Chapter 1

Lignocellulose Biodegradation and Applications in

Biotechnology

Badal C. Saha
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Fermentation Biotechnology Research Unit, National Center

for Agricultural Utilization Research, Agricultural Research Service,
U.S. Department of Agriculture, 1815 North University Street,
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Peoria, IL 61604

Lignocellulosic biomass such as agricultural and forestry

residues and herbaceous energy crops can serve as low cost
feedstocks for production of fuel ethanol and other value-added
commodity chemicals. However, development of efficient
pretreatment and cost-effective enzymatic conversion of any
lignocellulosic biomass to fermentable sugars is a key issue. In
this overview chapter, various pretreatment options (dilute acid,
steam explosion, alkaline peroxide) and enzymes (mainly
cellulases and hemicellulases) involved in lignocellulose
degradation are presented. Mixed sugars generated by
lignocellulose biodegradation are fermented to fuel ethanol,
xylitol, 2-3-butanediol and other value-added products. Recent
advances in the developments on lignocellulose biodegradation
and applications in biotechnology are reviewed.

2 U.S. government work. Published 2004 American Chemical Society

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In 2003, about 2.81 billion gallons of ethanol are produced annually in the
United States, with approximately 95% derived from fermentation of com starch.
With increased attention to clean air and oxygenates for fuels, opportunities exist
for rapid expansion of the fuel ethanol industry. Various lignocellulosic biomass
such as agricultural residues, wood, municipal solid wastes and wastesfrompulp
and paper industry can serve as low cost and abundant feedstocks for production of
fuel ethanol or value-added chemicals. It is estimated that approximately 50 billion
gallons of ethanol could be producedfromcurrent biomass wastes with the potential
to produce up to 350 billion gallons from dedicated energy farms in the USA (/).
At present, the degradation of lignocellulosic biomass to fermentable sugars
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represents significant technical and economic challenges, and its success depends
largely on the development of highly efficient and cost-effective enzymes for
conversion of pretreated lignocellulosic substrates to fermentable sugars. In this
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overview chapter, the author reviews the current knowledge on lignocellulose

biodegradation and use of lignocellulosic hydrolyzates as feedstocks for developing
bio-based products and processes.

Structure and Composition of Lignocellulosic Biomass

Lignocellulosic biomass includes various agricultural residues (straws, hulls,
stems, stalks), deciduous and coniferous woods, municipal solid wastes
(MSW, paper, cardboard, yard trash, wood products), waste from pulp and paper
industry and herbaceous energy crops (switchgrass, barmudagrass). The
compositions of these materials vary. The major component is cellulose (35-50%),
followed by hemicellulose (20-35%) and lignin (10-25%). Proteins, oils and ash
make up the remaining fraction of lignocellulosic biomass (/). The structures of
these materials are complex with recalcitrant and heterogeneous characteristics and
native lignocellulose is resistant to an enzymatic hydrolysis. In the current model
of the structure of lignocellulose, cellulose fibers are embedded in a
lignin-polysaccharide matrix. Xylan may play a significant role in the structural
integrity of cell walls by both covalent and non-covalent associations (2).
Cellulose is a linear polymer of D-glucose units linked by 1,4-B-D-glucosidic
bonds. Hemicelluloses are heterogeneous polymers of pentoses (xylose,
arabinose), hexoses (mannose, glucose, galactose), and sugar acids. Unlike
cellulose, hemicelluloses are not chemically homogeneous. Hardwood
hemicelluloses contain mostly xylans, whereas softwood hemicelluloses contain
mostly glucomannans (5). Xylans of many plant materials are
heteropolysaccharides with homopolymeric backbone chains of 1,4-linked P-D-
xylopyranose units. Besides xylose, xylans may contain arabinose, glucuronic acid
or its 4-O-methyl ether, and acetic, ferulic and p-coumaric acids. The frequency
and composition of branches are dependent on the source of xylan (4). The
backbone consists of O-acetyl, a-L-arabinofuranosyl, a-1,2-linked glucuronic or 4-
O-methylglucuronic acid substituents. However, unsubstituted linear xylans have
also been isolated from guar seed husk, esparto grass and tobacco stalks (5).

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Xylans can thus be categorized as linear homoxylan, arabinoxylan, glucuronoxylan

and glucuronoarabinoxylan.
Xylansfromdifferent sources, such as grasses, cereals, softwood and hardwood,
differ in composition. Birch wood (Roth) xylan contains 89.3 % xylose, 1%
arabinose, 1.4% glucose and 8.3% anhydrouronic acid (6). Rice bran neutral xylan
contains 46% xylose, 44.9% arabinose, 6.1% galactose, 1.9% glucose and 1.1%
anhydrouronic acid (7). Wheat arabinoxylan contains 65.8% xylose, 33.5%
arabinose, 0.1 % mannose, 0.1 % galactose and 0.3% glucose (8). Cora fiber xylan
is one of the complex heteroxylans containing P-(l,4)-linked xylose residues (9).
It contains 48-54% xylose, 33-35% arabinose, 5-11% galactose and 3-6%
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glucuronic acid (10). About 80% of the xylan backbone is highly substituted with
monomeric side-chains of arabinose or glucuronic acid linked to 0-2 and/or 0-3 of
xylose residues and also by oligomeric side chains containing arabinose, xylose and
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sometimes galactose residues (7 /). The heteroxylans, which are highly cross-linked
by diferulic bridges, constitute a network in which the cellulose microfibrils may
be imbedded (12). Structural wall proteins might be cross-linked together by
isodityrosine bridges and with feruloylated heteroxylans, thus forming an insoluble
network (13). Ferulic acid is covalently cross-linked to polysaccharides by ester
bonds and to components of lignin mainly by ether bonds (14). In softwood
heteroxylans, arabinofuranosyl residues are esterified with /7-coumaric acids and
ferulic acids (75). In hardwood xylans, 60-70% of the xylose residues are
acetylated (16). The degree of polymerization of hardwood xylans (150-200) is
higher than that of softwoods (70-130).

Pretreatment of Lignocellulosic Biomass

The pretreatment of any lignocellulosic biomass is crucial before enzymatic
hydrolysis. The objective of pretreatment is to decrease the crystallinity of cellulose
which enhances the hydrolysis of cellulose by cellulases (17). Various pretreatment
options are available to fractionate, solubilize, hydrolyze and separate cellulose,
hemicellulose and lignin components (1, 18-20). These include concentrated acid
(21), dilute acid (22), S 0 (25), alkali (24, 25), hydrogen peroxide (26), wet-
2

oxidation (27), steam explosion (autohydrolysis) (28), ammonia fiber explosion

(AFEX) (29), C 0 explosion (30), liquid hot water (31) and organic solvent
2

treatments (32). In each option, the biomass is reduced in size and its physical
structure is opened. Some methods of pretreatment of Lignocellulose is given in
Table I.
The effectiveness of dilute acids to catalyze the hydrolysis of hemicellulose to
its sugar components is well known. Two categories of dilute acid pretreatment are
used: High temperature (> 160°C) continuous-flow for low solids loading (5-10%,
w/w) and low temperature (< 160°C) batch process for high solids loading (10-40%,
w/w) (33). Dilute acid pretreatment at high temperature usually hydrolyzes
hemicellulose to its sugars (xylose, arabinose and other sugars) that are water

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Table I. Methods for pretreatment of lignocellulosic biomass

Method Example

Autohydrolysis Liquid hot water, steam pressure, steam

explosion, supercritical C0 explosion
2

Acid treatment Dilute acid (H S0 ), Concentrated acid

2 4

(H S0 )
2 4

Alkali treatment Sodium hydroxide, lime, ammonia, alkaline

hydrogen peroxide
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Organic solvent with water Methanol, ethanol, butanol, phenol

