversion of ascorbic acid and
polyphenols into di- or polycarbonyl compounds are examples of oxidative reaction
(Hodge, 1953).
1.4.4 lignin
Most research on chemical structure, metabolic pathways and decomposition
reactions of lignin have been carried out on woody materials (Karina et al., 1992;
Hishiyama and Sudo, 1992). Grass lignins are comparatively much less studied than
wood lignins.
Lignin structure has been discussed previously (Section 1.3.3.1). In order to
elucidate lignin structure and reactivity several studies on lignin acidolysis have been
completed (Lundquist and Hedlund, 1967; Lundquist and Lundgren, 1972; Wayman and
Obiaga, 1974; Hishiyama and Sudo, 1992; Karina et al., 1992).
Lignin disruption under acidolysis is essentially attributed to the cleavage of
various ether bonds, the most important being of the arylglycerol--aryl ether bond
(Lundquist and Hedlund, 1967; Lundquist and Lundgren, 1972) (Fig 1.4.4.1).
Chua and Wayman (1979) reported that lignin-lignin and lignin-carbohydrate
bonds are more sensitive to temperature than acidity. Therefore, under auto-hydrolysis
o
conditions (t>160 C in absence of exogenous catalyst) one could expect greater lignin
o
depolymerization as compared to mild temperature acid hydrolysis (100-140 C) with
31
exogenous acid).
32
33
Fig 1.4.4.1 The susceptible bonds (-aryl and -O-4) in lignin molecule for cleavage
under acidolysis reaction.
34
Nimz (1974) isolated and identified a number of low-molecular weight

degradation products of the wood lignin under acid-hydrolysis conditions. During acid
hydrolysis of lignin considerable amounts of monomeric, dimeric and oligomeric
phenols are formed, the former being found in much smaller proportion. This is due to
the presence of C-C linkages between the monomers which prevent their degradation to
lower molecular weight phenolic compounds (Lundquist and Hedlund, 1967; Lundquist
and Lundgren, 1972). The rate at which phenolic monomers are released during
acidolysis depends upon the lignin structure. For example, acidolysis of birch wood
yields more monomers than spruce wood. This is due to the fact that phenylpropane
units in birch lignin are syringyl in type in which the 3 as well as the 5-position is
occupied by methoxy groups able to link to adjacent units by ether linkages (Lundquist
and Lundgren, 1972) (Fig 1.4.4.1).
Chua and Wayman (1978) reported the effect of auto-hydrolysis treatment on
o
aspen lignin. When aspen wood was subjected to auto-hydrolysis at 195 C it was
observed that treatment reaction time significantly affected the degree of lignin
depolymerization/repolymerization
(Fig
1.4.4.2).
Lignin
depolymerization
was
quantitatively more important than repolymerization reactions with treatment up to 30

min. Longer treatment shifted this to a greater rate of repolymerization reaction
compared to depolymerization. It has been postulated that hemicellulose degradation
products, e.g. furfural and its precursors, can react with lignin during auto-hydrolysis.
This has accounted for the apparent increase in lignin content (Chua and Wayman,
35
1978). The nature of such lignin is different from native lignin as it is more condensed
36
Fig 1.4.4.2 Effect of reaction time on depolymerization/repolymerization of lignin

(Wayman and Chua, 1979)
37
compared to original lignin (Wayman and Chua, 1979b). Auto-hydrolysis causes an

increase in the proportion of C to O/H and a decrease in content of methoxy group
(Chua and Wayman, 1978).
1.5 Protein precipitating capacity of phenolic compounds

Phenolic acids are known to inactivate enzymes (Martin and Akin, 1988) through
decreasing enzyme solubility (Sharma, 1985) and/or formation of a soluble and inactive
enzyme inhibitor complex at very low concentration (Zanobini et al., 1967). The extent
of enzymic inhibition by phenolic acids depends on the type of enzyme (Vohra et al.,
1980) and the phenolic compound involved (Sharma et al., 1985).
The ability of tannins to bind and precipitate proteins has been well documented
(Makkar et al., 1987; Makkar, 1989; Makkar et al., 1993b). This precipitating property
is explained by the ability of phenolic hydroxyl groups to form cross-links with proteins
(Feeny, 1969; Swain, 1979). Although tannins are considered to be toxic to rumen
microbial activity (Waghorn et al., 1990) their precipitating capacity may have a positive
effect by preventing bloat in animals feeding on legume pastures and protecting proteins
from microbial degradation (Driedger and Hatfield, 1972).
It is reported that proteins bound to tannins are more likely to escape rumen
degradation than unbound proteins (Driedger and Hatfield, 1972). The protein-tannin
complex is dissociated at abomasal pH (Oh and Hoff, 1987; Waghorn et al., 1990), thus
releasing protein to be digested in the lower tract. This property has been a matter of
38
research on protein protection for ruminants for several years (Nishimuta and Boling,
1973).
Beltrame et al. (1992) observed a significant increase in the extent of enzymic
hydrolysis of cellulose after removal of the lignin fraction from steam-treated wheat
straw.
Kawamoto et al. (1992) reported a protein-precipitating capacity in lignin
extracted from steam-exploded LM. Such protein-precipitating capacity of phenolics
was attributed to the formation of hydrogen bonds between lignin hydroxyl groups and
protein carboxyl groups.
1.6 The effect of steam treatment-associated toxic compounds on rumen microbial

activity
1.6.1 furfural and 5-hydroxymethyl furfural

As mentioned previously in this chapter, formation of toxic end-products during
steam treatment may be a major negative effect of treatment. These products can inhibit
yeast growth (Fireoved and Mutharasan, 1986) and methane production (Baugh and
McCarty, 1988). In these two reports such a toxic effect was attributed mainly to
furfural.
Furfural is the most widely distributed simple furan in nature (Dean, 1963). It is
formed during production and storage of fruit juices (Robertson and Samaniego, 1986)
39
and wines (Simpson, 1980), and is thus very important in the food industry. Furfural is
also a major degradation by-product of the steam treatment of LM (Sproull et al., 1985).
Sanchez and Bautista (1988) observed that both furfural and HMF were toxic to
yeast. Furfural has shown a greater toxicity compared to HMF. It has been reported that
such aldehydes are first converted into furoic acid and further reutilized by the yeast. In
another study, Van Tran and Chambers (1985) reported that furfural inhibited respiration
of Pichia stipitis at a concentration of 2g/l and the metabolic by-product furfuryl alcohol
reduced its growth rate.
The effects of furfural on animal performance has been also investigated. The
effect of furfural on food intake has been associated with its antimicrobial action and
mucosal irritation (Kyuma et al., 1991). Furfural depressed dry matter consumption rate
and dry matter intake by goats up to 4 h after feeding. However, there was no effect on
the total daily feed intake and digestibility. Cellulose digestibility in vitro using rumen
liquor as inoculum was depressed when furfural addition was greater than 200 ppm.
Such a depression was more marked between 4 and 12 h incubation. Contrary to Kyuma
et al. (1991), Castro et al. (1995) observed that furfural was not toxic to rumen microbial
activity in vitro up to a concentration of 1,300 ppm. It was also demonstrated that rumen
microbes can almost completely degrade both furfural and HMF within 6 h fermentation
in vitro.
40

Britton (1978) reported a positive effect of removing inhibitory materials from
steam-treated sawdust by ethanol solutions on microbial activity in vitro. Unfortunately
the nature of such inhibitory materials was not investigated in that study. Castro et al.
(1995) reported inhibition of methane production in vitro from acid-hydrolysed wheat
straw. Such an effect was attributed to lignin degradation products.
According to Clark and Mackie (1984) phenolic compounds derived from lignin
depolymerization inhibited yeast activity. These authors reported a greater inhibitory
effect from low molecular weight lignin-based phenolics than from carbohydrate
degradation products.
Production of materials inhibitory to microbes and cell-free enzymes during
steam treatment of fibrous materials limits the value of this process for improving the
fermentability of these materials by microbes or enzymes. This problem has received
surprisingly little attention.
Regarding the advantages of steam treatment which were discussed before,
different aspects of this type of treatment, specially negative ones, need to be studied.
The ultimate goal would be to find a suitable method to reduce such negative effects or
to try to make good use of them such as using the inhibitory compounds to manipulate
ruminal fermentation.
In the present study the biological effects of such compounds were examined
41
with emphasis on ruminant nutrition. The inhibitory capacity of such compounds were
studied in two biological systems, i.e. rumen microbial fermentation and cell-free
enzymic hydrolysis. The ability of
phenolics extracted from steam-treated
lignocellulosics to protect proteins was also studied.
42
CHAPTER 2
CHARACTERIZATION OF WATER-SOLUBLE EXTRACT FROM

STEAM-TREATED WHEAT STRAW
2.1 Introduction
Hemicellulose solubilization (Carrasco et al., 1994; Eklund et al., 1995) and
lignin depolymerization (Wayman and Chua, 1979a) are two important changes that
occur during steam treatment. The extent of hydrolysis of cell wall polysaccharides
depends on treatment conditions (Forsberg et al., 1986). A significant increase in water
soluble extract (WSE) from steam-treated LM occurs due to depolymerization of
hemicellulose and lignin into soluble compounds (Matsuzaki et al., 1994). Treatment
conditions affect both degree of polymerization of soluble carbohydrates (Matsuzaki et
al., 1994) and sugar composition (Grohmann, et al., 1985).
Due to its high content of readily fermentable compounds, WSE can be used for
some biological purposes, e.g. production of alcohol (Van Tran and Chambers, 1985;
Delgenes et al., 1990), single cell protein (Qu et al., 1992) and animal feed. Both
chemical composition and degree of polymerization of soluble carbohydrates are
important aspects when considering nutritive value of WSE (efficiency and site of
digestion).
With respect to monogastric feeding, the presence of non-monomeric pentoses
43
may be a limiting factor as such compounds cannot be digested and absorbed in the
small intestine and may have negative effects on an animal (Longstaff et al., 1988), e.g.
vomiting and diarrhoea, at high levels of feeding (Yule and Fuller, 1992). Oligomers due
to presence of 1-4 linkages between sugar residues are not hydrolysed by the enzymic
system in the intestine. However, they can be degraded by microbes in the lower gut into
volatile fatty acids (VFA) which can be absorbed and metabolised by the animal.
Although microbial degradation is less efficient as compared to the enzymic process in
the small intestine (McDonald et al., 1988), the use of WSE containing large quantities
of oligomeric forms of sugar may have a potential market for animal feeding.
Ruminants are more tolerant to some of the constraints described above as they
can degrade oligomeric forms of sugars in the rumen.
Another important constraint for using the WSE as an animal feed is the presence
of material inhibitory to microbial activity (Clark and Mackie, 1984). Also relevant is
the presence of a significant amount of undegradable compounds (Maillard reaction
compounds) which make no contribution to the energy value of this fraction (Van Soest
and Mason, 1991).
Optimum treatment conditions (temperature, reaction time, moisture content and
presence of exogenous catalyst) are achieved when cell wall structure is disrupted in
such a way so as maximize bio-utilization of cell wall polysaccharides while producing
minimum quantities of toxic materials. As mentioned in Section 1.4.3, furans are endproducts of browning reaction of sugars under acidic conditions. Caramelization is
44
another type of reaction (Section 1.4.3) which also affects soluble carbohydrate (Hodge
,1953). The nutritive value and toxicity of the products of such reactions have not been
reported in steam-treated LM.
The aim of the work described in this chapter was to investigate the nature and
biological value of water-soluble extract from steam-treated wheat straw produced under
different treatment conditions. Various components from this fraction were tested by
both chemical and biological approaches and possible implications on animal digestive
efficiency are discussed.
45
2.2 Materials and methods
2.2.1 substrate
Wheat straw variety Riband was used in this experiment. An air dried sample was
hammer milled through a 1 mm screen and stored at room temperature.
2.2.2 steam treatment conditions

A laboratory scale 1.5 litre high pressure vessel designed at the Rowett Research
Institute was used. To standardize treatment conditions nine batches were first carried
out at 19 bar. Two heating up times (time required to reach 19 bar starting from
atmospheric pressure) (14 and 19 min) and three dry matter (DM) contents (40, 50 and
60%) were tested. The material was then held at this pressure for various reaction times,
including 0 min reaction time which indicates immediate termination of the pressure
treatment.
O
The vessel (Fig 2.2.2.1) was kept at 250 C prior to treatment. Water was added
to wheat straw to achieve the appropriate moisture content. A 500 ml aluminium beaker
containing boiling water was placed in the vessel and a second similar beaker containing
wet sample ( 30 g DM basis) was put on top. The system was closed by the top lid.
Rise of pressure was manually controlled by adjusting the ball valve. Treatment
reproducibility was assessed by producing a series of samples (n=4) treated at 15 and 19
bar.
46
Fig 2.2.2.1a The High Pressure Steam Treatment Vessel in operation.
47
Fig 2.2.2.1b Components of the High Pressure Vessel.
48
(c)
(d)
49
Fig 2.2.2.1 Putting the sample in the beaker (c) and transferring into the vessel (d).
50
After validation another experiment was designed to determine the effect of

pressure (15, 17 and 19 bar), reaction time (RT) and heating up time (HT) (14 and 19
min) upon the properties of the WSE as described below.
2.2.3 extraction of water soluble extract

o
A 1 g sample was soaked in 30 ml water and kept at 40 C for 2 h. The aqueous

mixture was filtered through a No. 2 sintered glass filter and was centrifuged (Sorvall
o
RC-SB) at 10,000 x g and 4 C for 10 min. The supernatant was freeze dried and kept in
dark at room temperature for further analyses.
2.2.4 statistical analysis

Effect of DM content on soluble carbohydrate and total extractable phenolic
contents of steam-treated wheat straw was evaluated by analysis of variance (ANOVA).
Differences in nutritive value of water soluble extract from steam-treated wheat straw as
compared to control sample was tested by Bonferroni T test p<0.05.
2.2.5 total neutral sugar analysis

The total neutral sugar content (monomeric and oligomeric forms) from WSE
was measured by Gas Liquid Chromatography (GLC) as their alditol acetates (Blakeney
et al., 1983). A 10 mg freeze-dried sample was placed in a screw-cap pyrex test-tube.
Then 0.5 ml 72% H2SO4 was added and the mixture vortex mixed. The mixture was
51
incubated at 25 C in water bath for 30 min with occasional vortex mixing. Five ml water
O
was added to the tube, which was then vortex mixed, incubated at 100 C for 2 h, cooled
and centrifuged at 3,000 x g for 5 min. One ml of allose/inositol standard (1 mg/ml) was
added to a 3 ml aliquot of the supernatant hydrolysate. The mixture was neutralized by
adding 0.75 ml concentrated ammonia solution (12 M). A 0.4 ml NaBH4 solution (100
mg/ml in 0.05 M NH4OH) and 1 drop of octan-2ol (antifoaming agent) were added to
o
the neutralized aliquot which was incubated at 40 C for 60 min. Four hundred l acetic
acid was added to acidify and destroy excess NaBH4. Two ml of acetic anhydride and
0.3 ml 1-methyl imidazole were added to 200 l of reduced hydrolysate. The reaction
mixture was mixed and allowed to react for 10 min. Five ml of water was added and the
mixture allowed to stand for 10 min to destroy any excess of acetic anhydride. The
alditol acetates were extracted by adding 2 ml of dichloromethane and vortex mixing for
15 seconds. After centrifuging (3,000 x g for 10 min) the organic layer was transferred
O
to a vial. The dichloromethane was evaporated off under a stream of N2 at 40 C. The

alditol acetates were dissolved in 150 l ethyl acetate and 1 l sample was injected into
the GC fitted with a flame ionization detector (FID) and a wide bore capillary column
(30m, 0.75 mm I.D., Supelco SP-2330). The following temperatures were used: injector
o
(t=250 C), column oven (t=180 C, temperature increased at a rate of 10 C/min until
o
220 C and time of 3 min and FID (t=275 C). Helium gas was used as carrier with a flow
rate of 7 ml/min.
Monomeric sugars in water extracts were measured by dissolving a 20 mg sample
52
in 5 ml water. A 0.4 ml inositol standard (internal standard) was added to a 2 ml aliquot.

