Biodegradation of cellulosic materials:

substrates, microorganisms,
enzymes and products
VIRENDRA S. B I S A R I A a n d T A R U N K. G H O S E

Bioch emical Engineering Research Centre, Indian In s titu te

of" Technology, Delhi, Hauz Khas, New Delhi 110016, India

Summary. Cellulosic materials are the only renewable Table 2 Potential availability of some agricultural residues
( 1 9 7 8 - 7 9 ) in India 2 4
resources available in large quantities which need to be
properly utilized to meet our needs o f energy, chemicals, Residues Availability (million tons)
food and feed for a long-range solution. A variety o f
lignocellulosic materials are available and microorganisms Rice husk 18.0
capable o f degrading either one or more o f the three main Rice bran 3.2
Rice straw 59.2
constituents, viz. cellulose, hemicellulose and lignin have
Bagasse 52.1
been studied. A t least three different enzymes o f the multi- Jute sticks 2.5
component cellulase system, i.e. cellobiohydrolase Cotton stal ks 12.0
endo-glueanase and (J-glucosidase are involved in the Cotton linters 1.2
degradation o f erystalline cellulose into glucose. Their Wood wastes 5.5
Coconut shell/coir dust 0.6
mode of a c t i o n and the manner in which they bring about
hydrolysis o f crystalline cellulose is discussed in detail. The
Table 3 Estimates of energy yields from crops s
involvement o f parallel enzymes for hemicellulose degrada-
tion is also known to some extent but needs to be studied Oven-dried Mega joules
more elaborately, independently and in combination with (tons/ha) (gross/ha)
cellulases. The potential o f cellulosic biomass as a source o f
Pasture 10.8 204 120
fuel and petroleum-sparing substances is also reviewed.
Lucerne 11 200 200
Winter green crop 20 36 200
Maize (grain) 8 164 700
Peas 4.4 83 500
Introduction Fodder beet 20 354 000
Sugar beet 9.6 168 960
It is being increasingly realized that, given the growth rate Gorse 10.6 196 100
of population and the stock of accessible reserves of coal, Oat straw 6.3 112 770
gas and oil, it will be impossible to provide as much energy Wheat straw 5.7 98 940
to the world economy towards the end of the present
century as is going to be demanded. Nuclear energy may
provide a solution but its use is always associated with understood and mastered for their optimal use. Biomass in
possible dangers of radioactivity and not everybody is the form of agricultural and forest wastes accumulates
enthusiastic about it. Bioenergy technologies, on the other every year in large quantities, resulting in a deterioration of
hand, are examples of what Lovins 1 calls 'soft energy paths', the environment and a loss of potentially valuable resources.
in spite of their own intrinsic complexities, which must be Some of the potential biodegradable agricultural and agro-
industrial residues and their availability in India are given in
Tables 1 and 2. All these represent energy-rich resources of
Table 1 Some potential agricultural and agro-industrial cellulosic
residues cellulosic biomass; the estimates of energy yield for some
crops have been computed (Table 3). The biological con-
By-products version of these residues into feed and fuel can be a practical
solution to the above-mentioned problems. According to
Crop Rice straw, wheat straw, maize stal k,
castor stem, tapioca stem, banana
one estimate, 6 the annual net yield of photosynthesis is
stem, coconut stem, cotton stick, 1.8 x 1012 tons of biological substances. If only a fraction
corn cobs, bamboo dust of these materials is to be converted into fuel, gas and feed
Agro-industrial protein, a significant contribution could be made to the
Rice-milling industry Rice husk, rice bran
overall problem of resource recycle and conservation.
Sugar industry Bagasse, molasses, pressmud
Cotton ginning industry Cotton tinters, cotton seed hull, Biomass in the form of cellulose, hemicellulose and
cotton gin waste lignin provides a means of collecting and storing solar
Jute industry Jute sticks, jute mill waste energy and hence represents an important energy and
Sawmill industry Sawdust material resource. However, before using this resource, it is
Coconut industry Coconut husk, shell and pith
necessary to convert it into a usable form, such as a gaseous

0141 --0229/811020090--1 5 $02.00

90 E n z y m e M i c r o b . T e c h n o l . , 1 9 8 1 , V o l . 3, A p r i l © 1981 IPC Business Press
 Biodegradation of cellulosic materials: Virendra S. Bisaria and Tarun K. Ghose
Table 4 Heats of combustion of various compounds that can be
derived from plants s
Compound AH c (25° C)
(cal/g)
Methane 13 200
a-Pinene 10 900
Oleic acid 9 500
Phenol 7 900
Ethanol 7 600
Lignin 7 100
Methanol 5700
Cellulose 4 200
CO 2 400
CO 2 0 Lignin, the third major component of lignocellulosic
Compound + 0 2 AHc~ CO2 + H20
(methane) or liquid (ethanol) feedstock that will fit into
our chemical economy. For this reason, a variety of
strategies are being explored which range from direct
thermal methods such as pyrolysis to biological methods
such as enzymatic hydrolysis and fermentation. However,
the high moisture content of biomass makes it less suitable
for most chemical treatments and more suitable for bio-
logical processing. Lignin is considerably higher in heating
value than cellulose or hemicellulose (Table 4), but this
heating value is not available to fermentation processes. 7
One of the widely used approaches towards biomass
utilization is the enzyme-catalysed hydrolysis of cellulose
and hemicellulose, constituting between 30-50% and
20-40%, respectively, of most lignocellulosic agricultural
residues, to low molecular weight compounds that can serve
as substrates for fermentation to fuels and chemicals. Much
work has been done on the use of enzymes produced by the
fungus Trichoderma reesei. At the Biochemical Engineering
Research Centre of lIT Delhi, studies are being pursued to
develop technically and economically feasible processes for
the production of fuels (ethanol), feed protein and chemicals
from agricultural residues available in the country. One of
the important steps in making the processes feasible is the
efficient degradation of the cellulosic and hemicellulosic
contents of these residues. An understanding of the basic
mechanism of enzymatic cellulose and hemicellulose
degradation is, therefore, an important aspect of the whole
problem. The main objective of this review is to consider
various aspects of the enzymatic degradation, such as the
constraints involved in cellulose breakdown, cellulase assay
procedures, and the mode of action of cellulase and hemi-
cellulase enzymes. Genetic means for enhancing cellulase
yield and the engineering improvements of the processes
leading to enhanced production of ethanol are also
discussed.
Lignocellulosic residues: s u b s t r a t e s for
bioconversion
Cellulose, a linear homopotymer of anhydroglucose units
linked by fl(1 ~ 4)-glucosidic bonds, does not occur in pure
form in any natural resource; even the seed hair of cotton,
the most pure form of cellulose readily available in nature,
contains about 10% by weight of non-cellulosic poly-
saccharides, proteins and mineral elements. In nature,
cellulose is always associated with a variety of other poly-
saccharides, such as starch, pectin, lignin and a variety of
hemicelluloses.
The hemicelluloses are polymers of galactose, mannose,
xylose, arabinose, other sugars and their uronic acids. These
are usually classified according to the sugar residue present,
e.g. D-galactan, D-mannan, D-xylan, L-arabinan etc. However,
they do not occur as homoglycans but rather as hetero-
glycans containing different types of sugar residues, often
as short appendages linked to the main backbone chain. 8
Some of the commonly occurring hemicelluloses are shown
in Figure 1.9 The hemicelluloses rank next to cellulose, as
the most abundant natural organic chemical in the biosphere.
The hemicelluloses are present in all layers of the plant cell
wall, but are concentrated mainly in the primary and
secondary layers, where they occur closely associated with
cellulose and lignin.
residues, is a natural polymeric product arising from an
enzyme-initiated dehydrogenative polymerization of three
primary precursors: trans-p-coumaryl alcohol, trans-
coniferyl alcohol and trans-sinapyl alcohol. 1° A typical
softwood (spruce) lignin contains approximately 80%
coniferyl alcohol, 14% p-coumaryl alcohol and 6% sinapyl
alcohol.11 Hardwood (beech) lignin, on the other hand,
contains similar amounts of coniferyl and sinapyl alcohol
and a minor amount ofp-coumaryl alcohol. A schematic
formula for representing spruce lignins is given by Adler 12'13
(Figure 2) and for beech lignins by Nimz. 14 According to
Kirk,1 s there are three major intermonomer linkages in
lignin: (a) the arylglycerol-/3-aryl ether type, (b) the phenyl
coumaryl structure and (c) the biphenyl structures. Diaryl
ether structures, pinoresinol structures and benzyl ether
bonds are also present. It must be pointed out that this
formula for lignin, like other formulae, shows only an
arbitrary sequence of phenylpropane units from a very
limited part of the lignin macromolecule.
The susceptibility of cellulose as a substrate for bio-
conversion processes is determined by its accessibility to
cellulase enzymes. Over the years, various pretreatment
methods have been proposed which make the links in the
cellulose molecule more accessible to attack by the enzymes.
The degree of polymerization (DP) of a cellulose molecule
can be anywhere from ~1000 (newsprint) to ~10 000
(cotton). During acid hydrolysis, the DP of the residual
cellulose drops very rapidly at first and then attains a rather
constant value of 100 to 200 corresponding to its levelling
off degree of polymerization (LODP). The easily hydro-
lysable portion of cellulose is often referred to as the
'amorphous' region and the residual resistant portion con-
stitutes the 'crystalline' region. Various models have been
proposed for the distribution of crystalline and amorphous
regions in cellulose fibres. A folding chain model proposed
by Chang 16 for cellulose crystallite structure conforms to
the experimental values of DP of the residual cellulose
during acid hydrolysis. Cellulose is known to exist in various
polymorphic formsJ 7 Cellulose I is the polymorphic form
that occurs in natural materials, i.e. cotton, ramie and wood
pulps. Cellulose lI is the crystallite modification which is
representative of the regenerated forms of cellulose, cotton
and wood pulps treated with sodium hydroxide and also
saponified cellulose derivatives. Rayon is a typical example
of cellulose II. Solvent pretreatment brings about a trans-
formation in the cellulose crystallite structure from that of
cellulose I to cellulose lI; this has been confirmed
experimentally.18,19
The changed crystallite structure of cellulose on solvent
pretreatment and the resultant enhanced hydrolysis can be
explained in terms of the folding chain model for the
cellulose structure. In native cellulose, the molecule folds
back and forth on itself with a fold length of DP ~ 160.
E n z y m e Microb. Technol., 1981, Vol. 3, A p r i l 91
Reviews