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soluble {IS). The residue contains cellulose and often much of the lignin. The
lignin can be extracted with solvents such as ethanol, butanol, or formic acid.
Alternatively, hydrolysis of cellulose with lignin present produces water-soluble
sugars and the insoluble residues that are lignin plus unreacted materials. Torget
et al. (34) achieved both high xylan recovery and high simultaneous saccahrification
and fermentation (SSF) conversion while applying extremely dilute H S 0 (0.07 wt
2 4

%) in a counter-current flowthrough configuration. A major problem associated

with the dilute acid hydrolysis of lignocellulosic biomass is the poor fermentability
of the hydrolyzates. A drawback of the concentrated acid process is the costly
recovery of the acid.
Steam explosion provides effective fractionation of lignocellulosic components
at relatively low costs (35). Optimal solubilization and degradation of hemicellulose
are generally achieved by either high temperature and short residence time (270°C,
1 min) or lower temperature and longer residence time (190°C, 10 min) steam
explosion (36). The use of S 0 as a catalyst during steam pretreatment results in
2

the enzymatic accessibility of cellulose and enhanced recovery of the hemicellulose

derived sugars (37). Steam pretreatment at 200-210°C with the addition of 1% S0 2

(w/w) was superior to other forms of pretreatment of willow (38). A glucose yield
of 95%, based on the glycan available in the raw material, was achieved. Steam
explosion can induce hemicellulose degradation to furfural and its derivatives and
modification of the lignin-related chemicals under high severity treatment (> 200°C,
3-5 min, 2-3% S0 ) (39). Boussaid et al. (40) recovered around 87% of the original
2

hemicellulose component in the water-soluble stream by steam explosion of

Douglas fir softwood under low severity conditions (175°C, 7.5 min, 4.5% S0 ). 2

More than 80% of the recovered hemicellulose was in monomeric form. Enzymatic
digestibility of the steam-exploded Douglas-fir wood chips (105°C, 4.5 min, 4.5%
S0 ) was significantly improved using an optimized alkaline peroxide treatment
2

(1% H 0 , pH 11.5 and 80°C, 45 min) (41). About 90% of the lignin in the original
2 2

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wood was solubilized by this procedure, leaving a cellulose-rich residue that was
completely hydrolyzed within 48 h, using an enzyme (cellulase) loading of 1 OFPU/g
cellulose. Saccharification of 100 g sugarcane bagasse with enzymes after steam
explosion with 1% H S0 at 220°C for 30 sec at water to solid ratio of 2:1 yielded
2 4

65.1 g sugar (42).

A pretreatment method involves steeping of the lignocellulosic biomass (using
corn cob as a model feedstock) in dilute NH OH at ambient temperature to remove
4

lignin, acetate and extractives (43). This is followed by dilute acid treatment that
readily hydrolyzes the hemicellulose fraction to simple sugars, primarily xylose.
The residual cellulose fraction of biomass can then be enzymatically hydrolyzed to
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glucose. Sugarcane bagasse, corn husk and switchgrass were pretreated with
ammonia water to enhance enzymatic hydrolysis (44). Garrote et al. (45) treated
Eucalyptus wood substrates with water under selected operational conditions
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(autohydrolysis reaction) to obtain a liquid phase containing hemicellulose

decomposition products (mainly acetylated xylooligosaccharides, xylose and acetic
acid). In a further acid catalyzed step (posthydrolysis reaction),
xylooligosaccharides were converted into xylose. Wet oxidation method can be
used for fractionation of lignocellulosics into solubilized hemicellulose fraction and
a solid cellulose fraction susceptible to enzymatic saccharification. Bjerre et al.
(46) found that combination of alkali and wet oxidation did not generate furfural
and 5-hydroxymethyl furfural (HMF). Klinke et al. (47) characterized the
degradation products from alkaline wet oxidation (water, sodium carbonate,
oxygen, high temperature and pressure) of wheat straw. ApartfromC 0 and water,2

carboxylic acids were the main degradation productsfromhemicellulose and lignin.

Aromatic aldehyde formation was minimized by the addition of alkali and
temperature control. Oxygen delignification of kraft pulp removed up to 67% of
the lignin from softwood pulp and improved the rate and yield from, enzymatic
hydrolysis by up to 111% and 174%, respectively (48). Palm and Zacchi (49)
extracted 12.5 g of hemicellulose oligosaccharides from 100 g of dry spruce using
a microwave oven at 200°C for 5 min.
Supercritical C 0 explosion was found to be effective for pretreatment of
2

cellulosic materials before enzymatic hydrolysis (50, 51). Zheng et al. (52)
compared C 0 explosion with steam and ammonia explosion for pretreatment of
2

sugarcane bagasse and found that C 0 explosion was more cost-effective than
2

ammonia explosion and did not cause the formation of inhibitory compounds that
could occur in steam explosion.
Phenolic compounds from lignin degradation, furan derivatives (furfural and
HMF) from sugar degradation and aliphalic acids (acetic acid, formic acid and
levulinic acid) are considered to be fermentation inhibitors generated from
pretreated lignocellulosic biomass (53). The formation of these inhibitors depends
on the process conditions and the lignocellulosic feedstocks (54). Various methods
for detoxification of the hydrolyzates have been developed (55). These include

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treatment with ion-exchange resins, charcoal or ligninolytic enzyme laccase,

pre-fermentation with the filamentous fungus Trichoderma reesei, removal of
non-volatile compounds, extraction with ether or ethyl acetate and treatment with
alkali (lime) or sulfite. Treatment with alkali (overliming) has been widely used for
detoxification of lignocellulosic hydrolyzates prior to alcohol fermentation.
However, overliming is a costly method which also produces low-value byproducts
such as gypsum (56). Softwood hydrolyzate, when overlimed with wood ash,
improved its fermentability to ethanol which is due to the reduction of the inhibitors
such as fiiran and phenolic compounds and to nutrient effects of some inorganic
components from the wood ash on the fermentation (57). Persson et al. (58)
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employed countercurrent flow supercritical fluid extraction to detoxify a dilute acid

hydrolyzate of spruce prior to ethanol fermentation with baker's yeast. Weil et al.
(59) developed a method for the removal of furfural from biomass hydrolyzate by
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using a polymeric adsorbent, XAD-4, and desorption of the furfural to regenerate

the adsorbent using ethanol. Bjorklund et al. (60) explored the possibility of using
lignin residue left after acid hydrolysis of lignocellulosic material for detoxification
of spruce dilute acid hydrolyzates prior to fermentation with Saccharomyces
cerevisiae. Treatment with the lignin residue removed up to 53% of the phenolic
compounds and up to 68% of the furan aldehydes in a spruce dilute acid
hydrolyzate. Up to 84% of the lignin-derived compounds can be extracted with
organic solvents (ethyl acetate and diethyl ether) from Eucalyptus wood acid
hydrolyzate (61). The phenolic compounds extracted by solvents showed
antioxidant activity.
Each pretreatment method offers distinct advantages and disadvantages. The
pretreatment of lignocellulosic biomass is an expensive procedure with respect to
cost and energy.