The remaining steps for measuring of monomeric sugars (derivatization to alditol acetate
derivatives, addition of internal standard, extraction of alditol acetates by
dichloromethane from aqueous solution and GLC conditions) were the same as
mentioned above.
2.2.6 DM loss
DM loss during steam treatment was directly measured by weight difference
before and after treatment.
2.2.7 ash content

Ash content was determined by incinerating 1 g sample in a muffle furnace at
o
550 C for 6 h.
2.2.8 nitrogen (N) content

N content was measured using a Macro N Analyzer (Foss Electric, UK). The
o
method is based on the oxidative combustion of a given sample (200 mg) at >1,000 C
converting the nitrogen in the sample to N2, which is detected by thermal conductivity.
2.2.9 total extractable phenolic analysis

The phenolic content of water extracts was measured by the Folin and Ciocalteu
53
reagent (Julkunen-Tiitto, 1985). Five ml 70% acetone solution (v/v) was added to a test
tube containing 100 mg freeze dried sample (Muller-Harvey and Dhanoa, 1991) to
extract phenolic compounds. Nine hundred l water, 0.5 ml 1 M Folin and Ciocauteu
phenol reagent and 2.5 ml 20% Na2CO3 were added to 0.1 ml extract. The mixture was
vortex mixed after the addition of each reagent. It was kept for 35 min at room
temperature and the absorbence at 725 nm (A725) was recorded. A standard curve was
constructed using 0, 10, 20, 40, 80 and 100 l of 0.5 mg/ml tannic acid (Sigma product)
in 70% acetone solution. Volumes were made up to 1 ml with an appropriate amount of
distilled water.
2.2.10 in vitro gas production

The nutritive value of samples were estimated by the in vitro gas production
technique of Menke and Steingass (1988). Samples were incubated in duplicate in 100
ml standard glass syringes (Haberle Labortechnik - Germany, model Fortuna). The
rumen liquor was collected from 2 sheep (1 h after feeding) fed with a mixed diet (50%
hay, 30% barley, 10% molasses, 9.1% fish meal, 0.6% NaCl and 0.1% commercial
mineral mixture) twice a day. The rumen liquor was filtered through four layers of
muslin and mixed at a ratio 1:2 with artificial saliva. Artificial saliva composition is
shown in Table 2.2.10.1. Resazurin was used as an oxygen indicator, and intermittent
flushing of CO2 in the artificial-saliva:rumen-liquor mixture during setting up the run
was applied in order to minimize oxygen contamination. Rumen liquor was dispensed
54
into syringes containing an appropriate amount of sample as described below. Syringes

o
containing sample + rumen liquor-artificial saliva were kept in a water bath at 39 C. The
syringes were gently shaken every hour during the first 6 h incubation and then repeated
at the time of reading of syringes. Gas production
55
Table 2.2.10.1 Composition of macro-mineral, micro-mineral and buffer solutions used

for preparing artificial saliva.
macro-mineral
micro-mineral
reagent
buffer
g/l H2O
Na2HPO4
5.9
KH2PO4
6.2
MgSO4.7H2O
0.6
CaCl2.2H2O
132
MnCl2.4H2O
100
CoCl2.6H2O
10
FeCl2.6H2O
NaHCO3
35.0
NH4HCO3
4.0
56
was recorded after 2, 4, 6, 9, 12, 24 and 48 h incubation. WSE from the straw steamtreated at 19 bar and 5 min reaction time was chosen as it contained the highest
concentration of browning reaction products. Samples were incubated as follows:
a) 200 mg water extract (sugar composition: xylose 26.8%, arabinose 2.50%, glucose
5.80, galactose 1.40%, sum of mannose, rhamnose and fucose 2.30%).
b) sugar control 1: from sugar analysis of WSE. Analar sugars (xylose, arabinose,
glucose, galactose) were added at similar quantities as present in a).
c) sugar control 2: Analar sugars were added in similar proportion but their total quantity
was equivalent to the organic matter present in a).
2.2.11 browning reaction products

The content of browning reaction products were estimated as follows:
1
Browning Reaction Products (%) = 100 - Sugar (%) - Phenolics (%) - Ash (%)
as browning reaction products can not be measured directly, their concentration has been
estimated by difference. The limitation of this equation should be taken into consideration as
ash was measured gravimetrically and a proper standard was used for estimation of sugars but
phenolics were measured colorimetrically as tannic acid equivalent. Estimation of phenolics
using colorimetry method may be biased as phenolic composition and properties are affected by
treatment conditions but the same standard (tannic acid) was used for measuring those
57
compounds produced under different treatment conditions.
58
2.3 Results and discussion
2.3.1 steam treatment procedure

Unlike normal practice, the steam-treatment system in the present study operated
without direct steam injection into a reaction chamber. As shown in Fig 2.2.2.1 steam
was produced by adding water to a pre-heated chamber. In this procedure two important
variables may affect treatment efficiency, a) time required to reach a given temperature
(pressure), b) moisture content which might affect heat transfer to the substrate.
The total extractable phenolic and total sugar contents of steam-treated samples
are presented in Table 2.3.1.1. Sample DM content (30, 40 and 50%) did not affect
(p>0.05) total sugar and total extractable phenolic contents. The lower quantity of sugar
and phenolics in the samples treated for 14 min HT is explained by a shorter exposure to
treatment. For further treatments 50% DM content was chosen as a standard value
because preparation of homogenous samples at this moisture content was more
convenient.
Results of chemical analyses for total extractable phenolics and total sugars for
assessing vessel reproducibility are presented in Table 2.3.1.2. The results showed no
significant difference (p>0.05) between chemical composition and DM loss within the
replicates.
59
Table 2.3.1.1 Effect of sample DM content (prior to steam treatment) on chemical

composition of steam-treated wheat straw at 19 bar and 0 min reaction time.
1
sCHO
treatment
conditions
3
TEP
(%)
4
HT
DM %
14
40
14.4
50
14.6
60
14.9
40
17.7
50
17.3
60
19
SEM (n=2)
1
a5
3.93
4.47
4.79
6.40
6.25
17.0
6.72
0.509
0.390
b
b
d
d
d
soluble carbohydrate
total extractable phenolics measured as tannic acid
3
heating up time (min)
4
dry matter content
5
means on the same column with the same superscript are not significantly (p>0.05)
different
6
standard error of mean
2
60
Table 2.3.1.2 Sugar and phenolic contents and DM loss of wheat straw produced by
steam treatment.
1
TEP
sCHO
treatment
condition
3
(%)
4
PR
HT
REP
15
15
12.30
11.92
12.28
12.53
18.08
19.14
18.09
19
19
SEM (n=2)
DM loss
2.31
2.33
2.46
2.46
6.29
6.13
6.60
17.35
0.515
0.834
0.627
0.772
0.612
7.119
7.624
8.324
6.41
8.816
0.085
0.278
c
c
c
f
f
f
f
soluble carbohydrates
total extractable phenolics as tannic acid
3
pressure (bar)
4
5
replicate
6
means on the same column with the same superscript are not significantly (p>0.05)
different
7
2
61
2.3.2 soluble carbohydrates

Total sugar content and sugar composition of the WSE of steam-treated samples
are shown in Table 2.3.2.1. At 0 min reaction time increasing of pressure caused an
increase in the amount of soluble carbohydrates in steam-treated samples. Longer
o
reaction time at 17 and 19 bar (205 and 210 C) resulted in a decrease in soluble
carbohydrate concentration (Fig 2.3.2.1). Maximum values were obtained at 19 bar and
0 min reaction time. The reduction in recovery of WSE by increasing harshness of
treatment is explained by, a) transformation of sugars to volatile compounds (furfural
and HMF) as a consequence of destruction of monosaccharides (Tipson and Horton,
1988) and, b) binding of aldehyde reactive compounds and their intermediates to lignin
(Wayman and Chua, 1979).
Sugars were present in the water extracts in the following descending order:
xylose, glucose, arabinose, galactose, mannose, rhamnose and fucose for both total and
monomeric sugars. Increasing harshness of treatment caused an increase in the
proportion of monomeric sugars to total soluble carbohydrate content of WSE except at
19 bar and 0 min reaction time. This trend implies a decrease in the degree of
polymerization of soluble carbohydrates. Reduction in the proportion of monomeric
sugars to total insoluble carbohydrates at 19 bar and 0 min reaction time (the harshest
condition used in this study) could be due to destruction of most of sugars to browning
compounds. In the present study no exogenous acid was added prior to treatment
therefore the higher glucose concentration normally resulting from cellulose hydrolysis
62
was not observed.
63
Table 2.3.2.1 Neutral sugar composition of total and monomeric carbohydrates in water
soluble extract from steam-treated wheat straw at different conditions.
1
xyl
treatment
condition
9
ara
glu
gal
other
total
M/T
WSE
10
PR RT
15 0
11
18.85
5.95
14.90
3.10
2.10
44.90
0.29
1.52
0.80
0.23
0.57
3.40
32.85
4.85
12.00
2.30
1.25
53.25
1.61
1.59
0.53
0.41
0.36
4.48
25.90
2.20
11.75
2.15
1.65
43.65
2.93
1.41
0.59
0.47
0.85
6.25
27.90
5.30
12.55
2.65
1.55
49.95
0.60
1.58
0.52
0.27
0.35
3.32
31.30
2.90
9.85
1.75
1.10
46.90
1.91
1.47
0.47
0.35
0.20
4.39
15.35
1.30
8.65
1.20
1.40
27.90
6.41
2.54
1.14
0.55
1.09
11.72
42.50
5.00
6.45
1.95
2.30
58.20
2.10
2.26
0.36
0.43
1.35
6.49
25.95
2.35
5.60
1.50
2.10
37.50
7.97
2.62
0.77
0.65
1.53
13.53
9.70
1.80
5.75
0.75
3.35
21.35
2.22
1.24
2.22
0.51
0.80
6.98
0.723
0.185
0.392
0.126
0.141
0.959
0.030
0.030
0.022
0.091
0.032
0.101
12
10
17 0
10
19 0
10
SEM13 (n=2)
xylose
arabinose
3
glucose
4
galactose
5
sum of mannose, rhamnose and fucose
6
total carbohydrates
7
monomeric pentoses/total carbohydrates
8
water soluble extract recovery
9
pressure (bar)
10
reaction time (min).
2
64
30.15
7.57
40.90
8.40
36.73
14.31
41.02
6.64
38.85
9.35
28.84
41.99
40.63
11.15
32.33
36.08
25.97
32.69
11
total sugar
monomeric sugars
13
12
65
Fig 2.3.2.1 Effect of pressure and reaction time on soluble carbohydrate (sCHO) content
of the water soluble extract from steam-treated wheat straw (see data on appendix 1).
66
Despite the low cellulose hydrolysis during treatment the high sugar content
observed in the WSE indicates its potential use as an monogastric animal feed. Unlike
ruminants which are very efficient in degrading any form of soluble carbohydrates,
monogastric animals are not able to degrade 1-4 linked hemicellulosic carbohydrates
(Longstaff et al., 1988; McDonald et al., 1988). Thus, the proportion of monomeric
pentoses is an important factor which should be taken into consideration when using
WSE as a monogastric feed.
It is reported that xylose and arabinose are not efficiently utilized by the pig (Yule
and Fuller, 1992). Therefore, the choice of optimum treatment conditions for production
of water extract would be a compromise between different factors. Apart from those
discussed above, increasing of harshness of condition will reduce the recovery of water
extract.
Van Soest and Mason (1991) reported that heating a feed can cause production of
insoluble browning compounds, mainly through the Maillard reaction which causes an
increase in acid detergent fibre (ADF) content. Apparently, production of insoluble
browning compounds occurs at later stage of browning reaction. Presumably, the
browning reaction compounds formed at early stages of treatment tend to be more water
soluble than those formed at later stages.
Another very important aspect related to nutritive value of WSE is the capacity of
monogastric animals to absorb and metabolize pentoses. Yule and Fuller (1992) reported
health problems in pigs when arabinose was fed at a level of 750 g/day.
67
The highest concentration of arabinose in this study was 8.8% observed in

samples treated at 17 bar and 10 min reaction time. Even if the WSE is used as the only
energy source in pig nutrition, the quantity of arabinose in the ration will not reach to
harmful level reported by Yule and Fuller.
According to the same study xylose and arabinose were not efficiently
metabolised by the pigs after being absorbed in the small intestine. One way to avoid this
would be to prepare WSE in such a way as to contain sugars with a high degree of
polymerization to avoid intestinal absorption. Research on this respect is needed to
elucidate the effects of such compounds in monogastric nutrition.
2.3.3 nutritive value of water soluble extract

WSE composition is shown in Table 2.3.3.1. Presence of furans in steam-treated
samples is not at a high enough level (maximum measured is 1.44%) (Section 3.3.5) to
explain the reduction of sugars in samples treated under severe conditions. Regarding
the low concentration of N (Table 2.3.3.1) in the water extract, it is believed that
Maillard reaction is not the major reaction happening during steam treatment. As
mentioned earlier (Section 1.4.3) Maillard reaction is a type of browning reaction which
proteins or amino acids are involved. The caramelization reaction which involves only
sugars may also be occurring intensively.
Although browning compounds form an important proportion of the WSE, they
could be relevant if they were utilized to a reasonable extent. Utilization of such
68
compounds was assessed by comparing the nutritive value of sugars in steam-treated
69
Table 2.3.3.1 Chemical analysis of water soluble extract from steam-treated wheat straw.
1
BRC
TEP
treatment
conditions
6
ASH
ODML
(%)
PRS RT
15
17
19
29.9
7.03
2.29
16.5
0.83
26.2
7.47
1.53
12.9
10.3
10
37.5
8.28
1.35
9.4
15.9
26.6
6.90
1.73
13.6
4.8
31.4
7.36
1.46
12.5
13.2
10
34.4
12.8
0.816
24.6
20.5
22.9
6.51
0.803
12.7
7.1
42.1
11.2
0.816
7.1
17.7
10
40.6
12.2
1.22
25.1
22.1
0.557
0.031
0.573
SEM (n=2)
1
browning reaction compounds (see footnote on page 43)

total extractable phenolics
3
nitrogen
4
ash
5
original dry matter loss
6
pressure (bar)
7
reaction time (min)
8
2
70
samples with a control sample containing only sugar. Gas production data is illustrated
in Fig 2.3.3.1.
Gas production after 48 h incubation indicated no difference (p>0.05) between
WSE and sugar control 1. This indicates that browning compounds produced during the
steam treatment were not fermented by rumen micro-organisms.
The content of browning reaction compounds (see footnote on page 43) in that
sample was high enough (42%) to conclude that such compounds had no detrimental
effect to rumen microbial activity. Forsberg et al., (1986) reported that the caramelized
sugars may be utilized by microorganisms. The present study clearly showed that rumen
microorganisms are not able to utilize such compounds as a source of energy.
DM loss during steam treatment is considered to be an important nutrient loss as
the volatile compounds are mostly formed from hemicellulose degradation (Table
2.3.3.1). Results from this study showed that formation of browning reaction compounds
were the most important sources of nutrient loss during steam treatment.
Both low and high molecular weight compounds are generated by the Maillard
reaction. Only monomeric or low-molecular weight products have been well studied.
Low molecular weight Maillard products (condensates between sugar degradation
products and amino acids), when absorbed, cannot be utilized by the animal and are
consequently excreted in the urine (Van Soest and Mason, 1994). It should be noted that
at harsher conditions they are either transformed to insoluble materials (Van Soest and
Mason, 1991) or bind to lignin (Wayman, 1979).
71
Fig 2.3.3.1 Gas production from the water soluble extract from steam-treated wheat
72
straw (see data on appendix 2).
73
As mentioned in the Section 1.4, lignin depolymerization to lower molecular

weight phenolics occurs concurrently with solubilization of hemicellulose. Data for
extractable phenolics showed that no matter which treatment condition was used there
was always a positive correlation between extractable phenolics and harshness of
treatment.
The quantities of ash in the water extracts compared to ash content in the original
sample (7.09%) indicated the effect of steam treatment on ash solubilization. Ash
1
solubility was in a range from 32.4% to 100% from samples treated at 19 bar pressure,
5 min reaction and 17 bar pressure, 10 min reaction time respectively.
7.1 (Table 2.3.3.1) x 32.33 (Table 2.3.2.1) / 7.09 = 32.4
74
2.4 Conclusions
Level of soluble carbohydrates in WSE from steam-treated wheat straw reached
high level (>57%) at 19 bar and 0 min reaction time. Soluble carbohydrates were found
to be the main degradable component present in water extract which can be used as a
energy source by rumen microorganisms. Production of browning compounds during
steam treatment is the main source of nutrient loss though such compounds do not
restrict microbial activity. Monomeric pentoses in steam-treated LM were present at a
very low level ( 2.5%) thereby indicating low risk to animal health. Production of
WSE at milder treatment conditions (19 bar and 0 min reaction time) seems more
advantageous compared to harsher conditions (17 and/or 19 bar pressure and 5 and/or 10
min reaction time) due larger quantity of WSE and lesser production of browning
compounds.
75
CHAPTER 3
IDENTIFICATION OF INHIBITORY COMPOUNDS PRODUCED DURING

STEAM TREATMENT OF LIGNOCELLULOSICS
3.1 Introduction
Organic acids are important catalysts of chemical reactions that occur during
steam treatment (Baugh and McCarty, 1988). Under such conditions various inhibitory
compounds can be produced, e.g. phenolics and browning reaction products (Castro et
al., 1995).
The quantity of extractable phenolics detected in steam-treated LM are positively
correlated with harshness of treatment (Castro, 1994). Previous studies on the effect of
steam treatment upon the molecular size distribution of lignin derivatives have shown a
wide range of size of such compounds (Chua and Wayman, 1978). Nimz (1974) has
observed that under acidic hydrolysis conditions lignin derivatives can be detected with
various degrees of polymerization (monomers, dimers, polymers).
Furfural and HMF are considered to be reactive compounds able to react with
amino groups in protein and also with lignin derivatives (Wayman and Chua, 1979a).
Such reactions explain the high lignin apparent extractability observed in steam-treated
LM.
Production of extractable compounds inhibitory to yeast, fungi, rumen microbes
76
and enzymes have been reported in several papers related to steam treatment (Forsberg
et al., 1986; Kyuma et al., 1991; Castro et al., 1995).
Several efforts have been made to modify the in vitro gas production technique in
order to detect the toxicity of tannins in feeds (Khazaal and rskov, 1994; Makkar et al.,
1995). Such methods are based on the use of a polymer which make complex with the
active toxic compound (tannins) thereby negating its properties. Several polymers have
been used, polyvinylpolypyrrolidone (PVP) (10,000, 40,000 and 360,000 M.W.) or
polyethylene glycol (PEG) (2,000, 6,000 and 35,000 M.W.) (Khazaal and rskov, 1994;
Makkar et al., 1995).
Complexing agents were originally used to bind and inactivate polyphenolic
compounds (tannins) while extracting enzymes from plant cells (Badran and Jones,
1965; Loomis and Battaile, 1966). It is believed that these complexing agents bind to
phenolic compounds through hydrogen (H) bonding (Andersen and Sowers, 1968). PVP
is an inert compound so it does not affect microbial activity. Such properties were
considered to be useful in order to detect the presence of antinutritive compounds
(tannins) in animal feeds (Khazaal and rskov, 1994; Makkar, et al., 1995). Tannins
type and origin could have a great effect on their affinity for a specific polymer (Makkar
and Becker, 1993b; Makkar et al., 1995). Since polyphenolic compounds contain more
hydroxyl groups than do low molecular weight phenolics one would expect that PVP
will bind more strongly to the former.
With respect to the use of in vitro techniques to identify presence of compounds
77
toxic to rumen microbes, one has to consider the substrate:inoculum ratio. Under in vitro
conditions this ratio is normally much lower than those observed in vivo. This will
consequently affect any possible toxic response when inhibitory compounds are present.
The in vitro gas production technique was originally validated with 200 mg
substrate and 30 ml rumen-liquor artificial saliva mixture (1:2 ratio) (Menke and
Steingass, 1988). Although this ratio helps to maintain an optimum environment for
microbial fermentation, the concentration of inhibitory compounds may not be sufficient
to affect microbial activity (Castro, 1994). This aspect has been considered in previous
studies (Castro, 1994, Makkar et al., 1995) whereby the amount of substrate was
significantly increased (1,000 and 500 mg respectively). On the other hand, increasing
the quantity of substrate will increase the concentration of fermentation end-products
(i.e. VFA) thus depressing the pH and microbial activity.
With respect to formation of materials inhibitory to rumen microbial
fermentation, Britton (1978) reported that rumen microbial inhibitors were present in
steam-treated sawdust. However, it was not established what type of compound was
responsible for the observed depression in cellulose degradation. Clark and Mackie
(1984) observed that low molecular weight phenolic compounds were the main
inhibitors to yeast activity in steam-treated LM. Castro (1994) reported that phenolic
compounds in steam-treated wheat straw were responsible for depression in microbial
activity in vitro.
In Chapter 2 it was shown that soluble browning reaction compounds formed
78
during steam treatment had no detrimental effect on rumen microbial activity, therefore,
the inhibitory effect detected in previous experiments may well be related to the
presence of other compounds (phenolic compounds and furans).
The negative effect of natural phenolic compounds, i.e. monomers (Jung et al.,
1983; Akin and Rigsby, 1985; Eraso and Hartley, 1990), dimers (Hartley and Harris,
1981; Eraso and Hartley, 1990) and polymers (tannins) (Kumar and Singh, 1984;
Waghorn et al., 1990; Khazaal et al., 1993b) on rumen microbial activity has been well
described. Polyphenolic compounds have received more attention as they are considered
to be more toxic to microbes.
Toxicity of furfural to yeast (Sanchez and Bautista, 1988; Fireoved and
Mutharasan, 1986) and rumen microbes (Kyuma et al., 1991) has been also reported.
In this study the compounds formed during steam treatment that are inhibitory to
rumen microbes were investigated. A more detailed study was carried out on phenolic
compounds. The effects of low and high molecular weight phenolics and of the two
furans (furfural and HMF) on rumen microbial activity were also studied.
79
3.2 Material and methods
3.2.1 substrate
Wheat straw variety Riband, Carpinus duinensis (CD - a mediterranean browse),
and rye grass (early bloom) were used. Air-dried samples were ground through a 1 mm
screen and stored at room temperature.

