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Review

Enzymatic hydrolysis of cellulosic biomass

Biofuels (2011) 2(4), 421450

Bin Yang1, Ziyu Dai2, Shi-You Ding3,4 & Charles E Wyman4,5


Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to
economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic
biomass to fermentable sugars may be the most complex step in this process due to substrate-related and
enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher
yields, higher selectivity, lower energy costs and milder operating conditions than chemical processes, the
mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of
various glycosyl hydrolase components is not well understood. Consequently, limited success has been
realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key
substrate and enzyme features that influence performance, to better understand fundamental strategies to
advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are
summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of
polymerization and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric
xylan and lignin) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis.
We further discuss the diversity, stability and activity of individual enzymes and their synergistic effects
in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize
novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification
and over-expression of selected enzymes and modifications in lignocellulosic biomass are alsodiscussed.

Enzymatically based cellulosic ethanol production such technology was judged to be too high risk for
technology was selected as a key area for biomass industry to pursue at that time [1] . However, applica-
technology development in the 1980s, and the US tion of the emerging field of biotechnology offered the
Department of Energy (DOE) has actively supported promise for significant advances that could dramati-
the scale up of ethanol production since the Office of cally reduce costs and make cellulosic ethanol com-
Alcohol Fuels was created in the DOE after the energy petitive. Improvements in dilute acid pretreatment
crisis of the 1970s. Although biological conversion and cellulase produced by Trichodermareesei discov-
of cellulosic biomass to fuels and chemicals through ered during World War II led to most of the historic
enzymatic hydrolysis of cellulose offers the potential for cellulosic ethanol cost reductions in the 1980s [24] .
higher yields, higher selectivity, lower energy costs and Well-known T.reesei Rut C30 was derived at Rutgers
milder operating conditions than chemical processes, University through classical mutagenesis and strain

1
Washington State University, Center for Bioproducts & Bioenergy, Department of Biological Systems Engineering, 2710 University Drive, Richland,
WA99354, USA
2
Chemical & Biological Process Development Group, Pacific Northwest National Laboratory, 902 Battelle Boulevard, PO Box999, MSIN P860, Richland,
WA99352, USA
3
Biosciences Center, National Renewable Energy Laboratory, 1617 Cole Boulevard, Golden, CO 80401, USA
4
Bioenergy Science Center, Oak Ridge National Laboratory, PO Box2008, Oak Ridge, Tennessee, USA
5
Center for Environmental Research & Technology, Bourns College of Engineering University of California, 1084 Columbia Avenue Riverside, CA 92507,USA

Author for correspondence: Tel.: +1 509 372 7640; Fax: +1 509 372 7690; E-mail: binyang@tricity.wsu.edu