Cellulose Biodégradation
Effective hydrolysis of cellulose to glucose requires the cooperative action of
three enzymes: endo-1, 4-fl-glucanase (EC 3.2.1.4), exo-1, 4-B-glucanase
(EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21). Cellulolytic enzymes with
β-gîucosidase act sequentially and cooperatively to degrade crystalline cellulose
to glucose. Endoglucanase acts in a random fashion on the regions of low
crystallinity of the cellulosic fiber whereas exoglucanase removes cellobiose
(β-1, 4 glucose dimer) units from the non-reducing ends of cellulose chains.
Synergism between these two enzymes is attributed to the endo-exo form of
cooperativity and has been studied extensively between cellulases in T. reesei in
the degradation of cellulose (62). Besides synergism, the adsorption of the
cellulases on the insoluble substrates is a necessary step prior to hydrolysis.
Cellobiohydrolase appears to be the key enzyme for the degradation of native

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cellulose (63). The catalytic site of the enzyme is covered by long loops, resulting
in tunnel morphology (64). The loops can undergo large movements, leading to
the opening or closing of the tunnel roof (65). An endo type attack of the
polymeric substrates becomes possible when the roof is open and once entrapped
inside the catalytic tunnel, a cellulose chain is threaded through the tunnel and
sequentially hydrolyzed one cellobiosyl unit at a time. Kleywegt et al. ( 66)
revealed the presence of shorter loops that create a groove rather than a tunnel in
the structure of the enzyme EGI from T. reesei. In most organisms, cellulases
are modular enzymes that consist of a catalytic core connected to a cellulose-
binding domain (CBD) through aflexibleand heavily glycosylated linker region
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(67). The CBD is responsible for bringing the catalytic domain in an appropriate
position for the breakdown of cellulose. Binding of cellulases and the formation
of cellulose-cellulase complexes are considered critical steps in the hydrolysis of
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insoluble cellulose (68). B-Glucosidase hydrolyzes cellobiose and in some cases

cellooligosaccharides to glucose. The enzyme is generally responsible for the
regulation of the whole cellulolytic process and is a rate limiting factor during
enzymatic hydrolysis of cellulose as both endoglucanase and cellobiohydrolase
activities are often inhibited by cellobiose (69-71). Thus, β-glueosidase not only
produces glucose from cellobiose but also reduces cellobiose inhibition, allowing
the cellulolytic enzymes to function more efficiently. However, like B-
glucanases, most B-glucosidases are subject to end-product (glucose) inhibition
(72). C. pe/tataproduces a highly glucose tolerant β-glucosidase with a value
of 1.4 M (252 mg/ml) for glucose ( 73). The kinetics of the enzymatic hydrolysis
of cellulose including adsorption, inactivation and inhibition of enzymes have been
studied extensively (74). For a complete hydrolysis of cellulose to glucose, the
enzyme system must contain the three enzymes in right proportions. T. reesei
(initially called T. viride) produces at least five endoglucanases (EGI, EGII,
EGIII, EGIV and EGV), two exoglucanases (CBHI and CBHII) and two β-
glucosidases (BGLI and BGLII) (75). An exo-exo synergism between the two
cellobiohydrolases was also observed (76). The fungus produces up to 0.33 g
protein per g of utilizable carbohydrate (77).
Product inhibition, thermal inactivation, substrate inhibition, low product
yield and high cost of cellulase are some barriers to commercial development of
the enzymatic hydrolysis of cellulose. Many microorganisms are cellulolytic.
However, only two microorganisms (Trichoderma and Aspergillus) have been
studied extensively for cellulase. A newly isolated Mucor circinelloides strain
produces a complete cellulase enzyme system (78). The endoglucanase from this
strain was found to have a wide pH stability and activity. There is an increasing
demand for the development of thermostable, environmentally compatible,
product and substrate tolerant cellulases with increased specificity and activity for
application in the conversion of cellulose to glucose in the fuel ethanol industry.
Thermostable cellulases offer certain advantages such as higher reaction rate,

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increased product formation, less microbial contamination, longer shelf-life,

easier purification and better yield.
The cellulose hydrolysis step is a significant component of the total production
cost of ethanol from wood (79). Achieving a high glucose yield is necessary
(>85% theoretical) at high substrate loading (>10% w/v) over short residence times
(<4 days). Simultaneous saccharification (hydrolysis) of cellulose to glucose and
fermentation of glucose to ethanol (SSF) improve the kinetics and economics of
biomass conversion by reducing accumulation of hydrolysis products that are
inhibitory to cellulase and β-glucosidase, reducing the contamination risk because
of the presence of ethanol, and reducing the capital equipment requirements (80).
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An important drawback of SSF is that the reaction has to operate at a compromised

temperature of around 30°C instead of enzyme optimum temperature of 45-50°C.
Enzyme recycling, by ultrafiltration of the hydrolyzate, can reduce the net enzyme
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requirement and thus lower costs (8 J). A preliminary estimate of the cost of ethanol
production for SSF technology based on wood-to-ethanol process is $1.22/gal of
which the wood cost is $0.459/gal (82). A separate fungal enzyme hydrolysis and
fermentation process for converting lignocellulose to ethanol were also evaluated
(83). The cellulase enzyme was produced by the fungal mutant Trichoderma Rut
C-30 (the first mutant with greatly increased β-glucosidase activity) in a fed batch
production system that is the single most expensive operation in the process. The
conversion of lignocellulosic biomass to fermentable sugars requires the addition
of complex enzyme mixtures tailored for the process and parallel reuse and recycle
the enzymes until the cost of enzymes comes down. Enzyme recycling may
increase the rates and yields of hydrolysis, reduce the net enzyme requirements and
thus lower costs (84). As mentioned earlier, the first step in cellulose hydrolysis is
considered as the adsorption of cellulase onto cellulosic substrate. As the cellulose
hydrolysis proceeds, the adsorbed enzymes (endo- and exo-glucanase components)
are gradually released in the reaction mixture. The β-glucosidase does not adsorb
onto the substrate. These enzymes can be recovered and reused by contacting the
hydrolyzate with the fresh substrate. However, the amount of enzyme recovered is
limited because some enzymes remain attached to the residual substrate, and some
enzymes are thermally inactivated during hydrolysis. Poor recovery of cellulase
was achieved in the case of substrates containing a high proportion of lignin (85).
Addition of surfactant to enzymatic hydrolysis of lignocellulose increases the
conversion of cellulose to soluble sugars. Castanon and Wilke (86) reported a 14%
increase in glucose yield and more than twice as much recovered enzyme from
newspaper saccharification when Tween 80 was added. Karr and Holtzapple (87)
studied the effect of Tween on the enzymatic hydrolysis of lime pretreated corn
stover and concluded that Tween improves corn stover hydrolysis through three
effects: enzyme stabilizer, lignocellulose disrupter and enzyme effector. The
enhancement is due to reduction of the unproductive enzyme adsorption to the
lignin part of the substrate as a result of hydrophobic interaction of surfactant with

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lignin on the lignocellulose surface, which releases nonspecifïcally bound enzyme

(88) .
Cellolignin is an industrial residue obtained during the production of furfural
from wood and corn cobs when pretreated by dilute H S0 at elevated temperature.
2 4

It was completely converted to glucose by cellulase from T. viride and A. foetidus

(89) . The concentration of glucose in the hydrolyzate reached 4-5.5% with about
80% cellulose conversion. Kinetic analysis of cellolignin hydrolysis, using a
mathematical model of the process, has shown that, with product inhibition,
nonspecific adsorption of cellulase onto lignin and substrate induced inactivation
seem to affect negatively the hydrolysis efficiency. Borchert and Buchholz (90)
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investigated the enzymatic hydrolysis of different cellulosic materials (straw, potato

pulp, sugar beet pulp) with respect to reactor design. The kinetics were studied
including enzyme adsorption, inhibition and inactivation. The results suggest the
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use of reactors with plug flow characteristics to achieve high substrate and product
concentrations and to avoid back-mixing to limit the effect of product inhibition.
For efficient use of cellulases, a reactor with semipermeable hollow fiber or an
ultrafilter membrane was used, and this allowed cellulases to escape end-product
inhibition (91-94). A totally integrated biotechnology of rice straw conversion into
ethanol was reported (95). It dealt with (a) ethanol refining of rice straw to
segregate cellulose from pentose sugars and lignin, (b) preparation of highly active
mixed cellulase enzymes, (c) a novel reactor system allowing rapid product
formation involving enzymatic hydrolysis of cellulose to sugars followed by
microbial conversion of the later into ethanol and its simultaneous flash separation
employing a programmed recompression of ethanol vapors and condensation and
(d) concentration of ethanol via alternative approaches. Use of cellulase enzymes
improves ink detachment from old newspapers giving similar or better results in
place of classical chemicals (96).