Steam treatment conditions are shown in Table 3.2.2.1. For acid hydrolysis, 2%
H2SO4 (DM basis) was added to wheat straw prior to steam treatment. For extracting
materials using ethanol solutions, acid hydrolysed wheat straw was used. Acid
impregnated wheat straw was subjected to steam treatment at 19 bar pressure and 10 min
reaction time (the harshest conditions used in this study). Such treatment conditions were
applied because in previous studies (Castro, 1994; Britton, 1978) depression in microbial
activity were observed from the acid hydrolysed LM.
3.2.3 extraction of water soluble extract

o
CD and steam-treated wheat straw were soaked in water 1/30 (w/v ratio) at 40 C
for 1 h. The mixture was filtered through a sintered glass No. 2 filter and the filtrate was
o
centrifuged at 10,000 x g and 4 C for 10 min. The clear supernatant and residue were
o
stored at 4 C for further analyses.
80
Table 3.2.2.1 Steam treatment conditions applied to wheat straw.

treatment condition
pressure
heating up time
reaction time
(min)
(min)
(min)
15
auto-hydrolysis (no acid added)

15
5
10
17
19
17
0
5
10
5
7.30
0
5
10
5
2.30
8.30
8.50
19
10.50
11.30
acid-hydrolysis (2% H2SO4 on DM basis)
15
15
17
17
17
7.15
7.45
7.50
9
10
19
19
0
5
10
0
5
10
4.30
6
3
7.30
9
0
5
10
*
15
*
25
0
0
81
steam treatment with direct injection of steam
82
3.2.4 effect of addition of PVP on microbial fermentation

Biological activity of phenolic compounds in steam-treated wheat straw was
assessed at 600 mg substrate loading. Steam-treated wheat straw samples were
neutralized prior incubation by mixing 1 g sample with 10 ml water and neutralized by
0.5 M NaOH and then were freeze dried. Six hundred mg PVP was added to the gas
syringes prior to incubation. Gas production procedure was followed as described in
Section 2.2.10. Gas production was recorded at 3, 6, 9, 12, 24 and 48 h incubation.
3.2.5 extraction of phenolic compounds

Phenolic compounds were sequentially extracted from steam-treated samples. A
diagrammatic representation of extraction is shown in Fig 3.2.5.1. Details are found
below.
3.2.5.1 ethyl acetate extraction

The extract containing phenolics (Section 3.2.3) was mixed with ethyl acetate
(EA) (1:1 v/v), shaken manually and left for 5 min to separate the two phases. The upper
layer containing EA was collected. This procedure was repeated four times. The EA
o
fraction was dried under vacuum at 40 C using Rotary Evaporator. The dried materials
were dissolved in pure acetone and transferred into a flat glass tray. The acetone was
dried off under nitrogen and the phenolic compounds collected by scraping from the
dish.
83
Fig 3.2.5.1 Procedure for the extraction of phenolic compounds (EAEP and WIP) from
steam-treated wheat straw and Carpinus duinensis using ethyl acetate and acetone
84
solutions.
85
The efficiency of extraction of phenolic compounds from aqueous solution using

EA was tested by extracting phenolic compounds up to 5 times from the WSE from
steam-treated wheat straw (see Section 3.2.3). After each extraction the water soluble
phenolic compounds were measured by the Folin and Ciocalteu reagent (Julkunen-Tiitto,
1985). Standard procedure is described as four times extraction using ethyl acetate.
3.2.5.2 extraction of water insoluble phenolics

One g water insoluble residue (Section 3.2.3) was mixed with 30 ml 70% acetone
solution (1/30, w/v). The mixture was ultrasonicated (using a water bath ultrasound with
720 watts power) for 5 min, mixed and ultrasonicated again for 5 min. The mixture was
o
centrifuged at 4,000 x g and 4 C for 20 min and the clear supernatant separated off.
o
Acetone solution was evaporated under vacuum at 40 C using a Rotary Evaporator. The
dried materials were dissolved in pure acetone and transferred into a flat glass tray. The
acetone was dried off under nitrogen and the phenolic compounds collected by scraping
from the dish. This fraction was called water insoluble phenolics (WIP).
3.2.6 effect of extraction procedure by ethyl acetate on nutritive value of feed

Effect of EA on microbial fermentation in vitro was evaluated by measuring in
vitro gas production of EA treated and untreated sugar mixture (Section 2.2.10). Sugars
were mixed with EA (1/30, w/v) and kept for 30 min and the solvent was evaporated
o
under vacuum at 40 C using a Rotary Evaporator. The ethyl acetate treated sugar sample
86
was incubated in vitro and the gas production measured (Section 2.2.10).
3.2.7 HPLC analysis of phenolic compounds from steam-treated wheat straw

samples
Analysis of phenolic compounds was completed by reverse phase High
Performance Liquid Chromatography (HPLC). Acetic acid aqueous solution (2.5% v/v
basis) and 100% methanol were used for gradient elution at 2 ml/min. The linear
gradient started with 100% acetic acid solution and ended with 100% methanol after 45
min. A reversed phase column (Techsphere 25 Cm x 4.6 mm 5 m ODS column HPLC Technology) was used and the measurement was taken at 280 nm wavelength
(McMurrough, 1981). The phenolic compounds were extracted from steam-treated
wheat straw (150 mg on DM basis) using 80% methanol solution (v/v basis).
3.2.8 effect of ethyl acetate extractable phenolics and water insoluble phenolics on
rumen microbes
Equivalent amounts of extractable phenolics present in 200, 400 and 800 mg of
original sample from EAEP and WIP were added to 200 mg of a control feed (rye grass).
A similar procedure was used for the ethanol extracts (Section 3.2.9). All samples were
assessed by the gas production method (Section 2.2.10). Gas production was recorded at
3, 6, 9, 12, 24 and 48 h incubation.
87
3.2.9 extraction of phenolic compounds using ethanol solutions

Phenolic compounds were sequentially extracted from wheat straw acidhydrolysed at 19 bar pressure and 10 min reaction time using ethanol solutions as
described by Britton (1978). The extraction procedure is shown in Fig 3.2.9.1. A dried
acid-hydrolysed wheat straw sample (380 g) was mixed with 2 l pure ethanol. The
mixture was sonicated (using a water bath ultrasound with 720 watts power) twice, each
time for 5 min, with mixing in between and was filtered through a sintered glass No. 2
filter. The residue was mixed with 2 l 95% ethanol solution. The mixture was sonicated
and filtered as mentioned above. The same procedure was applied for extraction using
o
80% ethanol solution. The filtrates were left for 10 days at 5 C in the dark. The
supernatants and precipitates were separated by suction. The solvents were evaporated
o
under vacuum at 40 C using a Rotary Evaporator and the extracts were collected.
3.2.10 effect of EAEP and WIP at high substrate loading on microbial activity
Effect of the EAEP extract on microbial activity (gas production) using the
procedure described above was also assessed at high substrate loading. In Section 3.2.4
use of 600 mg substrate (steam-treated wheat straw) cause a low in vitro pH. Effect of
low molecular weight phenolics can not be detected by using complexing agents (e.g.
PVP) as PVP have more affinity to high molecular weight phenolics than to low
molecular weight ones. Effect of low molecular weight phenolics on rumen microbes at
similar pH was studied by increasing the level of substrate (rye grass) to 700 mg
88
Fig 3.2.9.1 Extraction of phenolic compounds from wheat straw acid-hydrolysed at 19

bar pressure and 10 min reaction time using ethanol solutions.
89
(Section 3.2.12). Amount of extract and gas production procedure were the same as
mentioned in Section 3.2.8.

Effect of phenolics (ethyl acetate extractable phenolics, water insoluble phenolics
and extracts using ethanol solutions) extracted from Carpinus duinensis and steamtreated wheat straw and also effect of levels of furans (furfural and HMF) on rumen
microbial activity were evaluated by Bonferroni T test.
3.2.12 optimum level of substrate

To find the level of substrate needed to change the pH to 6.0 after 24 h of
incubation, samples were incubated in vitro in 100 ml conical flasks. Four levels of rye
grass (200, 400, 600 and 800 mg in duplicate) were added in the flasks. Artificial saliva
composition and preparation as well as artificial saliva : rumen liquor ratio were the
same as mentioned in Section 2.2.10. A 1 ml sample was taken from the flask at 0, 3, 6,
9, 12, 24 and 48 h incubation and pH was recorded.
3.2.13 effect of furfural and HMF on rumen microbes

Effect of furfural and HMF on microbial activity was assessed by the gas
production procedure. Furans at 1, 2 and 4% (DM basis) were added to syringes
containing 200 mg rye grass. Gas production was recorded after 3, 6, 12, 24 and 48 h
90
fermentation. Gas production procedure was the same as mentioned in Section 2.2.10.
3.2.14 extractable phenolics

Total extractable phenolic content of samples were measured by the Folin and
Ciocalteu reagent (Julkunen-Tiitto, 1985) as described in Section 2.2.9.
3.2.15 total tannins

Two ml of extract as described in Section 2.2.9 was added to a test tube
containing 100 mg of PVP. The mixture was vortex mixed and kept in an ice bath for 10
o
min, occasionally mixed and centrifuged at 3,000 x g and 4 C for 10 min. The phenolic
content of the supernatant was determined as for total extractable phenolics (Section
2.2.9). Total extractable tannins were estimated as the difference between phenolic
content compounds before and after addition of PVP (Makkar et al., 1992).
91
3.3.1 effect of PVP on microbial fermentation

The low substrate:inoculum ratio used in most in vitro methods is a major
limitation for detection of inhibitory compounds (Section 3.1). Therefore, in this study
substrate level was increased from 200 to 600 mg and inoculum volume kept the same.
Results from in vitro gas production from steam-treated wheat straw and the
effect of adding PVP are presented in Table 3.3.1.1. There was no significant difference
(p>0.05) in gas production when PVP was added to samples.
Phenolic compound data from auto and acid hydrolysed samples are given in
Table 3.3.1.2. Total extractable phenolic content was increased by increasing the
harshness of treatment. Such a trend was not observed for tannin-like compounds. The
negative effect of tannins on feed digestion could be either through direct effect on
microbes or indirect effect by reduction in protein degradation (through precipitation and
protection of proteins) and thereby lowering N available to rumen microbes (Waghorn et
al., 1990). Such an effect have been observed in forages containing 2.5% (Sadanandan
and Arora, 1979) to 5% (Waghorn et al., 1990) tannins. Tannin contents for all treated
samples was much lower than the harmful levels reported for natural tannins.
Interestingly, the level of tannin-like compounds in acid-hydrolysed samples were lower
than that of auto-hydrolysed samples. The results imply that acid hydrolysis had a
negative effect upon formation and/or disruption of such compounds.
92
Table 3.3.1.1 Effects of steam treatment of wheat straw and addition of PVP on the in
vitro gas production after 12 and 24 h incubation.
incubation time (h)
12
treatment conditions
1
PR
HT
24
control
+PVP
control
+PVP
RT

19
11.5
2.5
57.5
55.0
ns
74.9
72.3
ns
10.5
55.1
56.5
ns
74.9
77.2
ns
8.5
59.5
59.3
ns
76.2
77.0
ns
8.5
7.5
56.4
55.7
ns
76.6
76.3
ns

19
17
SEM (n=2)
15
42.3
43.4
ns
65.3
66.2
ns
25
40.1
40.8
ns
61.6
62.2
ns
7.5
55.8
55.4
ns
80.6
79.8
ns
4.5
51.5
52.5
ns
78.0
78.2
ns
45.0
44.6
ns
72.1
71.5
ns
7.5
39.2
39.8
ns
67.3
67.2
ns
10
34.8
34.3
ns
63.2
62.6
ns
1.23
1.94
1.65
1.90
pressure (bar)
heating up time (time required to reach to a give pressure - min)
3
reaction time (exposure of sample to a specific pressure for known period of time)
4
not significantly (p>0.05) different from control
5
2
93
Table 3.3.1.2 Total extractable phenolic and total tannin content from auto-hydrolysed and
acid-hydrolysed wheat straw.
treatment condition
pressure
HT
(%)
RT
TEP
TT
2.64
0.30
5.36
0.70
10
8.06
0.65
0
5
3.84
7.19
0.44
0.67
9.88
4.99
6.82
6.38
9.18
10.1
7.11
5.14
0.97
0.37
0.56
0.51
0.87
0.82
0.71
0.44
4.78
6.75
5.94
4.95
5.84
6.57
6.35
7.32
5.86
8.17
8.14
5.88
6.37
6.23
6.23
6.46
0.735
0.29
0.72
0.40
0.24
0.49
0.32
0.45
0.54
0.56
0.66
0.68
0.59
0.46
0.21
0.57
0.75
0.243

15
17
15
17
10
5
7.30
19
0
5
10
10.50
5
11.30
2.30
15
15
0
5
10
17
17
0
5
10
17
7.15
4.30
7.45
6
7.50
3
9
7.30
10
9
19
19
0
5
10
0
15
0
25
5
SEM (n=2)
8.30
8.50
19
heating up time (time required to reach to a give pressure - min)

reaction time (exposure of sample to a specific pressure for a known period of time)
3
total extractable phenolics as tannic acid equivalent
2
94
4
5
total tannins as tannic acid equivalent

95
Results from this study refer mostly to high molecular weight phenolic compounds
which are believed to have a greater affinity to PVP. The results also suggest that high
molecular weight phenolic compounds derived from lignin depolymerization were not
toxic to rumen microbial activity.
3.3.2 effect of EAEP and WIP on rumen microbes

Apart from polyphenolic compounds, low molecular weight phenolics could be
another source of toxic material in foods (Akin and Rigsby, 1985; Martin and Akin
1988; Borneman et al., 1986).
EA has been used to extract low molecular weight phenolic compounds from
aqueous solutions (Toussaint et al., 1991; Beltrame et al., 1992). Since in this study
EA was used to extract compounds from steam-treated wheat straw, the effect of the
EA extraction procedure on food microbial degradation was assessed. Results from in
vitro digestion of a sugar mixture treated with EA (which was subsequently removed
by evaporation) is shown in Table 3.3.2.1. This procedure did not affect (p>0.05) gas
production indicating that digestibility of food has not been altered by ethyl acetate
extraction. Extraction efficiency of phenolic compounds by EA is presented in Fig
3.3.2.1. Data clearly indicate that four-times extraction was enough to remove most
(97% of the phenolics which were extractable by EA) of the extractable phenolics
present in the sample.
Chemical activity of phenolic compounds, in particular tannins, may be
96
depressed when exposed to light or during sample preparation (Broadhurst and Jones,
97
Table 3.3.2.1 Effect of ethyl acetate extraction procedure on sugar fermentation by

rumen microbes.
incubation time (h)
12
24
gas production (ml)
sugar mixture
50.7
EA-treated sugar
53.4
SEM (n=2)
ns
1
62.2
ns
ns
64.2
0.739
0.604
not significantly different from control (p>0.05)