~4) /9 [ Xylp-(l~4)-
/9 D- Xylp-(V--~4) - ,~ - o - X y l p - ( l - - ~ 4 ) - B-D- X y l p - (I --P'
3 5

I I

(1- L- Araf a- L Arof

L Arabino - D-xytan ( wheat flour) ([)

- - - P 4 ) - /9 o Xylp (I--4~4) /9 D- Xylp-(i--'~4)- /9 D Xylp-(I---~4) /9-D -Xylp-(I----'b

5 2
f
I I

a - L - Araf e - u - GlcpA
L-Arobino-D-Glucurono- P-xylan (gross) (E)
A~etyl
3r
---D'4) - /9 D -Xytp-(t-P"4}- /9 D- Xylp-(I---4"4) /9 D - Xylp- ( 1 ~ 4 ) - /9- l)- Xylp- (I-~
2 2
t
I
~
I

a D GIcpA ~ - o -GIcpA
D-Glucurono- D-xylan (wood) (]:E)
Figure 1 Structure of some c o m m o n l y occurring hemicelluloses. ~9 Araf = arabinofuranose, Xylp = Xylopyranoside, Glcp A = glucopyranosyI-
uronide

H20H OH
i
H C ~ O [CH20H ]

HC
CH OH
2
~OCH

HCO - -
~ 0
OCH3

CH
HCO--
~ OCH3
H2COH
CHj "1"
CHO .OH _ _
HCOH

HC~--O
--0
/o\/
0 CH
CH3 0 CH OH OCH3 H2i CH
I 2
HCOH
HC-- 0 HOC,H2 HC - - CH

I HO~
HCOH /#~/L- CH
HE ~ o / C H 2
~H20H I OCH3

0
CH ~ HCO~
CH3d" OCH3 ( ~ O C H
3
H C - - O

.OCH ~.. HOH C

~½~ CH30
oi
OOH

HCOH
HC 0

.~ OCH3
CH30 IH20H H21 OH
0 CH HC -0

HCOH C~-----O

OH OH
F i g u r e 2 Schematic formula for a section o f spruce lignin consisting of 16 units ~2'13

92 Enzyme Microb. Technol., 1981, Vol. 3, April

 Biodegradation of cellulosic materials." Virendra S. Bisaria and Tarun K. Ghose
The bonds at the folds are supposedly the major weak
links and are mainly responsible for chemically induced
chain scission. On solvent pretreatment, the width of the
crystallite is increased due to swelling as well, as the fold
length decreases to D P ~ 30 (ref. 20; see Figure 3). It has
been established that pretreatment with the solvent does
not cause an appreciable reduction in DP of the original
cellulose. Taking this fact into account, a decrease in fold
length is only possible if the number of folds per molecule
increases. Since solvent pretreatment increases the number
of folds per molecule, it is expected to enhance the hydro-
lysis rate, as observed experimentally. Moreover, the
swelling accompanying the solvent pretreatment makes the
folds within the crystallite more accessible to acids or
enzymes. Thus, the folding chain model for the crystallite
structure of cellulose can explain the observed enhance-
ment of the rate of cellulose hydrolysis on solvent
pretreatment.
King and Vessa121 cultivated T. reesei on cellulose
preparation of four crystal forms and then determined the
activation energies for the reaction of the enzymes pro-
duced on each substrate during its activity on all four
substrates. The results showed that this organism produced
enzymes that required minimum activation energy for
hydrolysis of the specific substrata on which it was culti-
vated (Table 5). The results were remarkable because they
indicated that the enzyme synthesizing machinery of this
fungus can adapt to the structure of the active site of the
enzyme so as to accommodate a specific crystal lattice
structure in its substrate.
dk
ii
g cellulase is a complex of enzymes or enzyme-like factors
,r . organism. The practical saccharification of cellulosic
Untreated Cellulose in Solvent
cellulose solution pretreeted
(folding chain model) cellulose
Figure3 Changes in crystallite structure of cellulose on solvent
pretreatment 2°
Table 5 A c t i v a t i o n energy o f cellulase enzymes (T. reesei) in
degrading different cellulose structures 21
Cellulose structure f o r m A c t i v a t i o n energy
used to produce enzyme on structure f o r m
(cal gmol -l K -l)
I II
Form I 3270 6940
Form II 6190 5430
Microorganisms involved in cellulose, h e m i c e l l u l o s e
and lignin-degrading e n z y m e s
The availability of high activity cellulase is the basis for a
successful process for the enzymatic conversion of cellulose.
Although many fungi and bacteria degrade cellulose, the
products of growth are microbial cells and metabolic
products such as carbon dioxide and methane. Only a few
fungi, such as Trichoderma reesei (formerly called T.
viride22), 23-29 Trichoderma koningii, 30-33 T. lignorum, 34
Sporotrichum pulverulentum (formerly called Chrysosporum
lignorum), 3s Penicillium funiculosum, 36'37 P. iriensis, 38
Aspergillus wentii, 39'4° Po lyporus adustus, 4] Fusarium
solani, 42 F. lini, 43 Sclerotium rolfsii, 44'45 Eupenicillium
javanicum, 46 Schizophyllum commune 47 and a few bacteria
such as Clostridium thermocellum, 48-s° Thermomono-
spora sp., sl Cellulomonas sp., 52'53 and Streptomyces
flavogriseus s4, are known for their ability to produce high
activity cellulase capable of extensively degrading insoluble
cellulose to soluble sugars in vitro. In addition, there are
many organisms that produce cellulase preparations which
degrade only soluble cellulose derivatives such as carboxy-
methyl cellulose (CMC) while a few organisms produce very
little residual cellulase of any type of despite their active
growth on insoluble cellulose, zs This is due to the fact that
and that not all components of the cellulase complex are
necessarily found in the culture fluid after growth of the
residues requires a stable cell-free enzyme preparation
with adequate levels of all essential components of the
enzyme. Rapid growth on and decomposition of cellulose
and/or production of high levels of enzymes capable of
degrading soluble cellulose derivatives only are not adequate
criteria for selecting organisms to be used as a source of
cellulase. Some thermophilic organisms, such as an asco-
mycete, Chaetomium thermophile var. dissitum,SS Humicola
sp. 56 and a Thermomonospora sp. 57, also produce a cellulase
enzyme system capable of degrading native cellulose.
Thermophilic organisms are being looked at as a source of
thermostable cellulases; however, cellulases from thermo-
philes may not necessarily be more heat-stable than
cellulase from mesophiles, s8
Most of the work on hemicellulases has been concerned
with xylanases, since their substrate xylans constitute the
largest proportion of hemicelluloses in pasture plants.
Xylanases have been detected in several rumen bacterial
species, such as Bacillus firmus, B. pumilus, Bacteroides
amylogens, Butyrivibrio fibrisolvens, Ruminocoecus albus,
Clostridium species, s9-63 Fungi such as Myrothecium
verrucaria, Aspergillus oryzae, A. niger, A. wentii, A. terreus,
Coniphora cerebella, Trichoderma reeseL Chaetomium
trilaterale and Penicillium janthinellum also produce
xylanase enzymes. 64'6s Xylanases and other glycanases
Enzyme Microb. Technol., 1981, Vol. 3, April 93
Hevlews