Hemicellulose Biodégradation
Hemicellulases are either glycosyl hydrolases or carbohydrate esterases. The
total biodégradation of xylan requires endo-P-l,4-xylanase (EC 3.2.1.8),
β-xylosidase (EC 3.2.1.37) and several accessory enzymes, such as
α-L-arabinofuranosidase (EC 3.2.1.55), α-glucuronidase (EC 3.2.1.131),
acetylxylan esterase (EC 3.1.1,72), ferulic acid esterase ( EC 3.1.1.73) and
/?-coumaric acid esterase, which are necessary for hydrolyzing various substituted
xylans (97). The endo-xylanase attacks the main chains of xylans and β-xylosidase
hydrolyzes xylooligosaccharides to xylose. The α-arabinofuranosidase and
α-glucuronidase remove the arabinose and 4-O-methyl glucuronic acid substituents,
respectively,fromthe xylan backbone. The esterases hydrolyze the ester linkages
between xylose units of the xylan and acetic acid (acetylxylan esterase) or between

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arabinose side chain residues and phenolic acids, such as ferulic acid (ferulic acid
esterase) andp-eoumarie acid (p-coumaric acid esterase). It is stated that hindrance
of lignocellulose biodégradation is associated with phenolic compounds (P5).The
phenolic acids are produced via the phenylpropanoid biosynthetic pathway (99).
They act as a cross-linking agent between lignin and carbohydrates or between
carbohydrates. β-Mannanase (EC 3.2.1.78) hydrolyzes mannan-based
hemicellulases and liberate β-1,4-manno-oligomers, which can be further degraded
to mannose by β-mannosidase (EC 3.2.1.25).
Many microorganisms, such as Pénicillium capsulatum and Talaromyces
emersonii, possess complete xylan degrading enzyme systems (100). Significant
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synergistic interactions were observed among endo-xylanase, β-xylosidase,

α-arabinofuranosidase and acetylxylan esterase of the thermophilic actinomycete
Thermomonospora fusca (101). Synergistic action between depolymerizing and
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side-group cleaving enzymes has been verified using acetylated xylan as a substrate
(102). Many xylanases do not cleave glycosidic bonds between xylose units which
are substituted. The side chains must be cleaved before the xylan backbone can be
completely hydrolyzed (103). On the other hand, several accessory enzymes only
remove side chains from xylooligosaccharides. These enzymes require a partial
hydrolysis of xylan before the side chains can be cleaved (104). Although the
structure of xylan is more complex than cellulose and requires several different
enzymes with different specificities for complete hydrolysis, the polysaccharide
does not form tightly packed crystalline structures like cellulose and is, thus, more
accessible to enzymatic hydrolysis (105).
Corn fiber, a byproduct of corn wet milling facility, contains about 20% starch
in addition to 15% cellulose and 35% hemicellulose (106). The xylan from corn
fiber is highly resistant to enzymatic degradation by commercially available
hemicellulases (22). Dilute acid (1% H S0 v/v, 15% solids) pretreatment at a
2 4

relatively low temperature (120°C, 1 h) to minimize the formation of inhibitory

compounds, followed by enzymatic saccharification of the cellulosic portion, is an
excellent workable process for generating fermentable sugars (85-100% yield)
from corn fiber (22). A partial saccharification of corn fiber was achieved using a
crude enzyme preparation from Aureobasidium sp. (107). Christov et al. (108)
showed that crude enzyme preparation from A. pullulans was only partially
effective in the removal of xylan from dissolving pulp. Two newly isolated fungal
cultures (Fusarium proliferatum NRRL 26517, F. verticillioides NRRL Y-26518)
have the capability to utilize corn fiber xylan as growth substrate (109-112). The
crude enzyme preparationsfromthese fungi were able to degrade cornfiberxylan
well but the purified endo-xylanases could not degrade corn fiber xylan. The
purified β-xylosidase released xylose from xylobiose and other short-chain
xylooligosaccharides. For effective hydrolysis of xylan substrates, a proper mix of
endo-xylanase with several accessory enzymes is essential. A. pullulans produces
a highly thermostable novel extracellular α-L-arabinofuranosidase that has the
ability to rapidly hydrolyze arabinan and debranched arabinan and release arabinose

In Lignocellulose Biodegradation; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
 12

from various arabinoxylans (113). Arabinose-rich lignocellulosic hydrolyzates can

be used for production of the enzyme (114). Spagnuolo et al. (115) reported that
incubation of beet pulp with α-L-arabinofuranosidase and end-arabinase produced
a hydrolyzate consisting mainly arabinose.
Ferulic acid esterase breaks the ester linkage between ferulic acid and the attached
sugar and release ferulic acid from complex cell walls such as wheat bran, sugar
beet pulp, barley spent grain and oat hull (116-119). The ability of Thermomyces
lanuginosus to produce high levels of cellulase-free thermostable xylanase has
made the fungus an attractive source of the enzyme with potential as a bleach-
boosting agent in the pulp and paper industry and as an additive in the baking
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industry (120).
Publication Date: July 29, 2004 | doi: 10.1021/bk-2004-0889.ch001

Lignin Biodégradation
Lignin is a long chain heterogeneous aromatic polymer (average molecular
weight 8,000-11,000) composed largely of phenylpropane units most commonly
linked by ether bonds. It effectively protects the woody plants against microbial
attack and only a few organisms including rot-fungi and some bacteria can degrade
it (121). The conversion of cellulose and hemicellulose to fuels and chemicals will
generate lignin as a by-product that can be burned to provide heat and electricity,
converted to low-molecular weight chemicals and used in the manufacture of
various polymeric materials. As lignin makes up 15-25% in some lignocellulosic
biomass, the selling price of lignin has a very large impact on ethanol price (35).
Efficient removal of lignin from lignin-carbohydrate complex (LCC) is important
in pulp and paper industry. The lignin barrier can be disrupted by a variety of
pretreatment rendering the cellulose and hemicellulose more susceptible to
enzymatic attack (122). There are many papers about microbial breakdowns of
lignin, the enzymes and the pathways (123-126). Several white rot fungi have the
ability to delignify kraft pulp. Their lignin-degrading capacity is attributed to
extracellular oxidative enzymes that function together with low molecular weight
cofactors (127). The degradation of lignin by the white rot fungus (WRF)
Phanerochaete chrysosporium is catalyzed by extracellular peroxidases (lignin
peroxidase, LiP, EC 1.11.1.14 and manganese peroxidase, MnP, EC 1.11.1.13) in
a H 0 -dependent process (128, 129). LiP seems to use veratryl cation radical as
2 2

mediator (130). The WRF Ceriporiopsis subvermispora produces laccase

(EC 1.10.3.2) and MnP isozymes, as well as hemicellulases and a poor complex of
cellulases lacking in cellobiohydrolase activity (131). Laccase is a family of
'blue-copper' oxidases containing four copper ions. It oxidizes the phenolic but not
the non-phenolic subunits of lignin. Redox mediators drive laccase towards the
oxidation of non-phenolic subunits, particularly the benzyl alcohol groups. Each
laccase may have a preferred low molecular mass mediator substrate, which may

In Lignocellulose Biodegradation; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
 13

represent a major secreted metabolite (J30). A laccase and mediators with NO,
NOH or HRNOH groups can be combined in a laccase-mediated system
(Lignozyme process) that are effective in delignifying wood in a pilot pulp and
paper process. A pre-oxidation of the cc-hydroxy-P-arylether subunits in wood pulp
by the laccase/violuric acid system appears to be promising for weakening the
network of lignin, thereby activating it towards subsequent oxydelignification
treatments (J32). It was demonstrated that the WRF Pycnoporus cinnabarinus
degrades lignin in the absence of both Lip and MnP (133). The fungus was found
to produce predominantly laccase, and neither LiP nor MnP was produced. The
factors involved in lignin biodégradation process are not yet fully understood (134).
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The biodégradation of lignocellulose by Rigidoporus lignosus was greatly

stimulated in solid state cultivation when compared with liquid culture (135). This
WRF produces laccase and MnP. The edible mushroom Pleurotus ostreatus
Publication Date: July 29, 2004 | doi: 10.1021/bk-2004-0889.ch001

degrades lignin efficiently and selectively (136). It can, therefore, serve to upgrade
lignocellulosic wastes. Lignin degradation by Agaricus bisporus accounts for a
30% increase in bioavailable holocellulose during cultivation on compost (137).
Table II lists some of the enzymes involved in lignocellulose degradation.