future science group 10.4155/BFS.11.116 ISSN 1759-7269 421


Review Yang, Dai, Ding & Wyman

Key terms selection from wild strains, such that must be charged for ethanol ($/gallonethanol)
Enzymatic hydrolysis: Multi-step as T.reesei QM9414 [5] . Cellulase to cover the cost for different enzyme loadings that
heterogeneous reaction in which 150L produced by Genencor was all achieve the same ethanol yield (data adopted from
insoluble cellulose is initially broken down very effective because of enhanced National Renewable Energy Laboratory report) [11] .
at the solidliquid interface via the
levels of b-glucosidase [6,501] . Thus, to meet the enzyme cost goal ($0.10/gallonetha-
synergistic action of endoglucanases and
exoglucanases/cellobiohydrolases. This Dramatic improvements in reduc- nol or less) of the DOE Biomass Program will require
initial reaction is accompanied by further ing glycosyl hydrolase costs by a that enzymes cost less than $2/kgcellulase protein or
liquid-phase hydrolysis of soluble factor of 20 to 30 was announced strategies must be developed to substantially reduce
intermediates, that is, short
celluloligosaccharides and cellobiose,
recently [7,8] . the loadings needed for high yields, or some of both
which are catalytically cleaved to produce It is noteworthy that many micro- [1214] . In addition, mechanisms of action and factors
glucose by the action ofb-glucosidase. organisms in nature, mostly bacteria limiting hydrolysis effectiveness are not well known,
Cellulose: Predominant polysaccharide and fungi, are capable of produc- and consequently limiting in many promising com-
that makes up approximately 4050% of ing biomass-degrading enzymes. mercial applications [15] . Improving the understanding
cellulosic biomass in the form of linear Cellulolytic microbes may evolve of the structure and function of both lignocellulosic
fibrils of approximately 3040 hydrogen-
bonded chains of b-(1,4) as individual degraders or as part materials and their degrading enzymes will be invalu-
glucopyranosides with a native degree of of a chain reaction in microbial able to determining the roles of biomass pretreatment,
polymerization of approximately communities of some ecosystems. hydrolysis and enzymes in influencing lignocellulosic
10,00015,000. Cellulolytic enzymes secreted by biomass conversion and in developing appropriate strat-
Pretreatment: The disruption of the such microbe(s) are classes of gly- egies to achieve high rates and yields with low amounts
naturally resistant structure of
coside hydrolases (GHs), including of enzyme.
lignocellulosic biomass to make reactive
intermediates (e.g., fermentable sugars) to lignin-modifying catalysts in some Enzymatic hydrolysis is influenced by both structural
biologicalprocesses. cases. Enzyme and microbe combi- features of cellulose and the mode of enzyme action.
Cellulase: Combination of enzymes that nations vary in different biomass- Due to the complexity of the cellulose substrate and the
catalyze the reaction of water with degrading ecosystems depending on cellulase system, the mechanism of hydrolysis of cel-
cellulose to release shorter chains and the initial biomass source and envi- lulose substrate is still not fully understood, although
ultimately soluble glucose sugar.
ronmental factors. With emerging detailed knowledge of some aspects of enzyme struc-
Glycoside hydrolases: Enzymes that biotechnology tools, there is great ture, enzyme molecular properties and the ultrastruc-
hydrolyze a glycosidic bond between
two adjacent saccharide groups or potential to develop new enzyme ture of cellulose have been obtained through extensive
between a carbohydrate and a sources that offer more desirable study over the last few decades. Thus, this paper focuses
noncarbohydrate moiety. enzyme features, including higher on a review of the current understanding of key features
Lignin: Makes up approximately 1528% specific activities with more bal- of the pretreated biomass and glycosyl hydrolases that
of lignocellulosic biomass; it is distinctly anced synergism, better thermal influence sugar release and suggests opportunities to
different from the other macromolecular
stability, better resistance to envi- further advance our understanding of lignocellulosic
components of lignocellulosic biomass. It
is an amorphous, cross-linked and 3D ronmental inhibitors and improved bioconversion by newly advanced technologies, such as
polyphenolic polymer that is synthesized combination of various enzymes genomics, proteomics and microscopy.
by dehydrogenative polymerization of (e.g., cellulase, hemicellulase, pec-
three types of phenyl propanoid units,
including monolignols: coniferyl, sinapyl
tinase and proteinase) activities that Substrate-related factors
and p-coumaryl alcohol. maximize sugar yields at lowcost. This section of the review targets updating of recent
Lignocellulosic biomass: Biomass
Unfortunately, cellulosic etha- advances in understanding structural characteristics
feedstock mainly containing cellulose, nol technologies have not yet been of biomass and related enzyme features, and provid-
hemicelluloses and lignin; usually commercialized, at least partly ing perspectives towards improvement in substrates
including agricultural residues, woody because releasing sugars from nat- for enzymatic hydrolysis. Lignocellulosic biomass has
crops, herbaceous energy crops and
municipal solid wastes. urally recalcitrant cellulosic mate- numerous structural features that make it very dif-
rials is difficult [9,10] . The result ficult to deconstruct enzymatically. The majority of
Genomics: Study of the genomes of
organisms. This includes intensive efforts is that high enzyme doses are biopolymers, including cellulose, hemicellulose and
to determine the entire DNA sequence of needed, with the cellulase loadings lignin, are not just individual units in a plant cell wall
organisms and fine-scale genetic of approximately 15FPUper gram but are intimately interconnected [16] . Lignin and car-
mapping efforts. Also includes studies of
cellulose typically used to achieve bohydrates (e.g., cellulose and hemicellulose) form
intragenomic phenomena such as
heterosis, epistasis, pleiotropy and other economically viable sugar yields lignincarbohydrate complexes [17] . Recent studies
interactions between loci and alleles with from pretreated biomass equivalent demonstrated that in grasses, polysaccharidelignin
thegenome. to approximately 30 g of enzyme crosslinking is mediated by ferulates attached primar-
perliter of ethanol made. Figure1 ily to arabinoxylans. Ferulated hemicelluloses pro-
illustrates the relationship between the cost of enzyme vide points of growth for lignin via ether bonds that
protein production (US$/kgenzyme) and the amount anchor lignin to plant-wall polysaccharides and could