have been found to be associated with cellulases in enzyme Table 6 Cellulase and hemicellulase assays
preparations of fungal origin. 66'67
Enzyme Substrate(s) Product(s)
Lignin-degrading microorganisms are also found among
both fungi and bacteria. Soft-rot fungi 68-70 belonging to Cellobiase Cellobiose Glucose
species of Paecilomyces, Allescheria, Preussia, Chaetomium Cellodextrins
and Stachybotrys, brown-rot fungi 68'71'72 such as Poria Xylobiase Xylobiose Xylose
monticola, P. cocos and Lenzites trabea, and white-rot Xylodextrins
Aryl-Ct-glucosidase/ o-Nitrophenyl o-Nitrophenol
fungi 73-77 belonging to species of Coriolus versicolor, /3-glucosidase /3-glucoside
Polyporus anceps, Phanerochaete chrysosporiurn, Sporo- Salicin Saligenin
trichurn pulverulenlurn, Aspergillus fumigatus, Polyporous /3-Xylosidase o-Nitrophenyl o-Nitrophenol
versicolor, P. hirsutus, Poria subacida, Polyporus abientinus, /3-xylopyranoside
Exo-13(1-~ 4)-glucanase
Gleoporus dichrous and Lentinus edodes have been found Cellobiohydrolase Crystalline cellulose Cellobiose
to have capacity to degrade lignin to varying degrees. (CBH, C 1) (cotton)
Bacteria capable of degrading lignin or lignin related Avicel
phenolic compounds include species of Nocardia, Strepto- Cellodextrins
Glucohydrolase Crystalline cellulose Glucose
myces, Pseudomonas, Flavobacterium, A erornonas, Bacillus, (cotton)
Agrobacterium and Xanthomonas. 78-82 Some detailed Avicel
reviews discussing the lignin decomposing groups of wood Cellodextrins
decay fungi and mechanisms of its microbial degradation Endo-~3(1 ~ 4)- CM-cellulose Reducing sugars,
have appeared. 68'69'83'84 The lignin model compounds glucanase (Cx) HE-cellulose and/or loss in
Amorphous cellulose viscosity
have been used for the study of lignin biodegradation. It 'Cellulase' (FPase, Filter paper (FP) Reducing sugars/
has been observed that microorganisms having the capacity Avicelase, Avicel loss in weight/
to degrade these model compounds often do not degrade hydrocellulase) Hydrocellulose reduction in
the lignin macromolecule; however, known lignin degraders Cellodextrins absorbance
Other cellulase assay
generally decompose lignin models. Swelling factor Cotton Uptake of alkali
Cellulase Dyed cellulose Release of dye
Thread Breaking strength
Assays and fractionation of enzymes Hemicellulases
One of the main difficulties with the assay of cellulolytic Xylanase Xylan Xylose/reducing
enzymes is the choice of suitable substrates. Although there Glucuronoxylan sugars
Arabinoxylan
is no doubt that pure cellulose is composed of very long
Mannanase Poly (galactomannan) Reducing sugars
chains of/3(1-+ 4)-glucose units, there is still much contro-
versy on how these chains are arranged in the microflbril
and how the latter, in turn, form the cellulose fibres found scientific research, and this seems to be missing in the area
in woody materials and which constitute cotton. The of cellulase research.
question as to whether regions of less well ordered cellulose From the preceding discussion, it is clear that if one
chains exist in the micro fibril is of fundamental importance wishes to discover all the different cellulolytic components
and is yet to be clarified. Moreover, cellulose samples of in a culture fluid, several different substrates are required.
different origin vary widely in chain length and the degree Another factor which renders the cellulase fractionation
of interaction between the chains. Cellulase is a complex of difficult is that the cellulase enzymes are nearly always
enzymes containing mainly exo-glucanases and endo- present as isoenzymes. These isoenzymes often differ only
glucanases, plus cellobiase. For the complete hydrolysis of slightly in isoelectric pH and are therefore difficult to
insoluble cellulose, a synergistic action between these separate. It is also not very certain whether the components
components is required. Since different cellulase prepara- resolved, for instance, by isoelectric focusing actually
tions vary widely in the proportions of the different com- represent species of the same type of enzymes, or if they
ponents, depending on source, growth conditions and differ in specificity.
harvesting and handling procedures, the rate and extent of Before describing the fractionation procedures used for
their hydrolysis of cellulose substrates also vary widely. isolating cellulase enzyme components, it is important to
Assay of cellulase components requires a variety of fairly mention briefly the components present in the multi-
complicated procedures. Native cellulose fibres such as membered cellulase enzyme system. The most acceptable
cotton are unsuitable since their rate of hydrolysis is very trend is to consider the cellulase system as follows.
low. Moreover, some cellulases do not have a readily deter- (i) Endo-/3(1-+4)-glucan glucanases (the Cx enzymes as
minable effect on crystalline cellulose. referred to by Reese) are present in several components
Commercial cellulose preparations such as microcrystal- varying in degree of randomness. One of these may be the
line Avicel are more rapidly hydrolysed but are nearly as enzyme that acts first on crystalline or highly ordered cellulose
poorly defined as cotton fibres. The most convenient sub- (ii) Exo-/3(1-+4)-glucanases are present in two major
strates are carboxymethylcellulose (CMC) and hydroxy- forms: (a)/3(1 -~ 4)-glucan cellobiohydrolase (CBH) removing
ethylcellulose (HEC). CMC is rapidly hydrolysed but can cellobiose units from the non-reducing ends of the cellulose
not be used as an universal substrate since it is not attacked chain. This CBH is being currently equated with the old C1
by all the enzymes present in cellulolytic culture fluids. enzyme by many workers, (b)/3(1~ 4)-glucan glucohydro-
A cellooligosaccharide such as cellotetraose is attacked by lase, which removes glucose units from the non-reducing
all known types of cellulases but is rather difficult to prepare. end of the chain.
As a result, a bewildering array of substrates, enzyme actions, 0ii) Cellobiase,/3(1 -+ 4)-glucosidase, which converts
units and activities have been in use (Table 6), partly due to cellobiose and other cellodextrins into glucose.
the tendency of workers to develop their own assay pro- The most intensive fractionation studies have been
cedures. A rational nomenclature is a very valuable tool in carried out on cellulases elaborated by T. reesei, 85-89