Fig 3.3.2.1 Efficiency of extraction by ethyl acetate of phenolic compounds from
98
water soluble extract of steam-treated wheat straw.
99
Table 3.3.2.2 Extractable phenolic, tannin and soluble carbohydrates data of the ethyl
acetate extractable phenolics and water insoluble phenolics from steam-treated wheat
straw and Carpinus duinensis.
(%)
1
STWS
SEM (n=2)
TT
23.1
1.1
10.2
44.8
6.5
20.8
EAEP
55.8
36.5
7.0
WIP
60.3
49.4
15.9
0.672
0.750
0.339
EAEP
WIP
CD
TEP
total extractable phenolics as tannic acid

total tannins as tannic acid
3
soluble carbohydrates
4
steam treated wheat straw
5
6
7
Carpinus duinensis (a browse)
8
2
100
sCHO
1978; Price et al., 1978). Chemical activity of the extracts using the procedure
described in Sections 3.2.5.1 and 3.2.5.2 were tested by extraction of EAEP and WIP
from CD and their effect on in vitro digestion of rye grass (control feed) was assessed.
Concentrations of extractable phenolics in EAEP and WIP extracts from steam-treated
wheat straw were lower than that from CD (Table 3.3.2.2). The reason may be that
some browning compounds and also sugars were also extracted due to their high
concentrations in steam-treated wheat straw. Gas production data for EAEP and WIP
extracts are shown in Fig 3.3.2.2. Gas production from CD showed a consistent
depression in food fermentation caused by addition of levels of extracts. Depression
was higher for WIP than EAEP fraction. This was expected due to higher
concentration of tannin in this fraction (Khazaal et al., 1993b). There was no
consistent reduction in
gas production from steam-treated wheat straw (Table 3.3.2.3). WIP contains high
molecular weight phenolic compounds which were expected to react with PVP.
Results from the effect of WIP on food fermentation agreed with those obtained in
Section 3.3.1.
Chesson et al. (1982) reported that monomeric phenolic compounds (pcoumaric and ferulic acids) are inhibitory to cellulolytic activity. Stimulatory effects of
some phenolic compounds on cellulose digestion have also been reported (Borneman
et al., 1986). These effects were observed when concentration was greater than 10
mM. Results of HPLC analysis of phenolics are given in Table 3.3.2.4.
101
Concentrations of ferulic and p-coumaric acids as well as total monomer content were
too low to affect microbial activity. According to Table 3.3.2.4, the sum of all
phenolic compounds
102
Fig 3.3.2.2 Effect of ethyl acetate extractable phenolics (EAEP) and water insoluble
phenolics (WIP) extracted from C. duinensis on rumen microbial activity (see data on
103
appendix 3).
104
Table 3.3.2.3 Effects of ethyl acetate extractable phenolics and water insoluble
phenolics from steam-treated wheat straw and Carpinus duinensis on in vitro gas
production.
gas production after 12 h (ml)
control (rye grass)
22.6
Carpinus
duinensis
EAEP
22.6
3
4
ns
22.6
21.4
ns
22.1
16.7**
20.7
21.7
17.8**
21.0
12.9**
21.7
1.13
1.15
1
2
WIP
steamtreated
wheat straw
SEM (n=2)
ns
ns
ns
ns
ns
ns
21.4
ns
ns
not significantly (p>0.05) different from the control

** significance of difference at p<0.01 level
1
2
equal amounts of phenolics (as tannic acid) present in 200 mg of original substrate
3
4
5
6
105
Table 3.3.2.4 Concentration of monomeric phenolic compounds in steam-treated wheat straw.

treatment condition
1
PRS
HT
(%)
RT
FER
COM
SUM
autohydrolysis (no acid added)

15
17
19
15
17
19
0.02
0.02
0.18
0.05
0.02
0.33
10
0.02
0.02
0.51
0.03
0.00
0.43
0.05
0.03
0.36
10
0.02
0.02
0.31
0.03
0.04
0.31
0.02
0.02
0.41
10
0.02
0.02
0.53

15
17
19
15
17
19
0.10
0.07
0.75
0.06
0.04
0.58
10
0.03
0.02
0.47
0.08
0.04
0.62
0.02
0.02
0.30
10
0.02
0.02
0.33
0.10
0.06
0.73
0.04
0.00
0.36
10
0.00
0.00
0.46
pressure (bar)
3
reaction time (min)
4
ferulic acid
5
coumaric acid
6
sum of phenolic compounds (p-OH-benzoic acid, p-OH-benzaldehyde, vanillic acid, syringic
acid, vanillin, syringaldehyde, coumaric acid, ferulic acid and benzoic acid)
2
106
measured by HPLC was much lower than that of extractable phenolics measured by the Folin
and Ciocalteu reagent (Julkunen-Tiitto, 1985) (in a range of 6.4 to 31.9 from autohydrolysed at
17 bar, 0 min reaction time and acid-hydrolysed at 15 bar, 0 min reaction time respectively).
According to Conchie et al. (1988) phenolic compounds derived from lignin could react with
the HPLC column packing preventing complete elution. It should be noted that the
aforementioned limitation for measuring phenolic compounds (see footnote on page 43) by
Folin and Cicalteu reagent also exist here. This comparison was made because of big
differences observed in quantity of phenolics estimated by the two methods.
3.3.3 effect of phenolic compounds from steam-treated wheat straw on rumen

microbes at high substrate loading
It has been reported that the affinity of polyphenolic compounds for PVP is
dependent on pH, with highest affinity at pH 3-4 (Andersen and Sowers, 1968; Makkar
et al., 1995). Low rumen pH (5.89) in animals fed steam-treated bagasse has also been
reported (Castro and Machado, 1990).
Although in Section 3.3.1 the effect of polyphenolic compounds was assessed at
high substrate loading (600 mg) no effect of the low molecular weight phenolic
compounds was detected.
The effect of substrate loading on pH is shown in Fig 3.3.3.1. The optimum level
of rye grass to produce pH6.0 after 24 h incubation was chosen as 700 mg. Gas
production results are presented in Table 3.3.3.1. EAEP did not affect (p>0.05) food
fermentation confirming the results obtained in Section 3.3.2.
107
108
Fig 3.3.3.1 Effect of substrate (rye grass) loading on pH (see data on appendix 4).
Table 3.3.3.1 Effect of ethyl acetate extractable phenolics at high substrate loading on
rumen microbes.
incubation time (h)
12
sample
gas production (ml)
control
1
EAEP
57.0
60.1
57.8
4
5
89.2
1
2
SEM (n=2)
24
ns
90.1
ns
ns
86.4
61.5
ns
90.9
1.82
1.62
ns
ns
ns

2
3
1
109
4
5
110
3.3.4 effect of ethanol extract from steam-treated wheat straw on rumen

microbial activity
Britton (1978) reported that extracted materials from steam-treated sawdust
using sequential extraction by ethanol solution were able to depress rumen microbial
activity in vitro. In the ethyl acetate and acetone extraction method (Section 3.2.6 and
3.2.8), substrate was exposed to water for extracting phenolic compounds. Although
the control phenolic compounds from CD were shown to be active enough to affect
microbial fermentation, one could argue that compounds in the steam-treated wheat
straw could have been inactivated to some extent during the extraction process.
Aqueous solutions of organic solvents are preferred for extracting phenolic
compounds because of higher recovery and lower oxidation. In this section, the
alcohol extraction procedure reported by Britton (1978) was applied to extract
phenolic compounds from steam-treated samples.
Gas production data are presented in Table 3.3.4.1. As is shown, neither
supernatants nor precipitates affected gas production.
In Britton's study some points have to be considered. Britton had used
hydrolysed hardwood. It has been reported that the natural durability of many tree
species is due to toxicity of certain substances (phenols) that are deposited during
hardwood formation (Cowling and Kirk, 1976). Price et al. (1980) demonstrated that
boiling high-tannin sorghum grain in water did not improve its nutritional quality.
Such effect was attributed to the difficulty in extracting condensed tannins. The
111
inhibitors in Britton's report could be attributed to natural tannins rather than other
toxic materials produced during steam treatment.
Another important point is the concentration of extracts. The DM extracted
from steam-treated wheat straw using ethanol solutions are reported in Table 3.3.4.2.
In that study, the highest inhibition of rumen microbial activity was observed from the
precipitate of the 95% ethanol solution. Quantity of similar extract in the present study
was only 0.19% as compared to the original substrate. Such a low quantity is very
unlikely to affect microbial activity. Another point is that if the precipitates are formed
due to coupling of the low molecular weight inhibitory compounds during storage
period, such compounds are expected to be inactivated due to polymerization (Lomax,
J. A., personal communication, R. R. I.).
3.3.5 effect of furfural and HMF on microbial fermentation

Toxicity of furans formed during steam treatment was described in Section
1.6.1. Table 3.3.5.1 indicates that production of furans depends on treatment
conditions confirming previous results reported by Castro et al. (1995). The effects of
furfural and HMF on rumen microbial activity is presented in Table 3.3.5.2. There
was no significant difference (p>0.05) between treatments and control indicating that
furans had no detrimental effect on ruminal fermentation. It should be noted that the
highest furfural concentration (1.4%) were observed from the samples treated at
harsh conditions (acid-hydrolysis at 17 and 19 bar for 5 min reaction time and 15 and
112
17 for 10 min reaction time). Testing the effects of 4% of either furfural or HMF on
113
Table 3.3.4.1 Effect of supernatants and precipitates obtained by ethanol solutions on in vitro
gas production from rye grass.
incubation time (h)
ethanol
solution %
substrate
12
24
gas production (ml)

control
extracts
19.1
SUP
PRE
ns
32.5
ns
32.8
ns
32.1
ns
35.0
ns
34.0
18.3
ns
32.5
0.930
0.737
100
18.0
95
19.3
80
17.4
100
20.1
95
19.5
80
3
SEM (n=2)
33.6
ns
ns
ns
ns
ns
ns
ns

supernatant
2
precipitate
3
1
Table 3.3.4.2. Amount of dry matter extracted (supernatants and precipitates) from 380 g
acid-hydrolysed (19 bar pressure and 10 min reaction time) wheat straw by ethanol extraction.
100 % ethanol
extract
95 % ethanol
80 % ethanol
g
1
supernatant
36.9 (7.08%)
30.0 (7.90%)
10.4 (2.73%)
precipitate
0.80 (0.21%)
0.72 (0.19%)
0.30 (0.08%)
114
figures in parenthesis are percentage of extracted materials from the substrate
115
Table 3.3.5.1 Production of furfural and hydroxymethyl furfural from steam-treated

wheat straw under different treatment conditions.
pressure (bar)
15
reaction
time (min)
17
19
15
auto-hydrolysis
(no acid added)
17
19
acid-hydrolysis
(2% H2SO4 on DM
basis)
(%)
furfural
HMF
1
2
0.03
0.12
0.22
0.49
0.96
0.68
0.27
0.46
0.85
0.99
1.39
1.44
10
0.65
0.70
0.66
1.40
1.40
1.32
0.02
0.04
0.09
0.18
0.36
0.25
0.09
0.14
0.27
0.38
0.61
0.60
10
0.19
0.33
0.46
0.60
0.75
0.85
reaction time (exposure of sample to a specific pressure for known period of time)
116
Table 3.3.5.2 Effects of furfural and hydroxymethyl furfural on gas production in vitro
from rye grass.
incubation time (h)
12
24
gas production (ml)

control
furfural
HMF
SEM (n=2)
19.9
34.5
1% furfural
20.8
ns
36.7
ns
2 % furfural
22.1
ns
37.4
4 % furfural
21.4
ns
37.9
1% HMF
20.6
ns
36.3
2% HMF
20.0
ns
35.2
4% HMF
19.8
ns
35.6
ns
ns
ns
ns
ns
0.652
0.995
ns

2
1
117
microbial activity was a clear indication that they had no effect on microbial
fermentation which agrees with the data reported by Castro et al. (1995).
118
3.4 Conclusions
The effects of lignin derivatives on rumen microbial activity was assessed using
different approaches. The effect of high molecular weight phenolics was assessed by
three methods, PVP addition in vitro, WIP extract and precipitates from ethanol
extracts. Low molecular weight phenolics were tested by two methods (EAEP extract
and supernatants from ethanol extract). None of these fractions affected rumen
microbial activity in vitro indicating that the phenolic compounds derived from lignin
depolymerization are not toxic to rumen microbes.
Assays completed with furans also indicated that such compounds are not
harmful to microbial fermentation.
119
CHAPTER 4
PROTEIN PRECIPITATING CAPACITY OF LIGNIN EXTRACTED FROM

STEAM TREATED WHEAT STRAW
4.1 Introduction
Among the chemical components of steam-treated lignocellulosics, lignin has
been the least studied in animal science. One reason may be that lignin does not
contribute to the energy value of the fibre (Baker, 1973; Hartley, 1972). Research on
lignin regarding animal nutrition has focused on methods of lignin disruption to improve
cell wall bio-availability (Morrison, 1983).
The most promising treatment for the exploitation of agricultural fibrous biomass
is based upon the auto-hydrolysis of the fibre (Focher et al., 1990). Although substrate
bio-availability to cell free enzymes is markedly increased after steam treatment
(Knappert et al., 1980; Brownell et al., 1986; Kuznetsov et al., 1990) studies on enzymic
hydrolysis have shown partial enzyme inactivation in the presence of steam-treated
lignin (Puls et al., 1985; Beltrame et al., 1992). Excoffier et al. (1991) suggested that
such enzyme inactivation may be related to the presence of active phenolic compounds
which have the ability to bind to enzymes.
The ability of tannins to bind to proteins (Makkar et al., 1987) and enzymes
(Feeny, 1969) has been well documented. Tannins are considered to negatively affect
120
rumen degradation (metabolism) as they can inhibit microbial activity (Terrill et al.,
1992). However the presence of tannins can also be positive to animal nutrition by
protecting protein from being degraded in the rumen (Waghorn et al., 1987; Barry,
1989) and also by preventing bloat on legume pastures (McLeod, 1974).
Kawamoto et al. (1992) reported that lignin degradation products formed during
steam treatment were capable of precipitating of bovine serum albumin. Such a
precipitating capacity was found to be greater in the lignin samples as compared to
immobilised tannins. Such an activity of lignin derivatives was attributed to formation of
hydrogen bonds between phenolic compounds and protein. Similarly one could expect
that enzymes may be adsorbed and thereby inactivated in presence of lignin from steam
treated LM.
It was concluded in Chapter 3 that lignin degradation products had no
detrimental effect on ruminal fermentation. Although harmless to rumen microbes these
compounds may have other important effects such as precipitation of proteins as has
been reported for tannins (Makkar et al., 1987; Makkar, 1989).
The aim of this study was to determine the effect of alkali extracted lignin from
steam-treated wheat straw on cell free enzymic activity.
121
4.2.1 substrate
Wheat straw var. Riband was used as a substrate for steam treatment. Sample
processing was completed as described in Section 2.2.1.

Tannic acid (Sigma, UK) and two lignin samples including Indulin (alkali lignin Sigma, UK) and lignin extracted from steam-treated (19 bar pressure and 0 min reaction
time) wheat straw. That sample was chosen because it contained the highest soluble
carbohydrate content among different steam-treated wheat straw samples (Chapter 2)
therefore using such sample would be more appropriate for different biological
purposes.
4.2.3 lignin extraction

The lignin extraction procedure has been illustrated in Fig 4.2.3.1. A 1 g steamo
treated sample was thoroughly washed with 30 ml water at 40 C to remove the water
soluble fraction. The solid residue was soaked in 1M NaOH (1/10, w/v) and kept for 30
o
min at 50 C. The mixture was filtered through a sintered glass No.2 filter. In order to
precipitate soluble lignin the filtrate was acidified to pH 2.5 with 2M H2SO4 and
o
centrifuged at 10,000 x g and 4 C for 10 min. The precipitate was recovered and
122
Fig 4.2.3 Extraction procedure of lignin from steam-treated (19 bar pressure and 0 min
reaction time) wheat straw.
123
washed with distilled water to remove excess reagent. The lignin sample was freeze
dried and kept at room temperature.
Lignin purification was completed according to Sutcliffe and Saddler (1986).
One g lignin was mixed with 20 ml acetic acid solution and mixed with 200 ml
o
water.The mixture was centrifuged at 10,000 x g and 4 C for 10 min and washed twice
with water. The lignin while suspended in water was neutralized with 0.5M NaOH
solution and then freeze dried. Lignin was kept at room temperature until further
analyses.
Indulin and tannin (tannic acid) were used as control samples. Indulin was
subjected to similar purification procedure as described above.
4.2.4 enzyme assays
4.2.4.1 effect of lignin and tannin on xylanase inactivation

Lignin was weighed (25, 50 and 100 mg) in duplicate into test tubes and then 4.5
ml of a mixture of xylan and sodium acetate buffer (0.2 M, pH 5.0) (10 mg/ml) was
added. Tannic acid (10, 5 and 2.5 mg) was dissolved in 0.5 ml sodium acetate buffer in
test tubes containing 4 ml xylan and sodium acetate buffer mixture. Then 0.5 ml of
4
enzyme (from Trichoderma viridi EC 3.2.1.8, sigma, UK) solution (5 times dilution
o
series - 0.202 IU) was added and incubated at 30 C for 30 min. Samples were heated at
o
100 C for 10 min to stop enzymic activity. Samples were centrifuged at 2,500 x g and
124
4 C for 20 min to precipitate the excess xylan. The supernatants were finally analyzed
for reducing sugars (Section 4.2.8).
4.2.4.2 enzymic inhibition by lignin extracted from steam-treated wheat straw

using gas production technique
Effect of lignin on cellulase activity was investigated using gas production
technique. The reason for using this method was to assess the effect of lignin extracted
from steam-treated wheat straw on rumen microbial activity as well because this aspect
was not studied in the previous chapter. Cellulase preparation (from Penicillium
funiculosum, EC 3.2.1.4, Sigma UK) was dissolved in artificial saliva (Section 2.2.10)
(5 mg/ml). Two hundred mg ground filter paper (Watman No.1 filter) was added to 100
ml standard glass syringes. Four levels (0, 25, 50 and 100 mg) of lignin were added
followed by an addition of 0.5 ml of enzyme (25 IU) solution. Twenty ml of artificial
o
saliva was added into the syringes and samples incubated at 37 C for 72 h. Ten ml
filtered rumen liquor (Section 2.2.10) was injected into each syringe. Samples were
o
incubated at 39 C and gas production was recorded at 2, 4, 6, 9 and 12 h incubation. Gas

production from blanks containing only filter paper was subtracted from treatments to
obtain gas production from sugars released on enzymic hydrolysis of cellulose.
4.2.5 effect of lignin extracted from steam-treated wheat straw on rumen microbial
125
activity
Gas production technique was used to detect the effect of levels of lignin on
microbial activity. Four levels (0, 100, 200 and 400 mg) were added into glass syringes.
Two hundred mg rye grass was added into the syringes containing the lignin. Procedure
of in vitro gas production technique was the same as mentioned in Section 2.2.10.