Direct Microbial Conversion

In direct microbial conversion of lignocellulosic biomass into ethanol that could
simplify the ethanol production process from these materials and reduce ethanol
production costs, Clostridium thermocellum, a thermoanaerobe was used for
enzyme production, hydrolysis and glucose fermentation (138). Cofermentation
with C. thermosaccharolyticum simultaneously converted the hemicellulosic sugars
to ethanol. However, the formations of by-products such as acetic acid and low
ethanol tolerance are some drawbacks of the process. Neurospora crassa produces
extracellular cellulase and xylanase and has the ability to ferment cellulose to
ethanol (139).
In nature, cellulosic materials are degraded with the cooperation of many
microorganisms. A mixed culture of one cellulolytic bacterium together with
another non-cellulolytic bacterium was found to be effective for cellulose
degradation (140, 141). Recently, Haruta et al. (142) obtained a microbial
community from rice straw compost that had the capability of degrading 60% of
rice straw within 4 days at 50°C. The community structure consisting of both
aerobic and anaerobic bacteria remained constant after multiple subcultures
exceeding 2 years.

In Lignocellulose Biodegradation; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
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Table IL Enzymes involved in lignocellulose degradation

Enzyme Systematic name EC number Mode of action

Endo-1,4-P-glucanase 1,4-P~D-Glucan-4-glucanohydrolase 3.2.1.4 Endo-hydrolysis of 1,4-P-D-glucosidic

linkages
Exo-1,4-P-glucanase 1,4-P-D-Glucan cellobiohydrolase 3.2.1.91 Hydrolysis of 1,4-P-D-glucosidic linkages
releasing cellobiose
β-Glucosidase β-D-Glucoside glucohydrolase 3.2.1.21 Hydrolyzes cellobiose and short chain cello-
oligosaccharides to glucose
Εηάο-1,4-β- xylanase 1,4-p-D-Xylan xylanohydrolase 3.2.1.8 Hydrolyzes mainly interior P~l,4-xylose
linkages of the xylan backbone
a-L-Arabinofuranosidase α-L-Arabinofuranoside arabinofurano- 3.2.1.55 Hydrolyzes terminal nonreducing
hydrolase α-arabinofuranose fromarabinoxylans
α-Glucuronidase 3.2.1.31 Releases glucuronic acid from
α-Glucuronoside glucanohydrolase glucuronoxylans
Acetylxylan esterase 3.1.1.6 Hydrolyzes acetylester bonds in acetyl
Acetyl-ester acetylhydrolase xylans
Ferulic acid esterase 3.1.1.1 Hydrolyzes feruloylester bonds in xylans

In Lignocellulose Biodegradation; Saha, B., et al.;

Lignin peroxidase Carboxylic ester hydrolase 1.11.1.7 Oxidation of benzilic alcohols, cleavage of
C-C bonds, cleavage of C-O bonds.
2+
Manganese peroxidase 1.11.1.7 Catalytically dependent on H 0 and M n
2 2

ions

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

Laccase Donar: hydrogen peroxide 1.10.3.2 Oxidizes phenolic subunits of lignin
oxidoreductase
 15

Applications in Biotechnology

Production of Fuel Ethanol

Lignocellulosic biomass can serve as low-cost feedstocks for production of fuel

ethanol. It generates a mixture of sugars upon pretreatment itself or in combination
with enzymatic hydrolysis. The sugar mixture may contain any combination of
xylose, arabinose, glucose, galactose, mannose, fucose and rhamnose depending on
the source. Although traditional S. cerevisiae and Zymomonas mobilis ferment
glucose to ethanol rapidly and efficiently, they cannot ferment other sugars such as
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xylose and arabinose to ethanol. Some yeasts (Pachysolen tannophilus, Pichia

stipitis, Candida shehatae) have the capability to ferment xylose to ethanol (143,
144). These yeasts have low ethanol tolerance and slow rates of fermentation and
Publication Date: July 29, 2004 | doi: 10.1021/bk-2004-0889.ch001

cannot be used in industrial application (145, 146). Xylose can be converted to

xylulose using the enzyme xylose isomerase and traditional yeasts can ferment
xylulose to ethanol (147, 148). However, the process is not cost-effective. Only a
few yeast strains can hardly ferment arabinose to ethanol (149, 150). Thus, no
naturally occurring yeast and bacterium can ferment mixed sugars to ethanol.
Some bacteria such as Escherichia coli, Klebsiella , Erwinia, Lactobacillus,
Bacillus and Clostridia can utilize mixed sugars but produce no or limited quantity
of ethanol. These bacteria generally produce mixed acids (acetate, lactate,
propionate, succinate) and solvents (acetone, butanol, 2,3-butanediol). Several
microorganisms have been genetically engineered to produce ethanol from mixed
sugar substrates by using two different approaches: (a) divert carbon flow from
native fermentation products to ethanol in efficient mixed sugar utilizers such as
Escherichia, Erwinia and Klebsiella and (b) introduce the pentose utilizing
capability in the efficient ethanol producers such as Saccharomyces and
Zymomobilis (151-154). Recombinant E. coli KOI 1, E. coli SL40, E. coli FBR3,
Zymomonas CP4 (pZB5) and Saccharomyces 1400 (pLNH32) strains fermented
cornfiberhydrolyzates to ethanol (21 -34 g/L) with yields of 0.41 -0.50 g of ethanol
per gram of sugar consumed (155-156). Increasing gene expression through the
replacement of promoters and the use of a higher gene dosage (plasmids)
substantially eliminated the apparent requirement for large amounts of complex
nutrients of ethanologenic recombinant E. coli strain (157). Ethanol tolerant
mutants of recombinant E. coli have been developed that can produce up to 6%
ethanol (158). The increased ethanol tolerance in the E. coli mutant LY01 appears
to result from increased glycine metabolism, increased production of the osmolyte
betaine from choline, loss of FNR function, increased production of mar drug
resistance proteins, increased metabolism of serine and pyruvate and decreased
production of organic acids (159). The recombinant Z. mobilis in which four genes
from E. coli, xylA (xylose isomerase), xylB (xylulokinase), tal (transaldolase) and
tktA (transketolase) were inserted, grew on xylose as the sole carbon source and