94 Enzyme Microb. Technol., 1981, Vol. 3, April

 Biodegradation o f cellulosic materials: Virendra S. Bisaria and Tarun K. Ghose

T. koningii 30'31'90-92 and S. pulverulentum 41 using
standard protein separation techniques such as solvent
precipitation, ion-exchange chromatography, molecular
sieve chromatography and isoelectric focusing. The frac-
tionation schemes for the separation of different cellulase
components from T. reesei and T. koningii are shown in
Figures 4 and 5, respectively. It is clear from these Figures
that all the components are present in multiple forms, often
as isoenzymes. A comparison of cellulases from different
microorganisms with respect to certain characteristic
protein parameters such as molecular weight, isoelectric pH
and carbohydrate contents is given in Table 7.
It seems difficult to make any generalization about the
size of cellulase components. The most common range of
their molecular weights is probably 12 000 to 80 000. The
smallest and largest molecular weights of endo-glucanases
have been reported to be 5300 and 145 000, respectively.88'93
/3-Glucosidases appear to have larger molecular weights than
exo- and endo-glucanases; the largest molecular weight of
/3-glucosidase found so far is 400 000. 91 The isoelectric
points of all the components so far investigated have been
found to be on the acidic side, pH < 6.3. The most common
range of isoelectric pH seems to be as follows: exo-
glucanase, 3.7 to 4.3, endo-glucanase, 3.3 to 6.2 and
/3-glucosidase, 5.5 to 5.9. Further, the cellulase components
may or may not be glycoproteins (Table 7).
Xylanases have been purified from T. reesei, 4°'94 T.
roseum, 94 A. niger 9s and other fungi and bacteria 9'96'97
by ammonium sulphate precipitation, gel filtration, ion
exchange chromatography, hydroxyapatite chromato-
graphy and isoelectric focusing. Xylanases of the endo type
[/3(1 -+ 4)-D-xylan xylanohydrolase, EC 3.2.1.8] are the only
types that have been characterized. Although exo-xylanases
[/3(1-+ 4)-D-xylan xylohydrolase] have been found, literature
on their purification and mode of action is not available, to
the best of our knowledge. The molecular weights of endo-
xylanases have been found to lie between 16 000 and
39 000. 95 The isoelectric pH of xylanases purified from
different sources have also, like cellulases, been reported to
be on the acidic side, between 3.9 and 4.5.
The third component of the xylanase enzyme system, i.e.
/3-xylosidase ~-D-xyloside xylohydrolase, EC 3.2.1.37),
which has a similar action to the/3-glucosidase component
of the cellulase system, causes hydrolysis of/3(1~4) bonds
at the non-reducing ends of xylooligosaccharides and
~(1 ~ 4)-aryl xylopyranosides./3-Xylosidase, which is widely
distributed in microorganisms such as Aspergillus niger 98
and Bacillus pumilus, 99 forms xylose from xylooligo-
saccharides produced by the action of endo- and exo-
xylanases on xylan. A 27-fold purification of/3-xylosidase
has been reported in 19% yield from A. niger using dif-
ferential ammonium sulphate precipitation and cation

Table 7 Characteristic protein parameters of cetlulase components isolated from different microorganisms

Organism Cellulose Molecular Isoelectric Carbohydrate Reference

components weight pH (%)

T. reesei Exo-glucanase 42 000 3.79 9 89

Endo-glucanase
I 12 500 4.60 21
II 50 000 3.39 12
3-Glucosidase 47000 5.74 0
T. koningii Exo-glucanase
I 62 000 3.80 N.a. 9 1 , 92
II 62 000 3.95 N.a.
Endo-glucanases
Cx 1 13 000 4.73 N .a.
Cx2 N .a. N .a. N.a.
Cx3 a 38 000 4.32 N.a.
Cx3 b 38 000 4.32 N.a.
Cx4 31 000 5.09 N.a.
Cx5 N.a. 6.28 N .a.
3-Glucosidase
I N.a. 5.53 N .a.
II N.a. 5.85 N.a.
S. pulverulentum Exo-glucanase 48 600 4.3 0 41
Endo-glucanases
T1 32 300 5.32 10.5
T2a 36 700 4.72 0
T2b 28 300 4.40 7.8
T3a 37 500 4.65 4.7
T3b 37 000 4.20 2.2
3-Glucosidase N.a. N.a. N.a.
Geotrichum candidum 3C Exo-glucanase N.a. N.a. N.a. 93
Endo-glucanase
I 82 000 3.75 N .a.
II 1 4 5 000 4.0 N .a.
III 36 000 4.0 N .a.
IV 11 0 0 0 4.0 N .a.
V 67 000 3.3 N .a.
Cellobiase 200 000 5.9 N.a.
F. solani Exo-glucanase 45 000 N .a. N.a. 91
Endo-glucanase 37 000 N .a. N.a.
~ Glucosidase 400 000 N .a. N.a.

N.a. = not available.

E n z y m e M i c r o b . T e c h n o l . , 1981, V o l . 3, A p r i l 95
Reviews

Crude cellulose
I
Molecular sieve chromatography ( 8io- gel P- I0 )
L
I I
Ion-exchangechromatography Ion- exchange
(DEAE-Sephadex A- 50) chromatography
(Arginine - Sepharose 6B )

I I
Ion- exchange chromatography Isoelectric focusing Isoelectric focusing
(SE- Sephodex C-50) (pH gradient 5.0-4.2) (pH gradient 4-5.2)

[
Biospecific chromatography
I
Isoelectric focusing
(Avicel) (pH gradient 5-7)

Isoelectric focusing Molecular- sieve Molecular - sieve Molecular-sieve

(pH gradient 2.5 - :5.5) chromatography chromatography chromatography
I (Big-gel P-60) (Big-gel P-60) (Big-gel P-f0)
Molecular- sieve chrom(Jtography I
(Big-gel P-60)

I Endo-g'ucan°se n l [ ,8-Glucosidose[ I Endo-glucanase] [ Endo-glucanaseIJ

Figure 4 Fractionation of T r i c h o d e r m a reesei cellulase 89

T konzng/)' exchange chromatography. 98

cellulose Little work has been reported on enzymes involved in
lignin degradation. The enzymes used by microorganisms
to degrade lignin model compounds have been found
SephadexG-75
q typically to be intracellular and highly substrate-specific,
Low-mol wt Cx and are unlikely to attack an extracellular complex polymer
CI +Cx + ,8 - glucosidase

~
Isoelectric such as lignin. Hence, the enzymes which attack the lignin
focusing
ephodex macromolecule may be very different from those that
Purified Iow-mol wt Cx attack model compounds. 84 An alcohol oxidase, purified
Cx~ (pl= 4.73)
Cx+,8-glucosidese from the culture filtrate of Fusarium solani 1°° after growth
trace Cxz
on DHPs has been reported to non-specifically oxidize a
. ~ h o d e x CI + D ~ E - wide range of lignin model compounds containing a, fl-
Sephadex unsaturated primary alcohol structures (such as coniferyl
alcohol) and macromolecular structures of various lignin
Cx ,8-Glucosidase CIf ~ ~- Cx2
~, Cx5 preparations. Another enzyme, cellobiose-quinone oxido-
Isoelectric ] I Isoelectric focusing
reductase has been found to take part in both lignin and
focusing J (pH 37-4.3 cellulose degradation.]°lAlthough the absolute requirement
of phenol oxidases in lignin degradation has been estab-
lished, 1°2 its exact role is still to be elucidated.
Cx/ ~CCx4 L CI (pl 3.95) " C I (pl 3.8)
(pl 4.32) (pl 509)
r. ~ Isoelectrc focusing Mode of action of cellulases and hemicellulases
j ,8-Gluc°sidasel ~ (pH 4-6)
LSE-Sephadex (pl 5.55) The degradation of crystalline cellulose is a complex process
requiring the participation of many enzymes. Reese and his
cJ. 5 colleagues l°a first suggested a route for the conversion of
native cellulose to soluble sugars based on a two-step
Isoelectric focusing sequential process (Figure 6). The C 1 enzyme was envisaged
~ pH 5 - 7
to be a hydrolytic factor (a hydrogen bondase) producing
an 'activated' or 'modified' cellulose which, in turn, was
capable of being attacked by the hydrolytic, Cx, enzymes
,8-Glucosidase Cx5 of the cellulase enzyme system; the x in Cx reflects the
(pl585] (pl 6.28) multicomponent nature of the enzyme. According to these
Figure 5 Fractionation scheme for purification of different workers, microorganisms which grow on soluble cellulose
cellulolytie enzymes from T. k o n i n g i i 92 such as CMC synthesize only Cx components whereas