Effect of levels of lignin on enzymic inactivation and rumen microbial activity
was evaluated by ANOVA. Difference in enzymic inactivation as compared to control
samples were evaluated by T test.
4.2.7 protein precipitating capacity

Bovine serum albumin (BSA - Sigma, UK) was used as a model protein for
measuring protein precipitating capacity of phenolic compounds (lignin and tannin
samples). One hundred mg lignin samples were weighed into test tubes and 5 ml BSA
solution (10 mg/ml in distilled water) was added. Half ml tannic acid solution (20
mg/ml) was also added into test tubes containing 5 ml BSA solution. The mixtures of
phenolic compounds and BSA solutions were gently shaken using an electric shaker
(Janke & Kunkel type VX1) for 30 min at room temperature. The mixtures were
o
centrifuged at 18,000 x g and 4 C for 15 min and supernatant was collected. The
supernatants were diluted 5 times using acetate buffer (0.2 M, pH 4.0) and centrifuged at
126
18,000 x g and 4 C for 15 min and subsequently analyzed for protein.
4.2.8 protein analysis

The Bradford method (Bradford, 1976) was used by Kawamoto (1992) to detect
protein adsorbing capacity of lignin samples. An initial assay was completed in order to
identify a possible interference of lignin in the protein measurement. One hundred mg
lignin was mixed with 5 ml distilled water, shaken for 30 min and centrifuged at 18,000
o
x g and 4 C for 20 min. The reading from a series (0, 20, 40, 60, 80, 100 l) of
supernatants was mixed with Bradford reagent and recorded at 595 nm. Since readings
were affected by lignin concentration Bradford's method for measuring protein was not
used in the present study.
An alternative method was found to be more suitable for the present study.
Protein concentration was measured by the method of Makkar et al. (1987).
Half ml supernatant (Section 4.2.6) was transferred to a test tube and dried in an
o
oven at 80 C followed by adding 0.6 ml 13.5 M NaOH and heated in an oil bath at
o
120 C for 20 min to hydrolyse protein to constituent amino acids. The sample was
cooled, and 1 ml acetic acid was added and then vortex mixed. A 0.1 ml from the
solution was added into a test tube containing 1 ml ninhydrin reagent (Sigma product,
o
UK). Samples were incubated in an oil bath at 100 C for 20 min, cooled and 5 ml water
was added and then absorbence was recorded at 570nm. The protein content was
calculated from the standard curve obtained from a series of a BSA solutions (0, 0.2, 0.4,
127
0.6, 0.8, and 1 mg/ml).
4.2.9 reducing sugar analysis

Samples were analyzed for reducing sugars using dinitrosalicylic acid method
(White and Kennedy, 1981). Half ml sample was added in duplicate into test tubes and
0.5 ml water was added. One ml of dinitrosalicylic acid solution (75 g sodium potassium
tartrate and 25 mg dinitrosalicylic acid dissolved in 50 ml of 2M NaOH and 250 ml
o
H2O) was added into the test tube. The mixture was incubated at 100 C for 10 min and
rapidly cooled to room temperature. The absorbence was recorded at 570 nm.
Concentration of reducing sugars was calculated from a glucose standard curve.
128
4.3.1 inhibition of enzyme by lignin extracted from steam-treated wheat straw

The inhibitory effect of tannins on enzymes has long been known (Badran and
Jones, 1965). The protein binding ability of tannins has traditionally been used to tan
leather (Mueller-Harvey and McAllan, 1992). Data from xylanase inhibition by lignin
and tannin are presented in Table 4.3.1.1. Tannin was considerably more effective than
lignin in inhibiting xylanase activity. Although it has been suggested that enzymic
inhibition in steam treated LM may be due to the lignin based phenolic compounds, this
aspect has not been studied in detail. Makkar et al. (1987) reported that 2.34 mg protein
was precipitated by 1 mg tannic acid. The extent of enzymic inhibition by tannin
observed in this study was much lower than the protein precipitating capacity reported
by Makkar et al. (1987). This aspect will be discussed in more detail in Section 4.3.2.
The gas production technique was used to demonstrate the inhibition of cellulase
activity by lignin. Gas production data showed a negative correlation (r=-0.98) between
lignin levels and fermentation of sugar from hydrolysis of cellulose (Fig 4.3.1.1)
indicating that lignin negatively affected cellulose hydrolysis.
Further assay demonstrated that lignin extracted from steam-treated wheat straw
did not affect (p>0.05) microbial activity (Table 4.3.1.2). This information together with
the information obtained in previous chapter (Chapter 3) on the effect of extractable
phenolics on rumen microbial activity is an indication that the phenolic
129
Table 4.3.1.1 Effect of lignin (extracted from steam-treated wheat straw) and tannin on
xylanase activity.
samples
wt (mg)
hydrolysis of xylan (%)
control (pure xylan)
9.28
lignin
25
5.46**
50
4.05**
100
0.29**
2.5
7.99**
4.45**
10
0.16**
tannin
SEM (n=2)
0.063
**
2
significantly different (p<0.01) from the control

130
Fig 4.3.1.1 Effect of lignin extracted from steam-treated wheat straw on cellulase
activity (see data on appendix 5).
Table 4.3.1.2 Effect of different levels of lignin extracted from steam-treated wheat
straw on gas production in vitro from rye grass.
incubation time (h)
6
12
lignin (mg)
7.8
100
6.5
ns
18.0
200
7.7
ns
18.1
400
7.4
ns
ns
48
gas production (ml)
0 (control)
SEM (n=2)
24
19.9
0.553
34.5
ns
33.4
ns
34.4
16.7
ns
0.694

131
44.3
ns
44.9
ns
ns
44.5
32.8
ns
43.1
1.19
2.28
ns
ns
compounds formed from depolymerization of lignin during steam treatment of wheat

straw are not toxic to rumen microbes.
Such data proves that the lower gas production observed when lignin was
added to a mixture of cellulose (filter paper) and cellulase was indeed due to an
inactivation of the enzyme. The advantage of filter paper as substrate was that because
of less gas production from substrate (because of long lag time in digestion due to time
needed for microbial colonization on cellulose fibres) it was possible to correct for the
gas production from only sugars released on enzymic hydrolysis of cellulose. It was
observed that 12 h incubation was enough for digestion of all soluble carbohydrates
released on enzymic hydrolysis by rumen microbes

The Bradford method (Bradford, 1976) used by Kawamoto for measuring
protein adsorbing capacity of lignin was tested in this study. The negative effect of
lignin on readings taken at 595 nm is clearly seen in Fig 4.3.2.1. Such results clearly
indicated that protein adsorbing capacity may be overestimated by the Bradford
method. This indeed happened in Kawamoto's study in which he reported higher
protein precipitating capacity from lignin as compared to tannin.
The way in which lignin per se affects the reading seems to be related to a
partial precipitation of the main reagent (Bradford reagent).
Testing the method of Makkar et al. (1987) used in this study revealed that
132
lignin did not interfere with this assay.
133
Fig 4.3.2.1 Effect of lignin on the Bradford method for determining protein
134
adsorption.
135
It was found in this study that the reported methods for measuring protein
precipitating capacity of tannins are not suitable for measuring such property in lignin.
It was observed that although lignin binds to protein, the lignin-protein complex does
not precipitate completely unless the pH of the lignin-protein solution is reduced to
4.0. It should be noted that such pH value is not an optimum pH for lignin
precipitation during extraction of lignin.
Tannin solubility is explained by the hydrophillic property of the hydroxyl
groups in the molecule. Solubility of lignin is due to hydrophillic property of hydroxyl
and acetyl groups (Provan, G. J. personal communication, R. I. I.). Solubility of ligninprotein complex is likely to be due to functioning of the acetyl groups. Precipitation of
lignin-protein complex at low pH (pH 4.0) is probably because of inactivation of the
acetyl groups in the lignin molecule.
Results of protein precipitation capacity from lignin, indulin and tannin are
presented in Fig 4.3.2.3. The highest effect was obtained from tannin (2.4 mg protein
per mg tannin) and was in agreement with the results reported by Makkar et al. (1987).
Results of both enzymic inhibition assays (xylanase and cellulase assays) do not
agree with the protein precipitating capacity of the tested phenolic compounds. Protein
precipitating capacity of both lignin and tannin were much higher than their inhibitory
effect on enzymic activity. For instance, 1 mg tannic acid could precipitate 2.4 mg of
BSA but this quantity of tannic acid inactivated only 0.002 mg of enzyme.
Inactivation of enzymes by tannin is explained by denaturation of enzyme due to
136
Fig 4.3.2.3 Protein (BSA) adsorbing capacity of 1 g of the three phenolic compounds.
137
binding to tannin and changing the conformation of enzyme molecule (Strumeyer and
Malin, 1970).
The explanation for such a difference may be related to a possible precipitation
of enzyme without leading to inactivation. Molecular weight and also structural
conformation of protein molecule are important factors for being precipitated by
tannins. Small molecules (e.g. xylanase, 20,000 Dalton compared to BSA, 68,000
Dalton) are more resistant to denaturation than large molecules. This implies that
although tannin binds to the enzyme, it can not totally inactivate it (Begbie, R. and
Wood, T. M. personal communication, R.R.I). The mode of action of phenolic
compounds on inactivation of enzyme is explained by formation of either a soluble
(Calderon et al., 1968) or insoluble (Hagerman and Butler, 1978; Mole and Waterman,
1985) enzyme-protein complex. According to that explanation, reduction in enzymic
activity cannot solely be explained by reduction in solubility of the enzyme. It is likely
that the precipitated enzyme is still active as reported by Goldstein and Swain (1965).
The strength of adsorption of phenolic compounds to complexing agents has
been reported to be dependent on the number of hydroxyl groups and the site of the
hydroxyl groups on phenolic compounds (Doner et al., 1993). It is likely however that
the site of hydroxyl groups is more important for inactivation of enzymes than the
number of hydroxyl groups. This may be the reason for complete inactivation of
enzymes by condensed tannins even after recovery of enzyme from tannin-enzyme
complex (Strumeyer and Malin, 1970). Lignin is not classified in this category as it
138
showed lower activity as compared to tannic acid.

Although lignin showed less activity compared to tannin, it can still
significantly depress enzymic activity in steam-treated LM because of the large
quantity of lignin present in these materials.
Measuring the extent of enzymic inhibition has been suggested as an approach
to measure the biological activity of tannins (Goldstein and Swain, 1965). Results
from this study agrees with Mark and Leighton (1987) in which the quantity of
inactivated enzyme (protein) cannot be extrapolated to other types of proteins.
Indulin showed a lower protein precipitating capacity. The large difference
between protein precipitating capacity between indulin and lignin extracted from
steam-treated wheat straw could be due to the method of lignin preparation or lignin
source. Such an aspect will be considered in the next chapter.
139
4.4 Conclusions
Lignin extracted from steam-treated wheat straw inactivated enzyme
polysaccharidase and precipitated protein. Such effects were even more intense when
tannic acid was used. Indulin was the least effective compound.
140
CHAPTER 5
EFFECT OF TREATMENT CONDITIONS AND SOURCE OF

LIGNOCELLULOSICS ON PROTEIN PRECIPITATING CAPACITY OF
LIGNIN
5.1 Introduction
From the nutritional point of view, lignin has always been considered to be one of
the major factors depressing the feeding value of mature forage plants (Jung and Fahey,
1983). Among different cell wall components lignin has been the least studied. There
has been little research into the chemical basis of the effects of lignin for animal
nutrition mostly due to the fact that lignin is not a nutrient.
Rotstein et al. (1981) reported that auto-hydrolysed aspen lignin reduced the
incidence of cholesterol gallstones in hamsters fed a diet deficient in essential fatty
acids. The observed reduction in enzymic hydrolysis of steam-treated LM has not been
fully studied (Puls et al., 1985) and the interesting work by Kawamoto et al. (1992) on
protein adsorbing capacity of lignin has not been followed up. Use of lignin for making
slow-release herbicides has also been reported (Cotrim et al., 1993). Since lignin has not
yet been used in any large-scale industrial application, the great majority of its
production is burnt as a fuel.
The two different lignins studied in Chapter 4 showed a significant difference in
141
protein precipitating capacity. Kay et al. (1979) observed that different lignin
preparations varied greatly in their affinity to bile acids. Kawamoto et al. (1992) also
found that steam treatment conditions affected protein adsorbing capacity of lignin.
Although the protein precipitating capacity of lignin is considered as a
disadvantageous property as it may affect enzymic hydrolysis of steam-treated LM, this
fraction could have useful applications, e.g. cleaning protein-rich industrial wastes and
inactivation of plant enzymes during storage period to protect preserved feeds from
reduction in nutritive value (Wetherall et al., 1995). The main problem in this respect
would be the low activity of lignin compared to tannins.
It has already been discussed in Section 1.4.4 that the extent of lignin
depolymerization depends on steam treatment conditions. It is also known (Wayman and
Chua 1979 a,b) that the chemical properties of lignin will vary according to treatment
conditions. So, one could expect that the biological activity of lignins would also vary.
The aim of this study was to investigate the properties of lignin produced under
different steam treatment conditions (pressure, reaction time and use of catalyst) and
from different sources (wheat straw, barley straw, rice straw and sugar cane bagasse).
It should be mentioned that it may not be possible to optimize treatment
conditions in such a way to effectively use all fractions of steam-treated LM. We may
have to sacrifice one fraction for another according to the importance of the product.
142
5.2.1 substrate
Wheat straw, barley straw, rice straw and sugar cane bagasse were used in this
study. Samples were air dried, hammer milled through a 1 mm screen and stored at room
temperature for further analysis.

Wheat straw was auto-hydrolysed at 15, 17 and 19 bar and for 0, 5 and 10 min
reaction time. Acid hydrolysed wheat straw was prepared by adding 2% H2SO4 (on DM
basis) to wheat straw prior to steam treatment.
Barley straw, rice straw and sugar cane bagasse were auto-hydrolysed at 17 and
19 bar and for 0, 5 and 10 min reaction time.
Acetic acid was added to acid detergent fibre (ADF) from wheat straw to obtain
50% DM content with 2% acetic acid concentration on dry matter basis. After acid
addition, ADF was steam treated at 15 and 17 bar, 0 min reaction times and at 19 bar, 0,
5 and 10 min reaction times.

A completely randomized block design was used to evaluate the effect of
exogenous acid (H2SO4), pressure and reaction time on protein precipitating capacity of
143
lignin extracted from wheat straw, barley straw, rice straw and sugar cane bagasse). The
data were analyzed by ANOVA.
5.2.4 effect of hemicellulose on lignin quality

The effect of hemicellulose degradation products on protein precipitating
capacity of lignin was studied by investigating lignin extracted from a hemicellulose-free
wheat straw. ADF was prepared according to the method of Goering and Van Soest
(1970). Sample (25 g) and 2.5 l of acid detergent solution [20 g cetyltrimethylammonium bromide (C16H33N(CH3)3Br in 0.5M H2SO4] together with silicone solution
(0.75% v/v, 25 ml), as an anti-foam agent, were added to a beaker. The reaction mixture
o
was boiled for 1 h and residue was filtered and dried in an oven at 50 C for 48 h.
5.2.5 lignin extraction and purification

Extraction and purification of lignin samples was carried out as described in
Section 4.2.3.

Protein precipitating capacity of lignin was measured as described in Section
4.2.7.
144

Protein analysis was carried out using the method described in Section 4.2.8.
5.2.8 water soluble fraction of lignin

Two hundred mg lignin was mixed with 20 ml water. The mixture was gently
o
shaken for 30 min at room temperature and centrifuged at 10,000 x g and 4 C for 20
o
min. The supernatant was collected and solid residue was dried at 50 C for 48 h.
5.2.9 total phenolic content

Phenolic content of lignin was measured by the Folin and Ciocalteu reagent
(Julkunen-Tiitto, 1985) using indulin as a standard. Lignin samples were dissolved in
70% (v/v) acetone solution (lignin is completely soluble in this solvent). The procedure
for phenolic analysis was as described in Section 2.2.9.
5.2.10 ash content

Ash content of lignin samples was measured using the procedure mentioned in
Section 2.2.7.
5.2.11 nitrogen content

Nitrogen content of lignin samples were measured by the procedure mentioned in
145
Section 2.2.8.
5.2.12 total sugar content