In Lignocellulose Biodegradation; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
 16

produced ethanol at 86% of the theoretical yield (Figure 1) (153). It was

demonstrated that phosphorylation is a vital step for metabolism of xylose through
the pentose phosphate pathway (160). The gene XKS1 (encoding xylulokinase)
from S. cerevisiae and the heterologous genes from XYL1 and XYL2 (from P.
stipitis) were inserted into a hybrid host, obtained by classical breeding of S.
uvarum and S. diastaticus, which resulted in Saccharomyces strain pLNH32,
capable of growing on xylose alone. Chromosomal integration of a single copy of
theATI/-ATI2-ATL5/cassetteein5. Cerevisiae resulted in strain TMB3001 (161).
This strain attained specific uptake rates (g/g.h) of 0.47 and 0.21 for glucose and
xylose, respectively, in continuous culture using a minimal medium. Martin et al.
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(162) studied ethanol production from steam exploded (205 and 215°C, 10 min)
enzymatic (cellulase, β-glucosidase) hydrolyzates of sugar cane bagasse using the
recombinant S. cerevisiae strain TMB 3001. The hydrolyzates were detoxified by
Publication Date: July 29, 2004 | doi: 10.1021/bk-2004-0889.ch001

treatment with laccase and also by overliming. The ethanol yield was 0.32-0.35 g/g
of total sugar from the detoxified hydrolyzates. Partial xylose utilization with low
xylitol formation was observed.
Sedlak and Ho (163) expressed genes [arab (L-ribulokinase), araA (L-arabinose
isomerase) and araD (L-ribulose-5-phosphate)]fromthe araBAD operon encoding
the arabinose metabolizing genes from E. coli in S. cerevisiae but the transformed
strain was not able to produce any detectable amount of ethanol from arabinose.
Zhang et al. ( 164) constructed one strain of Z. mobilis (PZB3 01 ) with seven plasmid
borne genes encoding xylose- and arabinose metabolizing genes and pentose
phosphate pathway (PPP) genes. This recombinant strain was capable of
fermenting both xylose and arabinose in a mixture of sugars with 82-84%
theoretical yield in 80-100 h at 30°C. Richard et al. (165) reported that
overexpression of all five enzymes (aldose reductase, L-arabinitol 4-dehydrogenase,
L-xylulose reductase, xylitol dehydrogenase and xylulokinase) of the arabinose
catabolic pathway in S. cerevisiae led to growth of S. cerevisiae on arabinose
(Figure 2). The recombinant S. cerevisiae produced ethanol from arabinose at a
very slow rate.
Microorganisms metabolically engineered with improved inhibitor tolerance
could reduce the need for detoxification process. Larsson et al. (166) developed a
S. cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in
lignocellulose hydrolyzates by heterologous expression of laccase.
Three technology options (concentrated acid process, CHAP; S0 /dilute acid
2

process, CASH; enzymatic hydrolysis process) were evaluated for ethanol

production from pine feedstock using a uniform platform (167). Even though each
process suffers from certain different disadvantages, none of the three processes can
be eliminated as less economical than the other two with an ethanol production
price of US $ 1.89-2.04/gallon. A feasibility study of using softwood forest thinning
as a biomass source for ethanol production in California was performed (168). A
two-stage dilute acid (190°C, 0.7% H S0 , 3 min; 220°C, 1.6% H S0 , 3 min)
2 4 2 4

In Lignocellulose Biodegradation; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
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Pentose metabolism pathway Entner-Doudoroff pathway

Xylose Glucose
jr-ATP
Xylose isomerase |
J** ADP
* 1
Glucose-6-P
Xylulose
1
I 1 Gluconolactone-6-P
I Xylulokinase |
k"" ATP
4
' ADP 6-P-Gluconate

Xylulose-5-P Ribulose-5-P Ribose-5-P 4

2-Keto-3-deoxy-6-P-gluconate
I
Transketolase | ••Glyceraldehyde-3-P

Sedoheptulose-7-P Glyceraldehyde-3-P 1,3-P-Glycerate

p"ATP
Transketolase | ^ ADP

Fructose-6-P —| 3-P-Glycerate
Erythrose-4-P • Fructose-6-P ——*
2-P-Glycerate ADP ATP
• Glyceraldehyde-3-P • Pyruvate
Transketolase
Phosphoenol pyruvate* U
Acetaldehyde + C 0 2

In Lignocellulose Biodegradation; Saha, B., et al.;

*
Ethanol

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

Figure 1. Proposed pentose metabolism and Entner-Doudoroff pathways in engineered Zymomonas mobilis. (Reproduced with
permission from reference 153. Copyright 1995.)
 18

hydrolysis process is used for the production of ethanol from softwoods using a
recombinant xylose-fermenting yeast, and the residual lignin is used to generate
steam and electricity. It is concluded that such a biomass to ethanol plant seems to
be an appealing proposition for California, if ethanol replaces MTBE, which is
slated for a phase out. With increased research efforts for developing a stable,
ethanol tolerant and robust recombinant ethanologenic organism capable of
tolerating common fermentation inhibitors generated during pretreatment,
competitive and economical production of fuel ethanol from lignocellulose holds
strong promise.
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Production of Xylitol
Publication Date: July 29, 2004 | doi: 10.1021/bk-2004-0889.ch001

Xylitol, a sugar alcohol, has potential use as a natural food sweetener, a dental
caries reducer and a sugar substitute for diabetics. It is produced by chemical
reduction in alkaline conditions of the xylose derived mainly from wood
hydrolyzate (J 69). The recovery of xylitolfromthe xylan fraction is about 50-60%
or 8-15% of the raw material employed. Drawbacks of the chemical process are the
requirements of high pressure (up to 50 arm) and temperature (80-140°C), use of an
expensive catalyst (Raney-Nickel) and use of extensive separation and purification
steps to remove the by-products that are mainly derived from the hemicellulose
hydrolyzate (770). The bulk of xylitol produced is consumed in various food
products such as chewing gum, candy, soft drinks and ice cream. It gives a pleasant
cool and fresh sensation due to its high negative heat of solution.
Many yeasts and mycelial fungi possess NADPH dependent xylose reductase
(EC 1.1.1.21), which catalyzes the reduction of xylose to xylitol as a first step in
xylose metabolism (/ 71). Xylitol can be subsequently oxidized to xylulose by the
action of xylitol dehydrogenase, which preferentially uses NAD as an acceptor
(172). In xylose fermenting yeasts, the initial reactions of xylose metabolism
appear to be rate-limiting (173). This results in accumulation of xylitol in the
culture medium, the degree varying with the culture conditions and the yeast strain
used (174). A surplus of NADH during transient oxygen limitation inhibiting the
activity of xylitol dehydrogenase results in xylitol accumulation (175). The
pathway for xylose utilization in microorganisms is shown in Figure 3. Some of the
natural xylose-fermenting yeasts that are known to produce xylitol are: C. boidini,
C. entomaea, C. guillermondii, C. peltata , C. tropicalis, C. parapsilosis and
Debaryomyces hansenii (176-179). Oxygen plays an important role in xylose
uptake by yeasts. Phosphate limitation stress induces xylitol overproduction by
D. hansenii (180). The highest xylitol concentration attained in microbial processes
using xylose as substrate have been in the range of 200 to 220 g/L (181-184).
Nakano et al. (185) reported very high xylitol (356 g/L) production by
C. magnoliae in fed-batch culture under a microaerobic condition maintained by

In Lignocellulose Biodegradation; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
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Ethanol + C 0 2