96 Enzyme Microb. Technol., 1981, Vol. 3, April


Crystalline CI Reactive Cx Soluble
cellulose ---~ cellulose =- sugars
Figure 6 Reese's c o n c e p t ~°3 ( 1 9 5 0 )
microorganisms growing on highly ordered forms of
cellulose produce both C1 and Cx. The latter group of
organisms includes T. reesei, T. koningiL F. solanL
S. pulverulentum, P. funiculosum and S. rolfsii. It was on
the basis of observations made with actively growing fungi
that the C1-Cx hypothesis was first formulated and it was
many years later that a cell-free filtrate from T. reesei was
isolated which was sufficiently active on highly ordered
cellulose for it to be concluded that a C 1 component was
present. 1°4
There is substantial evidence for the existence of a C l
component in the multi-membered cellulase enzyme
system of true cellulolytic microorganisms. However,
controversy still exists about the specificity of C 1 com-
ponent(s) and the original two-step C r C x mechanism.
There are contradictory claims in the literature on this
matter; some of the important observations are discussed
in the following paragraphs.
Selby and Maitland l°s isolated a C 1 component, a Cx
component and a cellobiase fraction from the culture of
T. reesei using Sephadex and ion exchange chromatography.
The C 1 fraction by itself did not support any cellulose
degradation but it was active in the presence of the Cx
fraction. The important finding of the study was the
striking synergism between C1 and Cx. Two possible hypo-
theses were proposed: that C1 makes cotton fibre more
susceptible to Cx action or that C1 is inhibited by products
of its own reaction unless these are removed by the action
of Cx. The second possibility is supported by the fact that
additional synergistic action of the cellobiase fraction was
observed. Furthermore, C 1 activity was found to increase
by 50% when simultaneous dialysis against water was
carried out. Halliwell 9° clearly demonstrated that although
the relatively weak action of the C 1 component against
cellulose can be promoted considerably by the Cx com-
ponent, this was achieved not by what has so far been
considered the main enzymic activity of this Cx component
but was attributed to cellobiase. The increased activity of
C 1 was due to the removal of its inhibitor cellobiose,
which was hydrolysed by cellobiase into glucose. Recent
advances in the elucidation of the nature and mechanism of
C1 action suggest it to be a cellobiohydrolase acting as an
exo-glucanase against native and derived forms of
cellulose . 3 3 , 4 2 , 8 9 , 9 2
Many reports which contradict the proposed hypothesis
that C1 initiates attack on crystalline cellulose have
appeared in the literature. Wood 30'31 purified a C1 com-
ponent from cellulase system of T. koningii and F. solani.
The purified C1 component was found to have limited
ability to produce reducing sugars from CMC but was not
able to attack highly ordered cellulose by itself. Acting
alone, it attacked oligosaccharides (such as cellotetraose,
cellohexaose) and produced cellobiose as the sole reaction
product. Similar results were also reported by Pettersson
and his coworkers 1°6 withpurified C1 component from
T. reesei. Nisizawa et al. 101 _reported that the purified
CMC-cellulase fraction decreased the degree of polymeriza-
tion (DP) of cotton at a much faster rate than purified C1
fraction. Streamer et al. 3s observed that if dewaxed cotton
was pretreated with endo-glucanase, the exo-glucanase
released far more degradation products than from an
Biodegradation of cellulosic materials: Virendra S. Bisaria and Tarun K. Ghose
Cellobiase
untreated cotton. All these observations strongly support
~- Glucose the alternative theory that Cx (endo-glucanases) acting
randomly over the cellulose chain open up the chain,
allowing subsequent action by C 1 (exo-glucanase) on the
exposed chain ends.
Wood 91'108 studied the properties of purified C1 and
Cx components by reconstituting them in the proportions
originally present in a culture filtrate of T. koningiL It was
observed that a recombination of C 1 with either Cx or with
/3-glucosidase was not as effective as a recombination of all
the three, and that all five components must be present in
their original proportions to account for the cotton-
solubilizing activity of the unfractionated culture filtrate.
Similar results on the synergistic effect of a C1, Cx and
cellobiase component have been reported for the manifesta-
tion of maximum activity with celluloses from T.
reesei,S9'l°S P. funiculosum 36 and F. solani. 42 A strong
synergistic response was observed for the five endo-
glucanases and an exo-glucanase isolated from the culture
filtrate of S. pulverulentum in the degradation of dewaxed
cotton and Avicel, but not of acid-swollen cellulose. 41
Also, endo-glucanase pretreatment increased the production
of degradation products from cotton by exo-glucanase,
supporting the theory that endo-glucanase initiates the
attack by creating open chain ends where exo-glucanase can
act.
This evidence strongly supports the mechanism of
enzymatic cellulose degradation, described by Pettersson, 89
as follows.
(a) Regions of low crystallinity in the cellulose fibre
are attacked by endo-glucanases and free chain ends are
created.
(b) Exo-glucanases start the degradation from the chain
ends by hydrolytically removing cellobiose.
(c) Cellobiose is hydrolysed to glucose through the
action of/3-glucosidase.
This kind of mechanism was first suggested by Eriks-
son. t°9 It is now understood that the endo-glucanases act
randomly over the cellulose chain and the exo-glucanases
act on exposed chain ends by splitting off celloboise 91 or
glucose, 110 the endo- and exo.-glucanases having a strong
synergistic action 42 (Figure 7). This hypothesis, which
enjoys considerable support, is just the reverse of the
hypothesis proposed by Reese. 1°3
In the light of these developments, Reese 111,112 has
now modified his original C1-Cx concept in which there is
some initial swelling or modification of the crystalline
cellulose followed by synergistic attack of endo- and
exo-glucanases (Figure 8). This concept retains the idea of
a swelling action (disruption of hydrogen bonds) as
originally assigned to CI but now accepts that a covalent
linkage is split and that this act is accompanied by splitting
of hydrogen bonds. Thus C 1 becomes a member of the
endo-glucanases (Cx). But it is a very special member
having properties not possessed by most endo-glucanases,
e.g. activity on crystalline cellulose, disruption of H-bonds,
lack of action on CMC and inability to act on products of
its own reaction. Reese has correlated this first step with the
enzymatic hydrolysis of other highly ordered structures such
as DNA and collagen, which are affected by enzymes whose
function is to produce some disorder in the structures as a
preliminary to hydrolysis by other enzymes. Such enzymes
are highly specific for ordered molecules and these are
unable to act on the disordered structures that they pro-
duce. Other randomly acting cellulose components lack the
specific properties required for initiation of the reaction
Enzyme Microb. Technol., 1981, Vol. 3, April 97
Reviews
Cx3.) and (Cl and Cx4 ) that have formed a loose complex
on t]ae surface of the cellulose crystallite (Table 8).
Crystalline Cellulose enzymes
regions Amorphous The involvement of oxidative enzymes in the breakdown
. . . . . . ~J. regions of cellulose has also been studied by some groups. Eriksson
and coworkers 110 observed that cell-free filtrates of the
fungi S. pulverulentum, Polyporus adustus, Myrothecium
I verrucaria and T. reesei solubflized cellulose at a slower
a Endo-/3- gluconase ( EG=Cx ) rate under anaerobic than under aerobic conditions. Accord-
ing to Wood and McCrae, 92 the extent of involvement of an
oxidase in T. koningii was markedly less, causing only 5%
reduction in weight loss (Table 9). The results with F. solalli
and P. fimiculosum showed that oxidation was not always a
/f / F factor in enzymatic cellulose degradation. In S. pulverulenmm
I the oxidative enzyme has been found to oxidize cellobiose to
cellobionic acid. The cellobiono-3-1actone and cellobionic
b Cellobiohydrolose ( CBH: CI ) acids formed by its action are thought to prevent the trans-
glycosylation reaction from taking place where high cello-