Total sugar content of lignin samples were measured using GC. The procedure
was as mentioned in Section 2.2.5.
146
5.3.1 optimization of steam treatment conditions for production of lignin from

wheat straw
The protein precipitating capacity of lignins extracted and purified from autohydrolysed and acid-hydrolysed wheat straw are illustrated in Fig 5.3.1.1 and 5.3.1.2
respectively. All lignin samples showed a high protein precipitating capacity. Effect of
pressure and reaction time on lignin quality (protein binding capacity) showed
significant differences (p<0.01) between auto-hydrolysis and acid-hydrolysis conditions.
Use of exogenous catalyst (H2SO4) during steam treatment accelerates cell wall
hydrolysis (Castro, 1994) but no beneficial effect (p<0.01) on lignin quality was
observed. No significant correlation was detected between protein precipitating capacity
and either solubility of lignin (low molecular weight of phenolics) or total extractable
phenolics in the steam-treated samples. Such results indicated that although disrupting
lignin to smaller fractions is a key factor for generating its binding ability to proteins, it
is not the only factor affecting this lignin property. This was clearly seen in the present
study as further lignin depolymerization at harsher treatment conditions did not improve
(p<0.01) the lignin quality. Differences in the quality of lignin samples is likely to be
related to formation of different molecular structures containing variable number of
hydroxyl groups.
Hagerman and Butler (1978) have described the similar effect of depolymerizing
147
condensed tannins to catechin and observed that its protein precipitating capacity was
148
Fig 5.3.1.1 Effect of steam treatment conditions on protein precipitating capacity of

lignin extracted from auto-hydrolysed wheat straw (see data on appendix 6).
149
Fig 5.3.1.2 Effect of steam treatment conditions on protein precipitating capacity of

lignin extracted from acid-hydrolysed wheat straw (see data on appendix 7).
150
not improved. Kawamoto et al. (1992) also did not find any correlation between
molecular size and protein precipitating capacity from lignin samples which indicated
that this property is likely to be related to internal lignin characteristics. Cotrim et al.
(1993) observed that the affinity of lignin to herbicides was positively correlated with
total hydroxyl content of lignin.
It has been reported that depolymerization of lignin during mild acid-hydrolysis is
due to cleavage of -aryl position (Fig 1.4.4.1) (Nimz, 1974). Under such conditions
only partial lignin depolymerization occurs. According to Freudenderg and Neish (1968)
extensive depolymerization of lignin can only be achieved when -O-4 bond (Fig
1.4.4.1) are disrupted. It is also known that a condensation reaction (increasing of carbon
content and decreasing of hydrogen and oxygen content in lignin molecule by formation
of carbon-carbon bonds and elimination of water at long reaction time. e.g. 30 min
reaction time). takes place under more severe treatment conditions (Chua and Wayman
1978; Lora and Wayman, 1978). It is likely that the position of cleavage in the lignin
molecule is an important factor which will affect its protein precipitating capacity.
No further improvement occurred in protein precipitating capacity of lignin when
harsher treatment conditions (i.e. use of exogenous acid) were applied. This could be
due to reaction between furfural and HMF with lignin (Chua and Wayman, 1978) or to
increased condensation.
In general it can be suggested that higher pressure could improve lignin quality
from wheat straw at 0 min reaction time.
151
5.3.2 lignin quality from barley straw, rice straw and sugar cane bagasse
The optimization work on lignin quality from wheat straw (Section 5.3.1)
showed that the use of an exogenous catalyst had no beneficial effect on protein
precipitating capacity of lignin. On the other hand, 15 bar pressure was shown to be
insufficient for production of good quality lignin. Therefore, a series of other LM were
steam-treated at 17 and 19 bar pressures and 3 reaction times (0, 5 and 10 min). Protein
precipitating capacity of lignins from barley straw, rice straw and sugar cane bagasse are
presented in Table 5.3.2.1.
Increasing pressure did not show a positive effect on lignin quality while
increasing of reaction time improved lignin quality from the all LM studied except for
barley straw at 19 bar pressure. The results suggest that the method for lignin preparation
from one LM may not be suited to other types of LM. Increasing of harshness of
treatment conditions (reaction time and pressure) causes more depolymerization of
lignin and consequently production of lower molecular weight lignin. No consistent
results in protein precipitating capacity of lignin samples and further depolymerization
of lignin from different lignin samples are in agreement with Kawamoto's (1992) report
that lignin molecular size is not correlated with its protein precipitating capacity.
There was a considerable variation in lignin quality amongst the different LM
studied.
152
Table 5.3.2.1 Protein precipitating capacity of lignin extracted from auto-hydrolysed

wheat straw, barley straw, rice straw and sugar cane bagasse at different treatment
conditions.
reaction time (min)
pressure (bar)
10
(%)
wheat straw
17
64.45
83.47
93.3
19
91.40
99.48
94.00
17
68.6
94.8
97.1
19
21.4
75.7
11.4
17
98.7
99.1
98.7
19
94.3
94.2
93.3
sugar cane
17
99.8
100.0
99.6
bagasse
19
5.7
20.5
94.5
0.982
0.727
0.814
barley straw
rice straw
SEM (n=2)
1
2
precipitated protein from a 50 mg protein (BSA - 5 ml, 10 mg/ml) by 100 mg lignin

153
5.3.3 lignin properties from different agricultural by-products

Different characteristics of lignin samples were studied in order to explain the
protein precipitating capacity of lignin and possibly find a way to increase its quality.
Characteristics of lignin extracted from auto and acid hydrolysed wheat straw and
from different agricultural by-products are presented in Table 5.3.3.1 and Table 5.3.3.2
respectively.
Increasing pressure and reaction time had a positive effect on quantity of
extracted lignin. Increasing harshness of treatment causes an increase in extent of lignin
depolymerization (Castro, 1994) and also affects the reaction between carbohydrate
degradation compounds and lignin (Chua and Wayman, 1978). It is not known whether
such reactions affects lignin quality. If pressure positively affects lignin quality (in wheat
straw), it could be speculated that artifacts (furfural and HMF) have detrimental effect
on this lignin property.
Harshness of treatment had a negative effect on sugar content of lignin. High
sugar content of lignin produced under mild treatment conditions could be mostly
attributed to cleavage of ether bonds at this stage of hydrolysis. It is known that ether
bonds are susceptible to acid hydrolysis (Lundquist and Lundgren, 1972). No correlation
(r=0.23) was observed between sugar content and protein precipitating capacity of lignin
samples. It is unlikely that the sugar content of lignins contributes to biological activity
of lignin samples. Thus the sugar content can be regarded as merely diluting the activity
of the lignin molecule.
154
Table 5.3.3.1 Characteristics of lignin extracted from auto-hydrolysed and acidhydrolysed wheat straw.
characteristics of lignin samples
1
WSF
EXT
TP
TEP
SUG
ASH
(%)
9
auto-hyd.
RT
15 bar
46.62
6.53
39.73
5.60
28.34
3.74
0.28
42.53
10.60
74.96
5.42
5.17
0.69
0.21
10
43.59
12.23
nd
8.02
1.02
0.54
0.21
42.05
10.43
58.69
4.16
13.36
1.04
0.21
34.03
12.71
81.54
7.27
1.88
0.89
0.25
10
29.16
16.46
81.84
10.37
1.05
0.99
0.11
29.05
11.37
53.81
10.44
2.70
0.93
0.36
34.74
15.02
61.95
9.84
0.98
1.00
0.25
10
29.87
17.89
57.21
11.15
0.83
1.01
0.02
35.88
8.72
55.37
4.91
1.20
0.94
3.33
32.44
9.56
91.51
5.93
0.33
1.35
2.69
10
28.75
12.52
83.61
6.64
0.94
1.15
3.65
34.71
10.29
73.33
5.84
1.17
1.21
3.56
33.06
10.70
74.37
6.62
0.92
1.34
3.08
10
29.32
10.78
76.62
7.81
0.89
1.26
3.54
29.00
10.95
80.70
5.70
0.97
1.30
3.03
30.56
12.01
81.82
7.43
0.68
1.00
3.38
10
28.50
9.01
81.14
6.39
0.84
0.85
3.66
1.262
1.758
0.445
0.155
0.107
0.108
17 bar
19 bar
acid-hyd.
15 bar
17 bar
19 bar
10
SEM (n=2)
1
water soluble fraction of lignin samples

percentage of extracted lignin relative to steam-treated samples
3
total phenolic content of lignin samples as indulin equivalent
4
total extractable phenolics in steam-treated samples as tannic acid equivalent
5
sugar (anhydrous) content of lignin samples
6
nitrogen content of lignin samples
7
ash content of lignin samples
8
relative to DM steam-treated wheat straw
9
reaction time (min)
10
2
155
Table 5.3.3.2 Characteristics of lignin samples from barley straw, rice straw and sugar
cane bagasse.
characteristics of lignin samples
1
WSF
EXT
TP
TEP
SUG
ASH
(%)
RT
barley straw
17 bar
19 bar
43.30
15.56
44.2
3.21
12.26
1.66
0.35
36.77
13.27
71.33
5.63
1.22
1.35
0.92
10
39.01
16.23
78.74
9.40
0.66
1.74
1.14
31.31
12.84
57.48
4.46
16.86
4.72
0.32
35.34
13.47
81.21
7.77
3.13
1.59
0.64
10
32.29
15.76
90.70
10.04
1.20
1.87
0.31
53.62
13.09
63.34
6.41
2.09
0.78
0.68
47.67
16.43
75.53
8.43
0.96
1.77
0.71
10
50.41
18.70
74.62
9.76
0.59
1.73
0.77
35.59
10.31
55.59
4.43
8.13
1.09
1.50
40.96
14.70
62.39
6.67
1.12
4.03
1.38
10
41.10
14.39
55.98
6.63
1.15
4.58
1.21
44.35
12.20
58.06
4.48
12.26
1.71
1.00
42.02
12.61
77.87
7.95
1.22
0.56
1.05
10
38.13
14.72
88.17
10.12
0.66
0.70
1.32
27.59
10.91
91.03
5.73
0.65
1.83
1.16
25.57
13.44
85.32
8.60
1.25
0.80
0.97
10
21.01
15.61
71.25
9.36
5.13
0.80
0.49
1.455
2.062
0.546
0.367
0.134
0.098
rice straw
17 bar
19 bar
sugar cane bagasse

17 bar
19 bar
10
SEM (n=2)
1
water soluble fraction of lignin samples

percentage of extracted lignin relative to steam-treated samples
3
total phenolics content of lignin samples as indulin equivalent
4
total extractable phenolics in steam-treated samples as tannic acid equivalent
5
sugar (anhydro) content of lignin samples
6
nitrogen content of lignin samples
7
ash content of lignin samples
8
relative to steam-treated LM
9
reaction time (min)
10
2
156
Results for the water soluble fraction from auto-hydrolysed wheat straw and other
LM did not show similar trends for different pressures and reaction times. Lignin
solubility from acid-hydrolysed wheat straw samples were consistently higher than the
auto-hydrolysed ones. Solubility of lignin samples was not in agreement with the results
of total extractable phenolics of original steam-treated (Table 3.3.1.2) LM. Such
discrepancy was more obvious for the acid-hydrolysed wheat straw samples.
Melting (Michalowics, et al., 1991; Toussaint et al., 1991) and repolymerization
(polymerization of phenolic compounds derived from lignin) (Schwartz, et al., 1989) of
lignin are associated with harshness of treatment conditions. High solubility of lignins
from acid-hydrolysed samples implies that melting of lignin may be a dominant change
occurred during the treatment. Therefore, the extraction and purification of lignin may
have caused the separation of lignin molecules thereby affecting its solubility.
Ash content of acid-hydrolysed samples were constantly higher than all the other
lignin samples. The presence of an acid catalyst positively affects ash content.
As mentioned in Section 1.3.3.1, several methods have been suggested for
measuring the lignin content of LM and several problems are associated with it.
Conjugation of browning reaction compounds with lignin was one reason for increasing
the quantity of extracted lignin. With the available methods it was difficult to precisely
measure lignin content and consequently estimate the conjugated browning compounds
with lignins.
No clear trend was observed between nitrogen content and protein precipitating
157
capacity of lignin samples. The nitrogen in all lignin samples is very likely to be in the
form of Maillard products (Van Soest and Mason, 1991).
5.3.4 Effect of hemicellulose removal on lignin quality

Formation of hemicellulose degradation products is an inevitable process which
is affected by the extent of exogenous acid and treatment conditions. The protein
precipitating capacity of lignin extracted from steam-treated ADF (from wheat straw) is
shown in Table 5.3.4.1. As is shown at mild treatment conditions hemicellulose removal
had a positive effect on lignin quality as compared to that of lignin extracted from
original steam-treated wheat straw. Dramatic reduction in lignin quality by harsh
treatment conditions implies that such a negative effect is more related to intermolecular
changes than reaction with browning compounds. Very negative effects of hemicellulose
removal on extraction of lignin was also observed in this study.
158
Table 5.3.4.1 Protein precipitating capacity of lignin and quantity of lignin extracted
from acid detergent fibre of wheat straw.
1
PRS
RT
PPC (g)
L-ADF
extraction (%)
5
L-STWS
15
0.58
0.25
1.00
17
0.68
0.32
1.26
19
0.31
0.46
1.29
0.31
0.50
2.56
10
0.20
0.47
2.71
0.008
0.012
SEM (n=2)
1
pressure (bar)
reaction time (min)
3
quantity of protein (g) precipitated by 1 g lignin
4
lignin extracted from acid detergent fibre of wheat straw
5
lignin extracted from steam treated wheat straw
6
2
159
5.4 Conclusions
All lignins extracted from steam-treated LM had some protein precipitating
capacity. Use of an exogenous catalyst did not improve protein precipitating capacity
of lignin. Both pressure and reaction time affected lignin quality. Different effects on
lignin quality were observed from different LM sources. Effect of treatment conditions
on lignin quality is attributed more to changes in molecular structure than to reaction
with browning compounds.
160
CHAPTER 6
THE USE OF LIGNIN TO PROTECT PROTEIN FROM MICROBIAL

DEGRADATION IN THE RUMEN
6.1 Introduction
Dietary protein is one important factor affecting the nutritional status of farm
animals. In ruminants, protein reaching the abomasum and small intestine can be either
microbial protein (Storm and rskov, 1984) synthesized in the rumen or dietary protein
which escapes from rumen degradation (rskov, 1982). Protection of protein from
microbial degradation in the rumen is one way to prevent losses caused by rumen
degradation (McDonald et al., 1988). The objective of protecting proteins from ruminal
degradation is to increase the supply of balanced essential amino acids to the animal.
There are basically two methods to protect proteins from microbial degradation in
the rumen, i.e. heat and chemical treatment (Mudgal and Sengar, 1984).
It has been reported that proteins bound to tannins are more likely to escape from
degradation in the rumen (Nishimuta et al., 1973). The main problems with such
methods is their cost and possible health problems.
Protection of amino acids has also been considered as a way of improving the
nutrition of high yielding animals. Studies on high yielding animals have shown that
quantity of amino acids normally supplied to intestine is not enough to meet the
161
production requirement (Fraser et al., 1991). Supply of limiting amino acids (methionine
and lysine) to the intestine by intragastric infusion increases milk synthesis (Fraser et al.,
1991). Protection of protein from microbial degradation may not help to solve this
problem as the response will also depend on biological value of the protein. Protection
of a large quantity of protein to supply the limiting amino acids to the intestine may
cause low concentration of nitrogen in the rumen which in turn affects rumen microbial
fermentation. Protecting individual amino acids from microbial degradation in the rumen
would not affect protein digestion and N supply to rumen microbes.
The use of tannin for protecting protein from microbial degradation was
previously discussed (Section 1.5). The protected protein must somehow be available to
the animal postruminaly. This is normally achieved by pH changes such as low abomasal
pH (Van Buren and Robinson, 1969). The concentration of 0.1 M hydrochloric acid in
abomasum could reduce pH to 2 (McDonald et al., 1988). Such an acidity has been
reported to be sufficient to dissociate protected protein from tannins (Oh and Hoff,
1987).
In the previous chapter, the protein precipitating capacity of lignin degradation
compounds was reported. This chapter considers protection of the proteins adsorbed to
lignin from ruminal degradation and also conditions for releasing the adsorbed protein
from lignin.
The objective was to quantify protein protection from rumen microbial
degradation as affected by addition of lignin extracted from steam-treated wheat straw.
162
Preliminary studies on the protection of amino acids were also carried out.
163
6.2.1 substrate
Wheat straw was used as a lignin source. Characteristics and method of
preparation of wheat straw were as described in Section 2.2.1.

Wheat straw was auto-hydrolysed at 19 bar pressure and 5 min reaction time.
This treatment condition was chosen because protein precipitating capacity of lignin
from this samples was higher compared to other lignin samples. Procedure of steam
treatment was as mentioned in Section 2.2.2.
6.2.3 extraction and purification of lignin

Extraction and purification of lignin was the same as mentioned in Section 4.2.3.
6.2.4 in vitro protein and amino acid degradation

An in vitro procedure was used to measure protein and amino acid degradation by
rumen microbes. Preparation of artificial saliva and rumen liquor, ratio of rumen liquor
to artificial saliva and incubation conditions were the same as in vitro gas production
technique mentioned in Section 2.2.10. Three levels of lignin (100, 200 and 400 mg)
together with 3.5 ml of the following solutions (16 mg/ml in artificial saliva): casein
164
(Sigma, technical, from bovine milk), methionine and cysteine, were added to 75 ml
conical flasks. Filter paper (144 mg) (Whatman No. 1) was added to each flask as an
energy source for rumen microbes. Rumen liquor was taken 1 h before morning feeding
from sheep fed on a hay and grass cube diet. Composition and method of preparation of
artificial saliva and rumen liquor are described in Section 2.2.10.
6.2.5 NH3 analysis

One ml rumen liquor was taken from each flask at 3, 6, 9, 12, 24 and 48 h
o
incubation. Samples were centrifuged at 18,000 x g and 4 C for 20 min. One part of
supernatant was mixed with 0.25 part of tri chloro acetic acid (3% Aqueous solution)
and the mixture diluted 5 times. NH3 concentration in the solutions was measured using
an autonanalyser (continues flow colorimetric system) (Whitehead et al., 1967). The
method is based on reaction of NH3 with Alkaline Sodium Phenate and Sodium
Hypochloric to produce the Inophenol Blue complex which absorbs light at 625 nm.
6.2.6 VFA analysis

Samples described in Section 6.2.6 were analyzed for VFA composition. To 1 ml
sample 250 l internal standard (20 mM 2-ethylbutyric acid in 20% w/v orthophosphoric
o
acid) was added and the mixture centrifuged at 18,000 g and 4 C for 20 min. The
supernatant fluid was analyzed by Gas Liquid Chromatography. A 1 l of supernatant
was injected into the GC fitted with a flame ionization detector (FID) and a capillary
165
column (10m, 0.53 mm I.D., Hewlet Packard). The following temperatures were used:
o
injector (t= 150 C), column oven (t= 75 C, temperature increased at a rate of 5 C/min
o
until 105 C and time of 7 min and FID (t=160 C). Helium gas was used as carrier with a
flow rate of 12 ml/min.

Effect of levels of lignin on protein (casein) degradation (NH3 and iso-acid
production) and also deamination activity (from methionine and cysteine) by rumen
microbes was evaluated by ANOVA.