422 Biofuels (2011) 2(4) future science group


Enzymatic hydrolysis of cellulosic biomass Review

contribute to recalcitrance[1820] . The complete struc- 1.5


ture and compositions of lignin, which binds cellulosic 1.4 15 FPU/g cellulose

Cost of cellulase, US$ per gallon of ethanol


fibers together in a composite structure and reduces 1.3 10 FPU/g cellulose
the accessibility of cellulose to enzymes [21] , is still not 1.2
fully understood. To completely deconstruct these het- 1.1
erogeneous structures in the plant cell wall requires 1.0
synergistic reactions of enzymes, such as cellulases, 5 FPU/g cellulose
0.9
hemicellulases, accessory enzymes and lignin-modify- 0.8
ing enzymes. Our current knowledge is insufficient to 0.7
understand the whole picture of enzymatic hydrolysis 0.6
of cellulosic biomass, and most evidence available to 0.5
date results from two approaches: purified enzyme(s) 0.4
acting on purified substrates or mixtures of enzymes 0.3
acting on thermo-chemically pretreatedbiomass. 0.2
0.1
Characteristics of cellulose 0.0
The main commercial purpose of enzymatic hydrolysis 0 5 10 15 20 25
Cost of protein, US$ per kg of protein
of cellulosics is to deconstruct cellulose and other car-
bohydrate polymers into fermentable sugars, including
Figure1. Cost of cellulase for ethanol production versus cost of protein at
glucose and/or oligomers that can be further converted
different loadings that all achieve the same ethanolyield.
into valuable products through biological or chemical
Data from [16].
approaches. Although enzymatic hydrolysis of cellulose
is complicated by existence of other components (e.g.,
hemicellulose and lignin) and their derivatives after pre- 66 CesA enzymes [28] . Each rosette synthesizes a
treatment, it is essential to understand the effects of key 36-chain CEF. The estimated dimensions of CEFs are
features of cellulose itself on the rate and effectiveness 35.5nm based on cellulose Ib structure, in agree-
of enzymatic hydrolysis. ment with direct AFM measurements. A number of
It is difficult to characterize native cellulose in the CEFs synthesized by rosettes with close proximity
plant cell wall, due to its small size and the matrix of may form a bundle, the macrofibril. The deposition of
polymers (mainly hemicelluloses and lignin) closely other wall polymers, mostly hemicellulose, during cell
interlinked with it. Cellulose can be considered as a growth, causes the macrofibril to split to form single
composite material built from nanometer-scale micro- microfibrils with hemicelluloses associated on their
fibrils. Recent studies using advanced imaging tech- surfaces [25,26] .
niques, such as atomic force microscopy (AFM), have Substrates used in cellulase assays are primarily
revealed precise measurements and detailed cellulose purified cellulose (e.g., Avicel or Sigmacell) with small
surface structure in its native stages. Based on AFM proportions of other polysaccharides, mainly hemicel-
studies of plant cell walls [2224] , the dimensions were luloses from higher plants. Regardless of the origin and
measured as 35 nm, consistent with the 36-chain purification methods used in their preparation, the
model of the cellulose elementary fibril (CEF) based structural characteristics of purified
Key terms
on the proposed cellulose-synthase complexes (the cellulose vary in crystallinity, degree
Proteomics: Large-scale study of
rosettes) that contain 36 cellulose synthases. One of of polymerization (DP) and surface proteins, particularly their structures,
the interesting findings from AFM imaging was mac- structure, which may significantly activities, modifications, localization and
rofibrils only observed on the uppermost layer of the affect enzyme hydrolysis. interactions of proteins in complexes.
primary cell wall. The macrofibril appeared to consist Substantial amounts of proteins/
enzymes are involved in the
of a bundle of CEFs that split at the end to form smaller Crystallinity lignocellulosic biomass degradation.
bundles and eventually a single CEF. Each microfibril Purified celluloses are micrometer-
Hemicellulose: Make up approximately
observed in mature primary cell walls contained only a sized particles composed of nano- 2030% of biomass, exhibit a much
single CEF and hemicelluloses associated with its sur- meter-sized microfibrils (Figure2) . broader distribution of sugars and are
face [25,26] . AFM images of maize cell walls from fresh Generally, these cellulose particles frequently branched and essentially
cells further confirmed this observation [27] . Figure2 are believed to consist of crystalline, amorphous polysaccharides. These
polysaccharides are usually associated
shows a schematic model of plant cell wall synthesis. paracrystalline (disordered) and with cellulose, often hydrogen bonded
In this model, at least three types of cellulose syn- amorphous structures. Historically, to cellulose. These branched
thases (CesA subunits, a1, a2 and b) are required amorphous cellulose has been polysaccharides are composed of 1,
4-linked b-d-hexosylresidues.
to spontaneously assemble the rosettes containing reported to be rapidly degraded to

future science group www.future-science.com 423


Review Yang, Dai, Ding & Wyman

Microfibril is a cellulose
elementary fibril surrounded
by hemicelluloses

Matrix polysaccharides
are associated to the surface
of cellulose elementary fibril

Macrofibril splitting and


hemicelluloses coating

Matrix polymers
are synthesized by
Golgi apparatus and
secreted to cell wall
Cell wall