"4-- biose concentrations build u p J 13

Reese and Mandels 114 used CO2 atmosphere to replace
air in order to investigate whether an oxidative mechanism
was involved in cellulose degradation using T reesei QM 9414
EG/CBH
and C30 strains. It was observed that C02 had no effect on
the hydrolysis rate with either enzyme under shaken or
C
unshaken conditions. On the other hand, CO2 did have a
favourable effect in unshaken flasks on the amount of
Avicelase activity and protein remaining in solution at the
end of incubation (Table 10).
Production of compounds inhibitory to the cellulase
/3- Glucosidose action in culture media may also affect the activity of the
d cellulase enzymes. It has been reported that culture filtrates
o • ~ = G~ucose obtained by growing a Fusarium sp. on media containing
high cellulose concentration, showed a decrease in hydro-
lytic activity towards cotton.11 s This was found to be due
Figure 7 Schematic representation of synergistic action of enzymes
in cellulolysis 42,tt°
to the increased synthesis of cellobiose dehydrogenase
enzyme with increased cellulose concentration. High
Endo-glucanases activity of this enzyme caused a corresponding accumula-
Oligomers
tion of lactones in the reaction mixture which are known
to inhibit cellulases. 116

Crystalline CI ?
ce~Wose
Modified
~ cellu%se
f Cellobiohydrolase ~Cellobiose There are relatively very few reports on the mode of
action of hemicellulases. Basically, like cellulase, hemi-
cellulases also occur in two basic forms, i.e. exo-type and
Table 8 Solubilization of dewaxed cotton by reconstituted
Gluco-hydrolase/ cellobiose/
/3 qlucosidose mixtures of the components of cellulose complex of T. koningii 92
- - ~ Glucose
Figure 8 Reese's Modified Concept H= (1977) Enzyme Solubilization of cotton
(%)
but are capable of catalysing the hydrolysis of the products
C1 + Cxl 2
of C 1 action. 112
C t + Cx3 a 34
Though synergism between exo- and endo-glucanases is C 1 + Cx3 b 2
clearly established, the recent discovery 3a'92 that C1 (exo- C 1 + Cx4 51
glucanase) acts synergistically only with certain Cx (endo- C 1 + Cx . + CxA 4- 1 3<jlucosidase 53
glucanases) suggests the possibility that more crystalline C 1 + Cx1 Cx3 a 4- Cx3 b + Cx4 72
Unfractionated complex 71
areas are effectively solubilized only by rapid sequential
action of those exo- and endo-glucanases that are capable
Table 9 Degradation of cotton cellulose by cellulase preparations
of forming an enzyme-enzyme complex on the surface of
from different fungi 92'1~°
cellulosic chains. It was found by Wood and McCrae 92 that
out of the six endo-glucanases isolated from T. koningii Source of cellutase Cellulose degradation (% w t loss)
(Figure 5), the lowest molecular weight component, Cx,,
was most random in its action, i.e. it produced the largest 02 atmosphere N 2 atmosphere
changes in viscosity of CMC and in DP of HaPO4-swollen 21.5
S. pulverulen turn 52.1
cellulose. But this most randomly acting component, which P. adustus 42.6 18.0
should show the highest capacity for solubilizing cotton, in M. verrucaria 33.6 17.0
fact did not show any synergism with Ca enzyme (Table 8). T. reesei 20.0 10.0
7:. koningii 43.0 38.1
It has, therefore, been suggested by these workers that 43.0
P. funiculosum 42.9
extensive hydrolysis of highly ordered cellulose is accom- F. solani 34.1 34.6
plished only by those pairs of hydrolytic enzymes (C1 and

98 E n z y m e M i c r o b . T e c h n o l . , 1981, V o l . 3, A p r i l
 Biodegradation of cellulosic materials: Virendra S. Bisaria and Tarun K. Ghose
Table 10 Effect of shaking and CO 2 on the enzymatic hydrolysis of Avicel cellulose after 70 h 114
Cellulase source Atmosphere % Hydrolysis
Shaken Unshaken
7-. reesei QM 9414 Air 42 49
CO 2 43 50
T. reesei C 30 Air 26 50
CO 2 23 51
endo-type, the endo-type being more common. Thus, there
are both types of u-arabinanases, D-galactanases, D-man-
nanases and D-xylanases among hemicellulose degrading
enzymes. The/3(1 -+ 4)-D-xylans are the most abundant
polysaccharides of terrestrial plant hemiceUuloses and the
enzymes, xylanases, capable of degrading this group have
been studied in some detail. 9'4°'94-97 Studies of the
purification and mode of action of exo-xylanases have not
been reported. Among the endo-xylanases produced by
fungi, two types are recognizable: those that release and
those that do not release L-arabinose from arabinoxylans
and arabinoglucuronoxylans. 9 Both types, however, are
capable of hydrolysing glucuronoxylans and/~(1 ~ 4)-D-
xylans.
The mode of action of xylanase depends on its source.
Kawanami 117 found that when wheat straw, barley straw,
rice straw and corncob xylans were exposed to prolonged
incubation with Chaetomium trilaterale, hydrolysis of
xylosidic linkage was ~92% in 24 h and xylose was the
main hydrolysis product. Detailed studies on the nature of
enzyme components involved in the breakdown of hemi-
cellulose are expected to lead to a better understanding of
their participation in the overall degradation of cellulosic
substances. 118,119 The authors 118 have studied the role of
xylanase action on the hydrolytic breakdown of bagasse. It
was observed that xylanase pretreatment had a definite
increased effect on the hydrolysis of bagasse. This sug-
gested that xylanase helped to create more accessible
cellulosic regions which could be acted upon by exo- and
endo-glucanases by hydrolysing the xylan component of
the bagasse, thereby resulting in higher sugar production.
Furthermore, xylanase action was found to be most effec-
tive when acting synergistically with other hydrolytic
components, viz. exo- and endo-glucanases in the case of
bagasse degradation. The mechanism of the synergistic
action has further been substantiated by the authors by
studying the adsorption characteristics of the exo-glucanase,
endo-glucanase, t3-glucosidase and xylanase components in
relation to the nature of cellulosic substance, i.e. native,
modified or spent bagasse. 118
A knowledge of the mechanisms involved in lignin
degradation, like that of cellulose and hemicellulose, is
also essential for the development of technical bioconver-
sion processes by which lignocellulosic materials can be
processed to sugars, ethanol, solvents or proteins. At
present, these processes are based primarily on knowledge
of enzyme mechanisms involved in microbial degradation of
cellulose and, to some extent, on hemicellulose. It is known
that degradation of lignin is the rate-limiting step in these
processes. A better understanding of the mode of action of
the lignin degrading enzyme is, therefore, clearly important
in making the bioconversion processes based on ligno-
cellulosic residues economical. Cellulase-deficient mutants
of S. pulverulentum may give some new insight into the
association of lignin with cellulose and hemicellulose in
lignocellulosics. 120
% Residual Avicelase % Residual protein
Shaken Unshaken Shaken Unshaken
21 32 44 49
21 50 43 56
1 49 16 64
0 57 13 67
Hypercellulaseproduction
The production of cellulase by a microbial cell is under
various strict genetic and biochemical control mechanisms.
Economy within the cell instructs the organism not to
oversynthesize enzymes and other metabolites unless their
production is useful to cell growth or survival. These con-
trols include induction, catabolite repression and end-
product repression. Each of these controls is operative under
cellulase production conditions, with the result that enzyme
yields are low. The mechanisms controlling the synthesis
and activity of each of the cellulase components are sum-
marized in Table 11. The aim of any microbial mutant
selection programme should, therefore, be to isolate
organisms which are no longer subject to these constraints.
Such mutants will drastically cut enzyme production costs
(which contributes to nearly 60% of the total process
cost 122) by increasing yields.
Several Trichoderma mutants have been isolated from
the present T. reesei QM 6a originally isolated at US Army
Natick Research and Development Command, Natick,
USA. The genetic lineage of current cellulase mutants of
T. reesei developed at Natick and Rutgers University is
shown in Figure 9. The wild strain QM 6a produces very
little cellulase because of the restrictions placed on cellulase
biosynthesis by genetic and metabolic controls. The mutant
strains NG 14 and MCG 77 with enhanced cellulase produc-
tivity are also subject to catabolite repression. With these
mutants the best yields were obtained when growth was
good but restricted by substrate limitation or by low pH.
The screening methodologies developed largely by Rutgers
group for the selection of high cellulase yielding mutants
are summarized in Table 12. A comparison of the cellulase
yields from different microbial mutants is shown in Table 13.
T. reesei NG 14 produces exo-glucanase component and fdter
paper degrading activity in higher yields, while endo-glucanase
and cellobiase are produced in better yields by S. rolfsii
UV 8 mutant. T. reesei is representative of a group of
mutants whose physiological characteristics in submerged
culture do not parallel those on agar plates. 121 NG 14 strain
is only partially resistant to catabolite repression. Another
strain, C 30, isolated from mutagenesis of NG 14, was found
to be resistant to catabolite repression and produced almost
as much filter paper degrading activity in the presence of 5 %
glycerol as it did during growth on cellulose alone. 121 T.
reesei C 30 was also resistant to catabolite repression by
5% glucose. Under controlled fermenter conditions, it gave
Table 11 Biochemical mechanisms controlling cellulase synthesis
and activity in T. reesei T M
Enzyme End product Catabolite Inducer
inhibitor represser
Cellobiohydrolase Cellobiose ~ Glucose or Cellulose
Endo-glucanase Cellobiose / metabolites Sophorose
Cellobiase Glucose of glucose Cellobiose
Enzyme Microb. Technol., 1981, Vol. 3, April 99
Reviews