Concentration of protein in the solutions was measured as described in Section
4.2.8.
6.2.9 adsorption of amino acids by lignin

Methionine (L-methionine), cysteine (L-cysteine) and lysine (L-lysine) were
dissolved in water (10 mg/ml). Five ml of amino acid solution was mixed with 100 mg
lignin. The mixture was shaken for 30 min at room temperature and diluted 5 times
o
using acetate buffer (0.2 M, pH 4.0) and centrifuged at 18,000 x g and 4 C for 15 min
and supernatant subsequently analyzed for amino acids. Concentrations of each amino
acid in the supernatants were calculated from the corresponding standard curves.
166
Adsorption of amino acid to lignin was calculated by subtracting the amino acid in the
supernatant solution from the original amount added.
6.2.10 amino acid analysis

One ml ninhydrin reagent was added to a test tube containing 50 l supernatant
o
(Section 6.2.4) and vortex mixed then incubated in an oil bath at 100 C for 20 min. The
tubes were cooled and 5 ml distilled water was added. The absorbence was recorded at
570 nm after the contents of the tubes were thoroughly mixed.
6.2.11 release of adsorbed protein from polyphenolic compounds

Forty mg lignin was added into a test tube. Two ml BSA solution (10 mg/ml in
distilled water) was added to the lignin and the mixture was shaken for 30 min at room
temperature. The mixture was diluted 5 times using acetate buffer (0.2 M, pH 4.0).
Tannin treatment was achieved by adding 0.5 ml of tannin solution (20 mg/ml) to 3 mls
BSA solution (10 mg/ml) and was shaken for 30 min at room temperature. Samples were
o
centrifuged at 18,000 x g and 4 C for 20 min and the clear supernatant was collected.
The solid fraction was mixed with 12 ml acetate buffer (0.2 M, pH 4.0 for lignin and pH
5.5 for tannin) and the pH was reduced slowly by adding 1 M hydrochloric acid.
Samples (1 ml) were taken at different pH values for further analysis. Samples were
o
centrifuged at 18,000 x g and 4 C for 20 min and the clear supernatants were transferred
into test tubes. The supernatants were analyzed for protein content.
167
6.2.12 specific gravity of lignin-protein complex

Functional specific gravity of lignin was determined by the volume displacement
method (Siciliano-Jones and Murphy, 1991). One g freeze dried lignin-protein complex
o
was added in duplicate to a 50 ml calibrated volumetric flask. Distilled water at 20 C

was added to complete the volume desired and the weight recorded. Specific gravity of
lignin-protein complex was measured as following:
S.G. = Wa/(Wc-Wb), where Wc is weight of water content of flask + sample weight, Wa
is sample weight and Wb is weight of volume of water contained by flask.
168
6.3.1 protection of protein by lignin

Rates of ammonia production from casein treated with different levels of lignin
are shown in Fig 6.3.1.1. All the figures have been corrected for the background
ammonia, i.e. NH3 from the inoculum. Ammonia concentration is a measure of the
proteolytic action of rumen microorganisms on protein. Rumen liquor was taken before
the morning feeding to minimise the amount of ammonia in the blank. Significant
differences (p<0.05) between control and treatments indicated the effect of lignin on
protecting protein from degradation. The higher the lignin concentration the lower the
ammonia concentration. From previous results on the protein precipitating capacity of
lignin it could be concluded that the bond between lignin and protein was strong enough
to prevent degradation.
Iso-acids (iso butyrate and iso valerate) were also used as an index of protein
degradation as these compounds are produced mainly from degradation of protein
(McDonald et al., 1988; Van Soest, 1994). The effects of lignin addition on iso-acid
production are presented in Table 6.3.1.1. Again, increasing the levels of lignin caused a
significant (p<0.01) reduction in iso acids production which indicated that inclusion of
lignin affected protein degradation by rumen microbes. It should be mentioned that
proteolysis, microbial NH3 adsorption for protein synthesis and also NH3 production
from lysis of microbes occur at the same time in such in vitro assays. To minimize such
169
effects more emphasis should be paid on NH3 production data at early stages of
170
Fig 6.3.1.1 Effect of levels of lignin extracted from steam-treated wheat straw on
degradation of casein by rumen microbes as indicated by production of NH3 (see data on
appendix 8).
Table 6.3.1.1 Effect of levels of lignin extracted from steam-treated wheat straw on isoacid production.
incubation time (h)
6
12
levels of lignin
24
iso-acid production (mM)
0 (control)
1.65
100
0.60
**
1.10
200
0.45
**
0.95
400
0.25
**
SEM (n=2)
48
2.55
0.050
3.55
**
1.40
**
1.10
0.50
**
0.152
171
3.75
**
2.25
**
**
1.70
1.10
**
1.50
0.035
0.152
**
**
**
1
Significantly (p<0.01) different from the control

172
incubation (e.g. 6, 12 and 9 h). The significant differences in NH3 and iso acid (p<0.05
for NH3 and p<0.01 for iso acid) concentration at 6, 9 and 12 h of incubation indicate
that protein has been protected to some extent from microbial degradation.
The functional specific gravity (FSG) of feeds is an important factor affecting
their retention time in the rumen (Ehle, 1984; Kaske and Engelhardt, 1990). It has
been shown that the maximum passage rate is achieved at FSG 1.35 ml/g (Ramanzin
et al., 1991). FSG of the lignin-protein complexes are shown in Table 6.3.1.2. The
mean FSG of the lignin-protein complexes was 1.31 g/ml thereby positively causing
a low retention time in the rumen.
6.3.2 adsorption of amino acids by lignin

The results of precipitation of methionine, cysteine and lysine showed that none
of them were adsorbed by lignin. It was concluded in Chapter 4 that the structure and
molecular weight of protein could affect its adsorption by polyphenolics. Data from
the present experiment indicates that the low molecular weight of amino acids as
compared to protein may be an important limitation to their affinity to lignin.
6.3.3 effect of lignin on deamination activity of rumen microbes

Effect of lignin on digestion of rye grass was studied in Section 4.2.5.
Although it was concluded that lignin did not affect food digestion, those results were
more related to cell wall digestion. Since amino acids were not absorbed by lignin it
173
was possible to assess the effect of lignin on deamination activity in rumen liquor. In
the
174
Table 6.3.1.2 Functional specific gravity of lignin-protein complexes.

treatment conditions
1
sample
PRS
RT
FSG (g/ml)
wheat straw
19
1.30
rice straw
17
1.31
barley straw
17
10
1.33
sugar cane bagasse
17
1.29
SEM (n=2)
0.009
pressure (bar)
reaction time (min)
3
2
175
present study degradation of methionine and cysteine solutions as affected by lignin

addition were investigated. Results (Fig 6.3.3.1) showed that lignin did not affect
deamination activity in the rumen apart from protein protection (Section 6.3.1).
6.3.4 postruminal availability of adsorbed protein by lignin

An important factor when considering the use of protected protein in ruminant
feeding is the availability of such proteins to intestinal digestion.
Protection of protein from ruminal degradation can be achieved by heat
treatment. One important constraint of such treatment is that treatment conditions
should achieve optimal protection within a narrow margin. Overheating of the protein
makes the fraction escaping degradation in the rumen less digestible in the intestine.
The effect of pH on protein concentration in a solution containing ligninprotein and tannin-protein complexes is presented in Fig 6.3.4.1. Protein adsorption to
lignin is a pH dependent reaction. Such an effect has also been reported for tannins
(Oh et al., 1985). The results obtained in the present study agree with the results of
Hagerman and Butler (1978). The similarities between lignin and tannin suggest that
the same H bonds as suggested for tannins (Doner et al., 1993) occur between lignin
and protein.
It has been reported that hydrophobic interactions could be also very important
during formation of tannin-protein complex (Oh et al., 1980; Hagerman and Butler,
1980). It is also believed that the process of complex formation is normally reversible
176
and both protein and polyphenols can be separated afterwards. A possible negative
177
Fig 6.3.3.1 Effect of lignin extracted from steam-treated (19 bar pressure and 5 min
178
reaction time) wheat straw on deamination activity (see data on appendix 9).
179
Fig 6.3.4.1 Effect of pH on releasing of adsorbed protein from tannin (tannic acid) and
lignin.
180
effect of the released lignin on enzymic digestion in the small intestine has still to be
investigated. It is worth noting that mineral concentration may also affect protein
binding ability of lignin as reported for tannins (Oh and Hoff, 1987).
181
6.4 Conclusions
Lignin has been shown to protect protein from being degraded by rumen
microbes. Lignin is inert to microbes and forms a pH dependent complex with protein.
This reaction enables the protein fraction to be released from the lignin-protein
complex at low abomasal pH.
Further work is necessary to improve lignin quality in order to maximize
protein protection while using a low quantity of lignin.
182
CHAPTER 7
GENERAL DISCUSSION AND CONCLUSIONS
7.1 Introduction
LM are the most available and renewable energy source in the world. The
potential for fuel and chemical production from this source has not seriously been
considered due to the low cost of fossil fuels.
Effective LM bio-utilization involves disrupting cell wall structure so as to make
it more susceptible to the action of enzymes or microorganisms. The main objective in
this respect is to loosen the structure of LM, so that a large surface area can be available
for the action of the cellulolytic enzymes. This can be achieved by several means such as
physical, chemical and biological techniques.
High pressure steam treatment has proved a promising method for upgrading LM
bio-availability to rumen microbes and cell free enzymes (Mathews and Peppers, 1978;
Brownell et al., 1986). Nowadays, alkaline treatment (e.g. by use of urea, ammonia or
sodium hydroxide) has been the most accepted method for upgrading agricultural byproducts for animal feed (Chesson, 1981; rskov et al., 1981; Sundstol and Owen,
1984). Alkaline treatment is cheaper than steam treatment because of the low price of
fossil fuels used to produce chemicals, e.g. urea and sodium hydroxide. Inexpensive
fossil fuel has discouraged extensive research for finding alternative sources. However
183
in the near future lack of fossil fuels may cause a turn to bioconversion of biomass to
fuels, chemicals and other useful products.
Although steam treatment has been identified as a physical process, it is clearly
chemical in nature when changes in the chemical composition of lignocellulosic
components are followed. Disrupting the chemico-physical structure of the cell wall
during steam treatment allows fractionation of the main components of the plant cell
wall, e.g. cellulose, hemicellulose and lignin (Overend and Chornet, 1987). Lignin has
received less attention for chemical production compared to polysaccharides and has
mostly been used as fuel (Wayman and Parekh, 1990).
In this study the constraints inherent in steam treatment of LM were investigated.
The results of this study suggest a novel application for lignin.
7.2 Constraints of steam treatment of lignocellulosic materials

Apart from the expensive equipment required for setting up a steam treatment
system, there are some disadvantages associated with this type of treatment. DM loss
(Beltrame, et al., 1992) and formation of toxic compounds (Mes-Hartree and saddler,
1983) are the main constraints.
7.2.1 nutrient loss

The main physical and chemical changes of lignocellulosics during autohydrolysis are illustrated in Fig 7.2.1.
184
Both chemical and physical characteristics of fibre are affected during steam
185
186
Fig 7.2.1 Physical and chemical changes of LM during steam treatment.
187
treatment thereby significant difference in bio-utilization is possible. Changes in

physical characteristics of fibre such as reduction in the degree of polymerization of
cellulose and reduction of particle size cause an increase in the active surface area of
cellulose making it more susceptible to enzymic hydrolysis (Michalowics, et al., 1991).
From a nutritional point of view, extreme changes in the physical characteristics, e.g.
reduction in particle size, may negatively affect fibre digestion by ruminants because
large quantities of potentially degradable substrate may leave the rumen after a short
period of fermentation (Poppi et al., 1981). Although such physical changes lower
potential degradation of cell wall polysaccharides, it may not be significant if other
changes such as DM loss and formation of indigestible compounds are taken into
consideration. Volatilization (formation of furans and organic acids) is an inevitable
process during steam treatment and could cause a considerable DM loss if harsh
treatment conditions are applied (Beltrame, et al., 1992). As is shown in Fig 7.2.1
hemicellulose is mainly affected by such process.
It was shown in this study (Table 3.3.5.1) that application of harsh treatment
conditions causes the formation of large quantities of indigestible browning reaction
compounds. Transformation of hemicellulosic sugars to browning products was more
important than DM loss in reduction of nutritive value of fibre. Even under mild
treatment conditions (15 bar pressure and 0 min reaction time) there was considerable
nutrient loss through formation of indigestible compounds (browning reaction
188
compounds - see footnote on page 43) ( 9%) while DM loss was insignificant (
1
0.8%) .
The extent of WSE is negatively affected by increase in harshness of treatment
conditions. The WSE has mostly been used for alcohol production (Wayman and
Parekh, 1990). This fraction can also be used as animal feed as it is rich in soluble
carbohydrates. Utilization of carbohydrates in this fraction for animal feed will depend
on their composition and site of digestion. Ruminants can effectively utilize almost any
type of sugar. On the contrary, monogastric animals may be affected negatively by
including high level of monomeric pentoses in diet (Longstaff et al., 1988; Yule and
Fuller, 1992). Production of monomeric pentoses in WSE is however much lower than
the harmful levels. In this respect the animals concerned are the monogastrics, e.g. pigs,
which can digest the hemicellulosic sugars in the hind gut and absorb the VFA. The
potential of this fraction as monogastric animal feed should be taken into consideration
due to its low production cost.
More work is needed to optimize steam treatment conditions for producing
animal feed and WSE. The important point for this purpose would be optimization of
steam treatment conditions so as to maximize soluble carbohydrate content while
minimizing formation of browning reaction compounds.
29.9 (Table 2.3.3.1)* 30.15 (Table 2.3.2.1)/100 = 9.01
189
7.2.2 identification of compounds toxic to rumen microbes in steam-treated wheat

straw
Research on upgrading LM by steam treatment has been oriented more towards
producing easily fermentable materials for alcohol production than producing animal
feed. There are several reports on formation of materials inhibitory to alcoholic
fermentation, e.g. acetic acid, furans and phenolic compounds, in steam-treated LM.
However, yeast can become adapted to tolerate these inhibitors (Wayman and Parekh,
1990).
Use of tannin complexing agent (PVP or PEG) has been the most widely
accepted method to inactivate of antinutritive phenolic compounds in feed (Khazaal and
rskov, 1994; Makkar et al., 1995). In this study the antinutritive effect of phenolic
compounds in steam-treated wheat straw was assessed by different methods.
High molecular weight extractable phenolics in feed (tannins) have shown greater
inhibitory effects on rumen microbes than low molecular weight ones. Addition of PVP
to steam-treated wheat straw did not alter in vitro gas production. Although steamtreated LM contains an appreciable amount of high molecular weight phenolics, they
did not show biological activity on rumen microbes. This result was in agreement with
the quantity of tannin-like compounds in steam-treated wheat straw.
Among the compounds inhibitory to yeast activity in steam-treated LM the low
molecular weight phenolics have shown greater toxicity than furans and acetic acid
(Clark and Mackie 1984). The phenolics extracted by the method described in this study
190
(Fig 3.2.5.1) did not affect in vitro feed digestion. Efficiency of the method (Fig 3.2.5.1)
had already been demonstrated by extracting phenolics from a browse and adding to a
control feed (Zahedifar et al., 1995) (Fig 3.3.2).
Britton (1978) reported a high depression in in vitro feed digestion by the extracts
obtained by sequential extraction of materials with ethanol solutions. The materials
extracted by the method described by Britton (1978) did not alter in vitro feed digestion
either. There is no indication of the nature of inhibitory compounds or of the quantity of
extracted materials in that report. It should be mentioned that metallic ions derived from
corrosion of reaction chambers can negatively affect microbial activity (Wayman and
Parekh, 1990).
The last method was to assess the effect of lignin extracted by alkali on in vitro
feed digestion. Lignin did not affect cellulolytic nor deamination activity of rumen
microbes. The very low amount of total extractable phenolics in untreated wheat straw
( 0.5%) indicates that almost all extractable phenolics in steam-treated straw originated
from lignin. Obtaining consistent results from all the methods used in this study is a
clear indication that the phenolic compounds formed from lignin depolymerization are
not toxic to rumen microbial activity.
Apart from phenolics, browning compounds are also present in considerable
quantities in steam-treated LM. No toxicity to rumen microbes was observed from
browning reaction compounds present in steam-treated wheat straw. According to
Kyuma et al. (1991) furfural depressed in vitro cellulose digestion but no effect on daily
191
food intake was observed by addition of furfural to the diet. The results of this study
showed that furans (furfural and HMF) are not toxic to ruminal fermentation. Similar
results have been reported by Castro et al. (1994). It should be mentioned that no further
increase in furfural and HMF production was observed when very harsh treatment
conditions were applied (Table 3.3.5.1). Further production of furans in a closed system
is inhibited by their presence in the system (Tipson and Horton, 1988). The highest
concentration of furans observed in this study was from the acid-hydrolysed wheat straw
treated at 19 bar pressure and 5 min reaction time.
In general it is indicated that no compound toxic to rumen microbes is produced
during steam treatment of wheat straw. Whatever browning reaction compounds are
produced during steam treatment are indigestible and should be considered simply as
nutrient loss.
7.3 Biological activity of lignin extracted from steam-treated LM

The definition of lignin can vary depending on the viewpoint. Biochemists, plant
physiologists and wood chemists tend to emphasize the importance of lignin in plant cell
wall development and the nature of the material and the objective of its use. The
nutritionist's view of lignin is also narrowly limited to its role in nutrient availability. In
this respect lignin is considered as the most significant factor affecting bio-degradation
of cell wall polysaccharides.
The potential of lignin in agriculture has been enormously underestimated.
192
Complexity in structure, indigestibility, negative effect on bio-degradation of cell wall

components have been the reasons for a negative attitude to lignin. Wherever lignin is
produced (as a by-product) there is a furnace to burn it. Although lignin is a good source
of energy, the main reason for burning is that not many large scale applications for lignin
have been found. Over 40 million tonnes of lignin is produced annually all over the
world. In North America and Europe only about 2% of the lignin is used for purposes
(Table 7.3.1) other than burning.
Removal of hemicellulose and lignin from LM is an important process for paper
1
making. There are principally two methods, the sulphite and the kraft processes. In
both of the methods LM subjected to very harsh treatment conditions. The Sulphite
process is cheaper than the Kraft process (Higham, 1971) but because of environmental
pollution industries are shifting to the Kraft process. No biological activity from
lignosulphonate or lignin obtained from the Kraft process has been reported. The
information in this respect is from the lignins isolated from steam-treated LM.
Lignin has long been known as an compound inert to rumen microorganisms
(Kamstra et al., 1958). Such inertness was also observed from lignin based phenolics.
In sulphite process the delignification is an acid solution of a bisulphite of calcium,

sodium or ammonium, which is added to wood chips in a reactor. The suspension is
o
heated to about 160 C for about 2 h during which time the lignin is sulphonated and the
salt of the sulphonic acid are dissolved. The softened chips are disintegrated and the pulp
so formed is filtered, the filtrate is being either evaporated and burned in a furnace, or
193
otherwise treated to recover lignosulphonate.