Figure 2. Redox cofactor requirement in L-arfibinose catabolism. L-Arabinose

conversion to equimolar amounts of C0 and ethanol is redox neutral, i.e.
2

anaerobicfermentation to ethanol should be possible. However, the conversion of

+
L-arabinose to D-xylulose requires NADPH and NAD and produces NADH and
+
NADP . NADPH is mainly regenerated in the oxidative part of the pentose
+
phosphate pathway, where the reduction ofNADP is coupled to C02 production.
The abbreviations are: G6p, glucose-6-phosphate; F6P,fructose6-phosphate;
X5P, D-Xylulose 5-phosphate; GAP, D-glyceraldehyde3-phosphate. (Reproduced
from Ref. 165 with permission from Elsevier Science)

simple fuzzy control with a yield of 0.75, which corresponded to 82% of the
theoretical yield..
The fermentation of sugarcane bagasse hemicellulose hydrolyzate to xylitol by
a hydrolyzate-acclimatized yeast strain Candida sp. B-22 was studied (186). A
final xylitol concentration of 94.74 g/L was obtained from 105.35 g/L xylose in
hemicellulose hydrolyzate after 96 h of incubation. C guilliermondii FTI 20037
was able to ferment a sugar cane bagasse hydrolyzate producing 18.4 g/L xylitol
from 29.5 g/L of xylose, at a production rate of 0.38 g/L.h (187). This lower value,
compared to that (0.66 g/L.h) of the synthetic medium, may be attributed to the
various toxic substances that interfere with microbial metabolism (e.g., acetic acid).
Dominguez et al. (188) studied different treatments (neutralization, activated
charcoal and neutralization, cation-exchange resins and neutralization) of sugar

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ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
 20

cane bagasse hemicellulose hydrolyzate to overcome the inhibitory effect on xylitol

production by Candida sp. 11-2. The highest xylitol productivity (0.205 g/L.h),
corresponding to 10.54 g/L, was obtainedfromhydrolyzates treated with activated
charcoal (initial xylose, 42.96 g/L). To obtain higher xylitol productivity, treated
hydrolyzates were concentrated by vacuum evaporation in rotavator to provide
higher initial xylose concentration. The rate of xylitol production increased with
increasing initial xylose concentration from 30 to 50 g/L, reaching a maximum of
28.9 g/L after 48 h fermentation. The decrease in xylitol production was dramatic
with further increases in the initial xylose concentration. Parajo et al. (189)
reported a xylitol production of 39-41 g/Lfromconcentrated Eucalyptus globulus
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wood acid hydrolyzate containing 58-78 g xylose/L by D. hansenii NRRL Y-7426

using an initial cell concentration of 50-80 g/L. Recently, Rivas et al (190)
achieved a xylitol concentration of 71 g/L and volumetric productivity of 1.5 g/L.h
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when D. hansenii NRRL Y-7426 (12 g dry mass/L) was grown semiaerobically
using detoxified corn cob hydrolyzate produced by autohydrolysis-posthydrolysis
at starting xylose concentration of 100 g/L.
Hydrolyzed hemicellulosic fractions of sugar cane bagasse and rice straw were
tested for xylitol production in batch fermentation by C. guilliermondii under
semiaerobic condition and compared these with synthetic medium containing xylose
(191,192). Simultaneous utilization of hemicellulosic sugars (glucose and xylose)
was observed, and the highest substrate uptake rate was attained in sugar cane
bagasse medium. Increased xylitol concentration (40 g/L) was achieved in synthetic
and rice straw media, although the highest xylitol production rate was obtained in
sugar cane bagasse hydrolyzate. Both hydrolyzates can be converted into xylitol
with satisfactory yields and productivities. Xylitol production by C. guilliermondii
was evaluated using rice straw hemicellulose hydrolyzate under different conditions
of initial pH, nitrogen sources and inoculum level (193,194). The xylitol yields
were 0.68 g/g for the medium containing ammonium sulfate at pH 5.3 and 0.66 g/g
with urea at pH 4.5. Under appropriate inoculum conditions, rice straw
hemicellulose hydrolyzate was converted into xylitol by the yeast with 77% yield.
Mayerhoff et al. (195) evaluated 30 different yeast strains belonging to 4 different
genera (Candida, Debaryomyces, Hansenula and Pichia) for xylitol production
from rice straw hemicellulose hydrolyzate. The best performer was C mogii
NRRL Y-17032, which yielded 0.65 g xylitol/g at 0.40 g/L.h over 75 h. Preziosi-
Belloy et al. (196) investigated the production of xylitol from aspenwood
hemicellulose hydrolyzate by C guilliermondii. The hydrolyzate was supplemented
by yeast extract, and the maximum xylitol yield (0.8 g/g) and productivity
(0.6 g/L.h) were reached by controlling oxygen input. A two-stage sequential
fermentation scheme for production of xylitol and arabitol from a mixture of sugars
by C. guilliermondii was developed (197). Following glucose consumption, cells

In Lignocellulose Biodegradation; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
 21

Xylito!
NAD*

D-Xylitol dehydrogenase
D-Xylose
+
NADH + H

ATP
D-Xylulose kinase
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ADP

D-Xylulose-5-phosphate
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Pentose phosphate pathway

Figure 3. Pathway for xylose utilization in microorganisms.

(Reproducedfrom reference 176. Copyright 1997 American Chemical Society.)

were removed from mixed sugar cultures and replaced with cells from cultures
grown on xylose alone. In the second fermentation stage, xylose and arabinose
were successfully fermented to xylitol and arabitol. Dilute acid hydrolyzate of corn
fiber after treatment with a mixed-bed deionization resin was suitable for the two-
stage fermentation process. Xylitol production was studied from barley bran
hydrolyzates by continuous fermentation with D. hansenii (198). The optimum
xylitol productivity (2.53 g/L.h) was reached at a dilution of rate of 0.284/h with
cell recycle after membrane separation. Xylitol was produced in a two-substrate
(xylose, glucose) batch fermentation by C tropicalis with cell recycling (199). The
optimized cell recycle fermentation resulted in xylitol yield of 0.823 g/g xylose
with a productivity of 4.94 g/L.h and a final xylitol concentration of 189 g/L.
Xylitol productivity up to 1.14 g xylitol/L.h with 86% conversion efficiency was
achieved with a strain of G guilliermondii in continuous culture using a membrane
bioreactor at a dilution rate of 0.03/h (200).
Carvalho et al. (201) achieved maximum xylitol concentration of 20.6 g/L with
a volumetric productivity of 0.43 g/L.h and yield of 0.47 g/g after 48 h fermentation
during batch xylitol productionfromconcentrated sugarcane bagasse hydrolyzate
by C guilliermondii cells, immobilized in calcium-alginate beads. Aeration rate
strongly influences xylitol production from sugarcane bagasse hydrolyzate by
immobilized cells of C. guilliermondii in a fluidized bed reactor (202).

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ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
 22

Ε. coli JM 109 was used as a host for xylitol production by expressing xylose
reductase gene (xyrA) of C. tropicalis IFO 0618 (203). When xylose (50 g/L) and
glucose (5 g/L) were added to IPTG-induced cells, 13.3 g/L of xylitol was produced
during 20 h of cultivation. A number of recombinant S. cerevisiae strains have been
created by expressing the xylose reductase gene (XYL1) from P. stipitis and C.
shehate, and production of xylitol from xylose by these recombinant strains in batch
and fed batch fermentations have been investigated (204-206). Lee et al. (207)
studied the fermentation characteristics of recombinant S. cereseviae containing a
xylose reductase gene from P. stipitis. Xylitol was produced with a maximum yield
of 0.95 g/g xylose consumed in the presence of glucose used as co-substrate for co-
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factor regeneration. However, addition of glucose caused inhibition of xylose