activity of 15 FP units/ml, nearly same as that of NG 14

strain. Thus, both C 30 and NG 14 mutants showed about
Tr/chodermo reese/ 1 5 - 2 0 fold increase in cellulase activity over the wild type
QM 6a strain (0.5-1.0 FP units/ml). 123 Both the mutants
V a-] NG 14 and C 30 produce ~ 2 0 mg/ml extracellular protein.
A yield of ~100 mg/ml as cellulase would greatly improve
the economics of enzymatic cellulose hydrolysis. Some new
Linear accelerator UV mutant strains producing as much as 80 units/l • h in a batch
culture system and 7 units/g cell- h in a continuous culture
system have been reported. 29 Further strain improvement
I o.9,2 1,969 .97 work is being carried out in several laboratories, including
our own.
Evaluation of microbial strains should be made not only
Linear accelerator Nitrosoguanidine for cellulase activity but also for stability and performance
during hydrolysis. An increase in enzyme stability would
make it possible to hydrolyse cellulose at higher tempera-
I I,7, tures, thus speeding up the process, and would use less
enzyme, so reducing the enzyme cost. T. reesei C 30 has
been found to possess the most stable exo-glucanase
(Avicelase) compared with other Trichoderma strains: 114
UV- Kabicidin UV
the enzyme inactivation for this strain is only 28% in 3 days
at 50°C and pH 5.0. One of the most intriguing effects
reported recently u4 is the inactivation of C 30 cellulase due
I TK°4'
1' 77, to shaking (Table 10): the hydrolysis in shaken flasks was
only half of that in unshaken flasks in 3 days. The QM 9414
cellulase was less affected by shaking. However, under
OV UV unshaken conditions, C 30 enzyme was more stable than
QM 9414 enzyme.
Another potentially active strain of T. reesei (VTT-D-
78085) capable of producing a 2 - 3 fold increase in cellulase
I activity and 6-fold increase in ~3-glucosidase activity over that
Notick Rutgers of QM 9414 in a fermenter has been isolated by mutation of
72. reesei QM 9414.124 Optimization of the cultural conditions
for this strain is expected to result in a further improvement
Figure 9 Genetic lineage of cellulase mutants of T. reesei. TM in enzyme production. Besides T. reesei, thermophilic
Years indicate the year of their isolation actinomyces, 125-128 thermophilic anaerobes,129yeasts 13°

Table 12 Screening methodologies for isolation of high yielding and catabolite repression resistant cellulase mutants T M

Enzyme Colony inhibitor Substrate Catabolite Visualization

repressor

Total cellulase complex 1.5% oxgall + Phosphon 13 Acid-swollen 5% glycerol Clear zones after incubation at 50°C
cellulose
Endo-glucanase 1.5% oxgall + Phosphon El Carboxymethyl 5% glycerol or Clear zones after flooding with quaternary
cellulose 5% glucose ammonium compounds
(5'-Glucosidase (1) 1.5% oxgall + Phosphon D Esculin (0.1%) 5% glucose Black rings in the presence of ferric
ammonium citrate
(2) 1.5% oxgall + Phosphon D Cellobiose (1%) 0.2% 2-deoxy Large vs. pinpoint colonies
glucose
(3) 1.5% oxgall + Phosphon El Any of above 5% glycerol Overlay with Glucostat + cellobiose,
50°C, 30 min, red spot

Table 13 Some important cellulase producing organisms and increase in their yields through mutation

Organism Filter paper activity Cellobiohydrolase Endo-glucanase Cellobiase Reference

(units/ml) (units/ml X 10 -3) (units/ml) (units/ml)

Pestalotiopsis westerdijkii QM 38 0.15 2.93 50 N.a. 58

Penicillium iriensis QM 9624 0.77 11.58 81 N.a. 58
Trichoderma reesei QM 6a 0.55 18.91 59 0.3 29, 58
T. reesei QM 9123 1.68 29.26 96 N .a. 58
Sclerotium rolfsii CMC 142 1.4 N.a. 200 11 44
S. rolfsE UV-8 2.0 N.a. 190 18 45
7". reesei QM 9414 9.5 57.0 110 0.5 26, 44
T. reesei MCG-77 10.5 60.8 100 0.9 26, 29
T. reesei NG-14 14.6 58.9 135 1.35 26, 29
T. reesei C-30 14.0 N.a. 150 0.3 29

N.a., not available

100 Enzyme Microb. Technol., 1981, Vol. 3, April

 Biodegradation o f cellulosic materials: Virendra S. Bisaria and Tarun K. Ghose

and other strains 4s have also been used for obtaining high Table 15 Rapid ethanol production from bagasse hydrolysate using
cellulase producing organisms and/or mutants. S. cerevisiae in batch and continuous cell recycle systems 145

Process parameter Bagasse hydrolysate (BERC process)