2
In kraft process, the reactive chemicals consist of sodium hydroxide and sodium
sulphide. The cooking liquor is added to wood chips and cooked for 2 to 4 h at about
o
150 C. The chips are washed and sent to the bleach plant or paper mill. The liquor is
concentrated in a evaporator to obtain 65% DM and then is heated in a furnace to
produce steam.
194
Table 7.3.1 Lignosulphonate sale in Europe, 1986 (Wayman and parekh, 1992).
1,000 tonnes
Binder for mineral palletization
50
Binder for soil stabilization and fertilizers
15
Binder for organic material (coal)
25
Binder for animal food
105
Binder as glue substitute
30
Concrete and cement additives
20
Dispersant for agricultural chemicals
25
Dispersant and use in ceramics
25
Drilling and additive
25
Dye dispersant, pigment
Miscellaneous
20
Total 343,000 tonnes
195
An interesting observation was the inhibitory activity of lignin on cell free

enzymes. It was shown in this study that lignin was responsible for partial inactivation of
enzymes during hydrolysis of cell wall polysaccharides of steam-treated LM. Although
lignin showed less activity than tannin in inactivation of enzyme, it could have a
significant effect on enzymic saccharification because of the large quantity of lignin
present in steam-treated LM. Using more enzyme and trying to recycle the
enzymes have been suggested to overcome this problem (Bungay, 1985). It was also
observed that lignin, similar to tannins, (Makkar, 1989) had protein adsorbing capacity.
Such property of lignin could provide the opportunity to use lignin (after completing
hydrolysis) to isolate and recycle enzymes.
Investigation of the lignin samples extracted from different agricultural byproducts showed that all the lignins had protein precipitating capacity. Very harsh
treatment conditions did not affect lignin quality greatly. When the sugar content of
lignin samples is taken into account it is revealed that increasing reaction time at low
pressure had little effect on lignin quality. In all cases increasing reaction time
consistently caused an increase in total lignin extraction. It implies that lignin
depolymerization was still taking place even at 19 bar and 10 min reaction time. This
was in agreement with the report of Chua and Wayman (1978) that lignin
repolymerization in aspen wood started after 30 min reaction time. It is not known
whether lignin repolymerization affects its protein precipitating capacity.
Protein precipitating capacity of lignin is affected by treatment conditions but no
196
clear trend in lignin quality was observed by increasing pressure. This result was
consistent in all LM studied except for autohydrolysed wheat straw. It implies that
although lignin depolymerization is necessary to generate its biological activity, it is not
necessary to strongly modify the original molecular structure of lignin to produce such a
property. The high protein adsorbing capacity of lignin samples extracted from the
samples treated at mild conditions supports this hypothesis.
Different characteristics of lignin samples were studied in order to find a simple
method to predict the biological activity of lignin. Contrary to expectation, no
correlations were found between different characteristics (solubility in water, total
phenolics as indulin equivalent, sugar content, nitrogen content and ash content) of
lignins and their protein adsorbing capacity which shows that more complex factors may
exist in this respect. Lignin extraction involves different steps which are time
consuming. Finding a reasonable correlation between protein adsorbing capacity of
lignin and a simple measurable characteristic (e.g. total extractable phenolics) of steamtreated LM would simplify research on the biological activity of lignin.
Reduction of ruminal protein degradation in high yielding ruminants has received
considerable attention and several methods have been developed (Nishimuta et al., 1973;
rskov, 1982; rskov et al., 1983; Gupta and Gupta, 1985; Wallace and Falconer, 1992)
(Fig 7.3.1). For this purpose heat and formaldehyde treatments have been the most
widely used (Virk et al., 1994). Carelessness in use of these methods may cause either
animal health problems or reduction in nutritive value of protein.
197
Studies on the effect of lignin on in vitro protein degradation showed that lignin
like tannin, was able to protect protein from ruminal fermentation. Protein dissociation
198
Fig 7.3.1 Methods for reducing protein degradation in the rumen.
199
from lignin at low pH makes it possible to use lignin as a method of protecting protein.
Lignin is an environmentally friendly compound without hazardous effect on animal
health and leaves no environmental hazardous waste.
The similarities between tannin and lignin suggest the presence of hydrogen
bonds and hydrophobic interactions between lignin and protein. If so, lignin, like tannin,
should be classified as a chemical suited to protect protein from ruminal fermentation.
The only problem with lignin would be its low activity compared to tannins.
Apart from protein protection, lignin could possibly be used to adsorb proteins
from slurries and recycle them into the animal feed chain. Use of lignin in the leather
industry and inactivation of plant enzyme during storage (specially silage) (Wetherall et
al., 1995) could be other applications.
One advantage of steam treatment is that the steam-treated LM can be easily
fractionated into its main chemical components (Overend and Chornet, 1987).
Production of lignin at mild treatment conditions could make it possible to use all
fractions of LM. In this case there is no need to sacrifice other cell wall fractions for
lignin.
The quality of lignin from other lignocellulosics e.g. softwoods and hardwoods,
needs to be studied. Other methods for lignin isolation, e.g. organic solvents, have to be
studied in order to reduce the cost of lignin isolation and also avoid environmental
pollution. It is likely that lignin molecules with a specific structure (higher in number of
-OH groups) may have greater biological activity. Isolation of these compounds could be
200
another possibility to increase its activity. Intensive research on lignin is required to

elucidate different aspects of its biological activity.
Lignin is a cheap product and abundantly available. It does not affect rumen
microbes and is an environmentally friendly compound.
Increased use of lignin in agriculture and industry will depend on producing
highly effective lignin.
201
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Appendix 1. Effect of pressure and reaction time on soluble carbohydrate

content of water soluble extract from steam-treated wheat straw.
reaction time (min)
0
pressure (bar)
10
(%)
auto hydrolysed
wheat straw
15
avg
17
avg
19
avg
224
44.3
52.0
43.4
45.5
54.5
43.9
44.9
53.0
43.7
51.4
47.4
28.4
48.5
46.4
27.4
50.0
46.9
27.9
56.9
38.8
20.8
59.5
36.2
21.9
58.2
37.5
21.35
Appendix 2. Data of gas production of water soluble extract from steam-treated

wheat straw and sugar mixtures.
incubation time (h)
2
12
24
48
gas production (ml)

1
WSE
avg
2
SC
avg
3
SCA
avg
6.1
13.4
19.5
23.5
26.2
29.8
32.2
6.3
13.4
19.0
22.5
25.8
29.6
31.8
6.24
13.4
19.3
23.0
26.0
29.7
32.0
4.3
13.9
21.0
25.0
26.5
29.1
30.3
3.8
13.3
20.0
24.4
25.4
28.0
29.3
4.1
13.6
20.5
24.7
25.9
28.5
29.8
6.3
19.2
33.2
48.1
52.5
59.8
63.4
5.5
17.5
31.0
46.4
50.4
58.2
61.8
5.9
18.4
32.1
47.3
51.5
59.0
62.6
water soluble extract steam-treated wheat straw at 19 bar pressure and 5 min
reaction time
2
sugar control containing the same quantity of sugar with the same sugar
composition as WSE
3
sugar control containing the same quantity of organic matter as WSE (WSE ash).
225
Appendix 3. Effect of ethyl acetate extractable phenolics (EAEP) and water

insoluble phenolics (WIP) extracted from Carpinus duinensis on gas production
from 200 mg rye grass.
incubation time (h)
8
10
12
gas production (ml)
24
48
avg
3.0
3.5
3.2
5.6
6.1
5.8
8.9
9.4
9.1
13.2
13.6
13.4
18.1
19.1
18.9
23.0
24.0
23.5
35.4
36.3
35.9
45.7
44.2
45.0
avg
2.5
3.0
2.7
5.9
5.4
5.7
9.8
8.9
9.3
13.7
12.3
13.0
18.7
17.2
17.9
23.6
21.6
22.6
35.4
34.4
34.9
44.7
44.3
44.5
avg
2.9
3.0
3.0
5.4
5.4
5.4
7.9
8.9
8.4
10.8
13.3
12.1
15.2
16.2
15.7
19.6
23.1
21.4
32.9
35.4
34.2
44.2
44.8
44.5
avg
2.0
2.9
2.5
4.9
4.9
4.9
7.4
7.9
7.6
9.8
10.3
10.1
12.7
13.7
13.3
16.2
17.2
16.7
29.4
31.9
30.7
42.2
43.7
42.9
avg
2.5
3.0
2.7
5.4
5.4
5.4
8.9
8.9
8.9
12.3
11.8
12.1
17.2
16.3
16.7
21.6
21.7
21.7
33.9
33.5
33.7
43.8
43.9
43.8
avg
2.9
3.4
3.2
5.4
5.9
5.6
7.8
8.3
8.1
9.8
10.8
10.3
12.7
14.2
13.5
17.2
18.6
17.9
31.4
32.4
31.9
41.7
43.6
42.7
avg
2.0
2.0
2.0
4.9
4.9
4.9
6.9
6.9
6.7
8.8
8.8
8.8
10.3
10.8
10.5
12.7
13.2
12.9
25.5
27.0
26.2
37.7
39.2
38.5
control
EAEP-1
EAEP-2
EAEP-4
WIP-1
WIP-2
WIP-4
effect of equivalent amounts of EAEP present in 200 mg Carpinus duinensis

3
4
effect of equivalent amounts of WIP present in 200 mg Carpinus duinensis
5
2
226
227
Appendix 4. Effect of substrate (rye grass) loading on pH in vitro.

incubation time (h)
0
12
24
48
pH
levels of rye
grass
200 mg
avg
400 mg
avg
600 mg
avg
800 mg
avg
6.95
6.89 6.85
6.71
6.63
6.62
5.56
6.95
6.86 6.87
6.72
6.65
6.65
5.50
6.95
6.88 6.86
6.72
6.64
6.64
6.58
6.95
6.85 6.81
6.63
6.51
6.46
6.30
6.95
6.86 6.82
6.65
6.55
6.49
6.34
6.95
6.86 6.82
6.64
6.53
6.48
6.32
6.95
6.83 6.79
6.56
6.43
6.18
6.05
6.95
6.83 6.81
6.59
6.46
6.22
6.10
6.95
6.83 6.80
6.58
6.45
6.20
6.08
6.95
6.81 6.76
6.54
6.33
5.88
5.80
6.95
6.84 6.79
6.57
6.34
5.93
5.86
6.95
6.83 6.78
6.56
6.34
5.91
5.83
228
Appendix 5. Effect of levels of lignin extracted from steam-treated wheat

straw on cellulase activity.
incubation time (h)
2
12
gas production (ml)

control
avg
25 mg lignin
avg
50 mg lignin
avg
100 mg lignin
avg
5.5
7.5
8.3
9.0
9.9
6.2
7.1
8.0
8.6
9.3
5.9
7.3
8.2
8.8
9.6
5.0
6.4
6.8
7.0
7.3
5.8
6.9
7.0
7.0
8.0
5.4
6.6
6.9
7.0
7.6
4.5
5.4
5.8
6.4
6.9
4.6
5.3
5.9
5.7
6.0
4.6
5.3
5.8
6.01
6.4
3.4
3.6
4.4
3.9
5.1
4.0
4.2
4.2
4.5
4.7
3.7
3.9
4.3
4.2
4.9
229
Appendix 6. Effect of steam treatment conditions (pressure and reaction time)

on protein precipitating capacity from autohydrolysed (no acid added) wheat
straw.
pressure (bar)
15
reaction
time (min)
17
19
(%)
auto hydrolysis
(no acid added)
0
avg
5
avg
10
avg
230
50.6
62.4
92.2
49.7
66.5
90.6
50.2
64.5
91.4
67.4
85.1
99.9
69.9
81.8
99.1
68.7
83.5
99.5
90.4
93.7
92.9
89.8
92.9
95.1
90.1
93.3
94.0
Appendix 7. Effect steam treatment conditions (pressure and reaction time) on

protein precipitating capacity from acid hydrolysed (2% H2SO4 on DM basis)
wheat straw.
pressure (bar)
15
reaction
time (min)
17
19
(%)
acid hydrolysis
(2% H2SO4 DM basis)
0
avg
5
avg
10
avg
231
58.3
74.6
86.1
56.1
76.1
88.1
57.2
75.4
87.1
70.5
80.3
80.5
73.7
82.1
77.8
72.1
81.2
79.1
74.4
81.8
98.1
75.9
79.1
99.4
75.1
80.5
98.7
Appendix 8. Effect of levels of lignin extracted from steam-treated (19 bar

pressure and 5 min reaction time) wheat straw on degradation of casein by
rumen microbes as indicated by production of NH3.
incubation time (h)
3
12
24
48
NH3 production (mg/l)

levels of lignin
0 (control)
avg
100 mg
avg
200 mg
avg
400 mg
avg
12.2
81.0
119.8
178.8
218.7
355.6
6.2
70.9
113.2
165.0
233.3
377.1
9.2
76.0
116.5
171.9
226.0
365.8
19.7
49.3
76.9
99.5
146.0
225.5
26.9
46.2
71.0
116.3
139.4
208.4
23.3
47.8
74.0
107.9
142.7
216.9
35.0
45.8
86.9
78.2
113.1
169.4
28.8
34.9
77.6
58.3
115.7
189.2
31.9
40.3
82.3
68.3
114.4
179.3
15.7
30.6
57.3
59.5
88.1
128.3
16.9
23.5
42.5
50.8
97.7
134.7
16.3
27.0
49.9
55.1
92.9
131.0
232
Appendix 9. Effect of levels of lignin extracted from steam-treated (19 bar

pressure and 5 min reaction time) wheat straw on degradation of amino acids
(methionine and cysteine) indicated by NH3 production.
incubation time (h)
6
12
24
48
NH3 production (mg/l)

methionine
avg
methionine + lignin
avg
cysteine
avg
cysteine + lignin
avg
20.3
33.4
65.1
128.4
10.9
21.7
68.8
136.3
15.6
27.6
67.0
132.4
22.3
25.6
72.8
138.4
24.8
41.7
60.4
123.7
23.5
33.7
67.0
132.4
3.2
22.4
69.9
95.7
0.0
16.6
74.2
98.6
1.6
19.5
72.0
97.2
5.9
14.9
73.9
110.2
8.2
22.6
68.4
101.7
7.0
18.7
71.1
106.0
233

Novel Uses of Lignin and Hemicellulosic Sugars From Acidhydrolysed

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NOVEL USES OF LIGNIN AND HEMICELLULOSIC SUGARS FROM ACIDHYDROLYSED LIGNOCELLULOSIC MATERIALS

(B.Sc., Animal Husbandry - University of Tehran)

Thesis submitted for the degree of Doctor of Philosophy

acid detergent fibre

I am extremely grateful to my supervisor Dr. E.R. rskov at The Rowett

My great appreciation to the Ministry of Jihad Sazandegi and the Ministry of

Lignocellulosic materials (LM) are an ever present renewable and available

(maximum 0.97% as tannic acid equivalent) to affect rumen microbes. Measurement of

for lignin production.

CHAPTER 1 - LITERATURE REVIEW

CHAPTER 2 - CHARACTERIZATION OF WATER-SOLUBLE EXTRACT

2.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

CHAPTER 3 - IDENTIFICATION OF INHIBITORY COMPOUNDS

3.2.6 effect of extraction procedure by ethyl acetate on

CHAPTER 4 - PROTEIN PRECIPITATING CAPACITY OF LIGNIN

4.2.4.2 enzymic inhibition by steam-treated lignin using

CHAPTER 5 - EFFECT OF TREATMENT CONDITIONS AND SOURCE OF

5.3.1 optimization of steam treatment conditions for lignin

5.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

CHAPTER 6 - THE USE OF LIGNIN TO PROTECT PROTEIN FROM

CHAPTER 7 - GENERAL DISCUSSION AND CONCLUSIONS

7.3 Biological activity of lignin extracted from steam-treated

Model of arrangement of components in wood cell wall

Relationship between lignin content and digestibility in rye

Illustration of primary and secondary cell walls

Distribution of cell wall polysaccharides (Worth, 1967) . . . . . . . . .6

Proposed arrangement of D-glucan chains within

Diagrammatic representation of cellulose microfibrillar

Hypothetical structure of plant xylan

Structure of three precursors of lignin

Proposed lignin structure in softwoods (Nimz, 1974) . . . . . . . . . . 14

Production of furfural from D-xylose

Production of 5-hydroxymethyl-2-furaldehyde from

The susceptible bounds (-aryl and -O-4) in lignin

Effect of reaction time on depolymerization/repolymerization

The High Pressure Steam Treatment Vessel in operation . . . . . . .36

Components of the High Pressure Vessel . . . . . . . . . . . . . . . . . .36

Putting the sample in the beaker (c) and transferring

Effect of pressure and reaction time on soluble

Nutritive value of the water soluble extract from

Extraction procedure of phenolic compounds (EAEP and WIP)

Extraction procedure by ethanol solution from

Efficiency of extraction by ethyl acetate of phenolic

Effect of ethyl acetate extractable phenolics and

Effect of substrate (rye grass) loading on pH . . . . . . . . . . . . . . . 82

Extraction procedure of lignin from steam-treated

lignin effect on cellulase activity . . . . . . . . . . . . . . . . . . . . . . . 101

4.3.2.1Effect of lignin on the Bradford method for determining

protein adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103

Protein (BSA) adsorbing capacity of 1 g of the three

Effect of steam treatment conditions on protein

Effect of steam treatment conditions on protein

Effect of levels of lignin extracted from steam-treated

Effect of lignin extracted from steam-treated

Effect of pH on releasing of protein from

Physical and chemical changes of LM during steam treatment . . .145

Method for reducing protein degradation in the rumen . . . . . . . . .155

Composition of macro-mineral, micro-mineral and buffer

Effect of sample DM content on chemical composition of

Sugar, phenolics contents and DM loss of wheat straw by

Neutral sugar composition of total and monomeric

Chemical analysis of water soluble extract from

Steam treatment conditions applied to wheat straw . . . . . . . . . . . .62

Effect of steam treatment and addition of PVP on