transport and accumulation of ethanol. By adopting a glucose-limited fed batch
fermentation where a high ratio of xylose to glucose was maintained, a xylitol
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concentration of 105.2 g/L was achieved with a 1.69 g/l.h productivity with this
recombinant yeast. Kim et al. (208) reported that S. cerevisiae containing multiple
XYL1 genes of P. stipitis on the chromosome is much more efficient for xylitol
production in the long term non-selective culture than S. cerevisiae harboring the
episomal plasmid containing the XYL1 gene. Such an improvement in the
integrated recombinant strain was supported by the fact that the mitotic stability of
the XR gene along with its high expression level worked in a cooperative manner
(209).
Continuous enzymatic production of xylitol with simultaneous coenzyme
regeneration in a charged membrane reactor was studied (210). An NADH
dependent xylose reductasefromC. tenuis catalyzed the reduction of xylose. This
was coupled to enzymatic oxidation of glucose by glucose dehydrogenase from
Bacillus cereus to make achievable an up to 10,000-fold regeneration of NADH per
cycle of discontinuous conversion. Under suitable conditions, 300 g/L of substrate
could be converted in yields above 96% in one single batch reaction.
The recovery of xylitol from fermented sugarcane bagasse hydrolyzate was
studied (211). The best clarifying treatment was found by adding 20 g activated
carbon to 100 ml fermented broth at 80°C for 1 h at pH 6.0. The clarified medium
was treated with ion exchange resins after which xylitol crystallization was
attempted. The ion exchange resins were not efficient but the crystallization
technique showed good performance, although the crystals were involved in a
viscous and colored solution. Recently, Faveri et al. (212) reported xylitol recovery
by crystallization from synthetic solutions and fermented hemicellulose
hydrolyzates. The method involves evaporation of dilute solution up to super
saturation, cooling of super saturation solution, separation of crystals by
centrifugation and final filtration. Using two sets of tests on xylitol-xylose synthetic
solutions and one set on fermented hardwood hemicellulose hydrolyzate, the best
results in terms of either crystallization (0.56) yield or purity degree (1.00) were
obtained with quite concentrated solutions of730 g/L at relatively high temperature

In Lignocellulose Biodegradation; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
 23

(-5°C). They concluded that xylitol separation by crystallization from fermented

hemicellulose hydrolyzate is feasible.
Thus, xylose rich hemicellulosic materials can serve as abundant and cheap
feedstocks for production of xylitol by fermentation. It is possible to introduce the
pathway for conversion of arabinose to xylitol in the xylitol producing yeast. In that
case, xylitol can be produced from both xylose and arabinose.

Production of 2,3-butanediol
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2,3-Butanediol, otherwise known as 2,3-butylene glycol (2,3-BD), is a valuable

chemical feedstock because of its application as a solvent, liquid fuel and as a
precursor of many synthetic polymers and resins. With a heating value of
Publication Date: July 29, 2004 | doi: 10.1021/bk-2004-0889.ch001

27,200 J/g, 2,3-BD compares favorably with ethanol (29,100 J/g) and methanol
(22,100 J/g) for use as a liquid fuel and fuel additive (213). Dehydration of 2,3-BD
yieldsthe industrial solvent methyl ethyl ketone which is much more suited as a fuel
because of its much lower boiling point. Further dehydration yields
1,3-butanediene, which is the starting material for synthetic rubber and is also an
important monomer in the polymer industry (214). During World War II, it was
needed for conversion to 1,3-butanediene. Methyl ethyl ketone can be
hydrogenated to yield high octane isomers suitable for high quality aviation fuels.
Diacetyl, formed by catalytic dehydrogenation of the diol, is a highly valued food
additive (275). A wide variety of chemicals can also be easily prepared from
2,3-BD (216, 217). There is an interest in industrial scale production of 2,3-BD
from various agricultural residues as well as logging, pulp and paper, and food
industry wastes (215).
2,3-BD can occur in two enantiomeric forms: D- (-) and L-(+) as well as an
optically inactive meso-form. B. polymyxa produces D-(-) 2,3-BD whereas
Klebsiella pneumoniae (Aerobacter aerogenes) produce meso-form and also some
of the L-(+) form. B. subtilis Seratia marcescens and A. hydrophia produce
y

mixtures of different forms (218). A newly isolated Enterobacter cloacae NRRL

B-23289 produces meso-2,3-butanediol (0.35-0.43 g/g sugar) from a variety of
sugar substrates including cornfiberhydrolyzates (219). The typical 2,3-BD yield
was 0.30-0.45 g/g sugar (214, 220). Ui et al. (221) cloned a gene fragment
including genes coding for three enzymes (α-acetolactate synthase, a-acetolactate
decarboxylase and meso2,3-BD dehydrogenase (D-acetoin forming) involved in the
formation of meso-BD of Κ pneumoniae IAM 1063 in E. coli JM 109 after its
insertion into pUCl 18. The resulting E. coli JM109/pBDOl 18 produced 17.7gof
meso-BD from 100 g of glucose per L.
Butanediol is produced during oxygen limited growth by a fermentative
pathway known as mixed acid-butanediol pathway (Figure 4) (222). The 2,3-BD
pathway and the relative proportions of acetoin and butanediol serve to maintain the
intracellular NAD/NADH balance in changing culture conditions. The theoretical

In Lignocellulose Biodegradation; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
 24

maximum yield of 2,3-BD from monosaccharides is 0.5 g/g (223). The efficient
biological conversion of all the available sugars in agricultural biomass residues to
fuels and chemicals is crucial to the efficiency of any process intended to compete
economically with petrochemical products (217).
The high boiling point of 2,3-BD, its high affinity for water, and the dissolved
and solid substances of the fermentation broth make it difficult for 2,3-BD to be
purified and recovered from fermentation slurry (224). Various methods such as
solvent extraction, liquid-liquid extraction and salting out have been used to recover
butanediol. Another feasible method to recover butanediol is by countercurrent
stream stripping (225).
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Production of Vanillin
Publication Date: July 29, 2004 | doi: 10.1021/bk-2004-0889.ch001

Ferulic acid is the major cinnamic acid found in a variety of plant cell walls.
Cornfibercontains about 3% ferulic acid. Wheat bran is another source of ferulic
acid (0.5-1%). Faulds et al. (226) developed a laboratory scale procedure to
produce free ferulic acid (5.7 g) from wheat bran (1 kg) by using a Trichoderma
xylanase preparation and A. niger ferulic acid esterase. Usingfilamentousfungi,
a two-stage process for vanillin formation was developed in which a strain of
A. niger was first used to convert ferulic acid to vanillic acid, which was then
reduced to vanillin by a laccase-deficient strain of Pycnoporus cinnabarinus (227).

Concluding Remarks
Lignocellulose biodégradation and its conversion to a wide variety of commodity
chemicals holds enormous potential. At present, the conversion of lignocellulosic
biomass to fermentable sugars is not cost-effective. Some of the emerging
pretreatment methods such as alkaline peroxide and AFEX generate solubilized and
partially degraded hemicellulosic biomass that need to be treated further with
enzymes or other means to produce fermentable sugars from them. With the
development of a suitable pretreatment method minimizing the formation of
inhibitory compounds for fermentative organisms and use of proper mixture of
cellulases and hernicellulases (enzyme cocktail) tailored for each biomass
conversion, this vast renewable resource can be utilized for production of fuels and
chemicals by fermentation or enzymatic means. Research emphasis should be to
develop efficient and cost-effective pretreatment method, enzymes for use in
cellulose and hemicellulose conversion in an industrial scale, robust efficient
microorganism to ferment lignocellulosic hydrolyzates in a cost-competitive way
and method for cost-effective recovery of fermentation products. Finally,
integration of various process steps such as biomass pretreatment, enzymatic

In Lignocellulose Biodegradation; Saha, B., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
 25

CHCHOHCOOH
3

Lactate

CH3C0-C0A
CH3CCOCH3
Acetaldehyde I
NADH <x| COCH 3

Acetolactate
2

NAD^I
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CH3CHO CH3CO-P
Acetaldehyde Acetyl Phosphate
NADH >J2
ADP \J Acetoin Dlacetyl
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NAD^I ΑΤΡ^Ί NADH ^1

2

NAD^I
CH CH OH CHCOOH
Ethanol
3 2 3

Acetate H3CHOH
2,3-Butanediol

Figure 4. Metabolic pathway of 2,3-butanediol production from glucose.

saccharification, detoxification, fermentation of the hydrolyzates and recovery of

products will greatly aid in reducing the overall cost of using lignocellulose for
practical purposes.

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