Fuels a n d c h e m i c a l s f r o m cellulosic b i o m a s s Batch Continuous Continuous
Over the last five years, there has been a widespread interest cell recycle cell recycle
and 02
in the fermentation of cellulosic residues into energy-rich sparging
products, chiefly ethanol, and other chemicals as a result of
developing petroleum shortages.131 - 139 Ethanol is an Fermentable sugar conc. 20 15 15
important feedstock for the manufacture of a large number (%, w/v)
of chemical derivatives, polymers, and solvents. In the Ethanol conc. 12.7 9.5 9.0
(%, v/v)
continuing oil crisis, ethanol can be used as a gasoline Product yield 97 97 95
diluent or even as a gasoline replacement. Some of the (% of theoretical)
potential uses of ethanol and other degradation products Fermentation time (h) 5.0 5.5 3.5
of lignocellulosic residues are listed in Table 14. Yeast cell conc. (g/I) 27.2 32.0 42.0
Productivity (g/I • h) 12 18.3 32.0
Many cellulosic substances contain as much hemicellulose
as cellulose. Unless the hemicellulose can be properly
utilized, biomass conversion based on cellulose utilization
enzymes than equivalent quantities of glucose or cellobiose
alone has little chance of becoming economically attractive
so that, under equal conditions, saccharification is greater
due not only to the lost revenue from hemicellulose but also
in the coupled system. This system also solves the problem
to the added waste disposal costs. Unlike cellulose hydro-
of preventing contamination in the hydrolysis reactor with-
lysate, which contains mainly glucose, the hemicellulose
out adding toxic or inhibitory chemicals. The temperature
hydrolysate contains mainly xylose together with minor
optimum for ethanol production with yeasts is ~30°C while
quantities of other sugars such as arabinose, rnannose,
that of the cellulase system is ~50°C. Zymomonas mobilis
galactose and their uronic acids. Xylose finds its use as a
bacterium, which has a faster specifc rate of ethanol pro-
source of furfural, as a source of xylitol for use as a
duction and is capable of growing at higher glucose and
sweetener, as an antidiabetic agent and, lately, as a substrate
ethanol concentrations at 37°C, has been successfully used
for conversion to ethanol by a yeast. 14° Ethanol can be
with T. reesei cellulase for the simultaneous production of
obtained by the enzymatic degradation of cellulose and
ethanol, ls4,lss Combined saccharification-fermentation of
subsequent fermentation of glucose by yeast. Significantly
cellulose has also been carried out with Thermomonospora
important contributions have been made to produce
ethanol in an efficient manner. 141-1s3 Direct production cellulase and xylose-positive, non-cellulolytic thermophilic
anaerobes; the amount of ethanol formed in the system was
of ethanol from cellulosic materials has also been achieved
~70% of theoretical. High ethanol yields from cellulose in a
by an anaerobic simultaneous saccharification-fermentation
system using cellulases and yeast concurrently in the same co-culture of Clostridium thermocellum and non-cellulolytic
organism are presumed to be due to the formation of
reactor and has been found to be more productive than a
recombinants.l s6
normal two-step saccharification-fermentation system. 142-144
In rapid fermentation of bagasse hydrolysate in batch
Ethanol has been found to be less inhibitory to the cellulase
culture, ethanol concentrations of 9.7% (w/v) have been
Table 14 Potential uses of products derived from lignocellulosic obtained in 5 - 6 h in our laboratory. The viability of the
residues yeast was >94% for a continuous 10 day steady-state
experiment without enriching the medium with oxygen,
Products Uses
unsaturated fatty acids or sterols. The ethanol productivity
Glucose Energy (alcohol, ATP) was further improved by using a cell recycle system and
Protein was found to be 4.5 times higher than in a normal con-
Chemicals tinuous process without cell recycle. This productivity was
Fructose (sweetener)
Xylose Ethanol
further increased by 7.5 times (32 g/l) by sparging air at
Xylitol (sweetener) low flow rates (0.125 win). Table 15 lists some signifcant
SCP data on rapid ethanol fermentation of bagasse hydrolysate
Antidiabetic agent obtained at this Centre (BERC process). 14s
Ethanol Chemicals
Direct conversion of cellulosic biomass to mainly ethanol
Solvents
Beverages (plus minute amounts of organic acids such as lactic and
Biological energy (ATP) acetic acids) has been reported for an anaerobic and thermo-
Petrochemicals synthesis (via philic organism, Clostridium thermocellum. 1s7 This organism
ethylene) degrades both cellulose and hemicellulose fractions into
Gasoline diluent and octane
enhancer soluble sugars such as cellobiose, glucose, xylobiose and
Food and feed xylose. To make this single-step process economically
Furfural and 2,3-butanediol Chemicals and solvents (such as feasible, isolates of C. thermocellum, through adaptation
methylethyl ketone) and mutation, have been selected which possess the pro-
Lignin degradation products: Chemicals and solvents
vanillin, dimethylsulphide,
perties of high ethanol productivity, tolerance towards
dimethylsulphoxide, methyl ethanol and a negligible yield of organic acids. Although
mercaptan, ferulic acid, catechol C. thermocellum effectively degraded hemicellulose, it did
Butanol/lignin slurry Furnace and diesel fuel not possess the capacity to metabolize xylose. In order to
Lignin sulphonates Chemicals utilize the hemicellulose fraction, another bacterium,
Additives in stabilizers, grinding
aids, binders C. thermosaccharolyticum, which is environmentally com-
patible with C. thermocellum, has been used in mixed

E n z y m e M i c r o b . T e c h n o l . , 1981, V o l . 3, A p r i l 101
Reviews
culture studies to achieve a single-step fermentation of
cellulosic biomass to ethanol, ls8
Besides ethanol, increasing emphasis is also being given
to fermentative synthesis of other chemicals from cellulosic
materials so as to reduce significantly the petroleum require-
ment. The bacterium Clostridium acetobutylicum has been
used for the production of acetone and butanol with simul-
taneous production of a gaseous mixture containing a high
percentage of hydrogen gas. 159 The same organism has also
been used in immobilized calcium alginate gels to produce
mainly butanol along with traces of acetone. Yields of 65 g
butanol per day per kg of gel have been reported using a
column containing alginate beads with a bacterial concentra-
tion of 1% dry weight of cells. 16°
A strain ofKlebsiella pneumoniae has been found to
convert all the carbohydrate of hemicellulose in nearly
100% theoretical yield into 2,3-butanediol in a final con-
centration of 100 g/1 of fermentation broth. 161 A selected
strain o f A e r o m o n a s sp. has also been reported to give
100% yield of ethanol and 2,3-butanediol mixture from
hemicellulose.161 With the chemical method of dehydration,
2,3-butanediol can be quantitatively converted to methyl
ethyl ketone, which is one of the major industrial solvents
currently derived from petroleum. Hemicellulose hydro-
lysate, being a crude mixture of many mono- and oligo-
saccharides, presents a far greater challenge than glucose;
for instance, diauxic and multiauxic growth patterns are
often observed in hemicellulose fermentation. Thus, in
order to optimize and control the fermentation processes
based on carbohydrate mixtures, the mechanisms of enzyme
repression, induction and inhibition need to be understood
more clearly.
Though still at a basic level, lignin biodegradation has a
considerable potential as a source for a variety of chemicals
many of which are presently derived from petroleum. Low
molecular weight lignin degradation products such as
vanillin, dimethyl sulphide, dimethyl sulphoxide, and
methyl mercaptan are produced by controlled chemical
hydrolysis of lignin sulphonates or Kraft lignin under high
temperature and pressure. All these processes are energy
intensive. The potential advantage of using microorgarfisms
in lignin degradation is that the processes are likely to be
less energy intensive. In addition, the use of microorganisms
capable of altering lignin in specific ways may make it
possible to obtain significant and economical yields of
useful chemicals.
Future scope
Apart from the development of economical process tech-
nology for various bioconversion schemes based on ligno-
cellulosics, a few basic areas of in-depth investigation in
biomass utilization research include the following
topics.
Biomass susceptibility
From the practical point of view, the chemical or
physical agents used to alter or open up the lignin carbo-
hydrate complex should be low-cost and effective. Bio-
logical methods making use of lignin-degrading organisms
need to be studied so that the breakdown products are of
some economic value. Perhaps an understanding of the
entire complex process of cellulose, hemicellulose and
lignin degradation together will give new insight as to
how these should be most effectively utilized.
102 Enzyme Microb. Technol., 1981, Vol. 3, April
Cellulases
Cellulolytic mutants possessing the following charac-
teristics should be developed.
(1) Constitutive cellulases. The present use of insoluble
cellulosic inducers substantially increases fermenter opera-
tional costs and complexity. The constitutive mutants will
circumvent the use of insoluble cellulose substrates and
would result in considerable savings.
(2) Increased yields. The current mutants T. reesei
NG 14 and C 30 produce ~ 2 0 mg/ml extracellular protein,
predominantly as cellulase. There is potential to increase
this yield by at least four fold.
(3) Catabolite repression resistant mutants.
(4) Thermostable cellulases.
(5) End product resistant cellulases.
Hernicellulases
The mode of action of hemicellulases towards hemi-
celluloses of different origins and their participation with
cellulases in the overall degradation of cellulosic substances
needs detailed investigation. Development studies on hemi-
cellulolytic mutants possessing the properties mentioned
above for cellulases will also be very valuable for efficient
degradation of hemicelluloses.
Liguins
The chemical make-up of lignin, assay procedure, mode
of action of microorganisms and enzymes in lignin degrada-
tion and its association with cellulose and hemicellulose
requires in-depth studies. Specific microbial strains should
be selected for the development of specific lignin biocon-
versions to derive useful chemicals.
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