Ribbon-like Microfibril
macrofibril is
synthesized Macrofibril
by arrayed
rosettes
36-chain nascent
elementary fibril
-1,4-glucan chains
emerging from rosettes

Arrayed rosettes

Cellulose synthase complex


(rosette)
Vesicles
Plasma membrane

Golgi apparatus
Microtubule

Figure2. Model of plant cell wall cellulose elementary fibril and its synthesis. The dimensions of cellulose elementary fibril are
estimated as 35.5 nm.
Adapted with permission from [29].

cellobiose by cellulases, while the hydrolysis of crys- Cellulose crystallinity was also reported to play a role
talline cellulose is much slower. Thus, some authors in enzyme adsorption, which can be correlated with
proposed that hydrolysis rates depended on cellulose hydrolysis rates and yields. Increased hydrolysis rates
crystallinity [2932] . Although rates have been found to and yields (>100 times) were shown to be related to the
slow with increasing crystallinity of cellulose in some higher capacity of amorphous cellulose than crystalline
studies [3335] , others found the opposite effect[3638] . cellulose for cellulases [35,39,5055] . Many results showed
It is expected that crystallinity should increase with that enzyme adsorption, including the complete glyco-
cellulose hydrolysis as a result of more paracrystalline syl hydrolase system, cellulose binding module (CBM)
and amorphous cellulose removal [3840] . However, and individual enzyme components, generally declined
no significant change in crystallinity during cellulose as cellulose crystallinity increased. Recently, Joeh and
hydrolysis was reported in some studies [41,42] . In some co-workers showed that crystallinity greatly impacted
reports, cellulose crystallinity was not considered to the adsorption of Cel7A (CBHI), leading to decreased
affect efficient hydrolysis [37,4349] . extent of hydrolysis [55] . Hall and co-workers indicated

424 Biofuels (2011) 2(4) future science group


Enzymatic hydrolysis of cellulosic biomass Review

that the initial enzymatic hydrolysis 100


rate increased with decreasing crys- 0h 1h
tallinity index, while the adsorbed 8 0

enzyme concentration stayed con- 60

CrI (%)
stant [42] . In addition, different cel-
lulase components have been shown 40

to have different adsorption capaci- 20


ties and activities for cellulose [50,51] .
Endoglucanase I (EGI), known to 0
0h 1h 2h 4h 5h 15 h
attack and adsorb preferentially on
amorphous cellulose, appeared to 2h 5h 15 h
have an average adsorption capac-
ity and activity greater than CBHI
on both types of cellulose studied.
A similar pattern was described
for EGI by Ding and Xu [56] .
Furthermore, Banka and Mishra
observed that crystallinity increased
adsorption of a nonhydrolytic pro-
tein named fibril-forming protein
Figure3. Features of solids resulting from interrupted hydrolysis of Avicel cellulose.
from T.reesei[57] . Such results indi-
Crystallinity index by x-ray diffractometer and atomic force microscopy for interrupted
cate that cellulose crystallinity has
hydrolysis samples at 0, 1, 2, (4), 5 and 15h.
important effects on nonhydrolytic
enzyme components, which can be
essential to effective enzymatic hydrolysis ofcellulose. elucidate the influence of crystallinity on the effective-
Cellulose crystallinity may not only affect cellu- ness of processive or pseudo-processive enzymes from
lase adsorption but may also impact the effectiveness various microorganisms.
of adsorbed cellulase components. The literature has
shown that cellulose crystallinity affects the synergism Degree of polymerization
among cellulase components [42,51,5866] . Hoshino etal. Several studies and literature reviews discuss the change
found increased synergism between CBHI and endoglu- in DP of insoluble and soluble cellulose after hydrolysis
canase II (EGII) from T.reesei with increased crystallin- with a complete set of cellulases or its purified com-
ity and the highest synergism between CBHII and EGII ponents [43,44,51,52,7480] . However, the understanding
at a crystallinity index approximately 1.0. In another of the impact of cellulose chain length on hydrolysis
study, Igarashi and co-workers showed that nature of is still limited. Sinistyn etal. showed that reduction
the crystalline cellulose polymorph affected the hydro- in DP of cotton linters by g-irradiation, while keeping
lytic activity of adsorbed CBHI [6769] . Moreover, crystallinity index constant, had negligible impact on
Mizutanietal. [70] and Gama and Mota [71] showed that the hydrolysis rate [35] . A recent kinetic study by Zhang
the impact of surfactant in enhancement of saccharifica- and Lynd [81] found that a decrease in cellulose DP had
tion is influenced by the crystallinity of pure cellulose. less effect in accelerating hydrolysis than an increase in
A few studies investigated the relationship between accessibility of b-glycosidic bonds as generally measured
cellulase processivity and crystallinity. The processiv- by the maximum amount of cellulase adsorbed on cel-
ity of CBHI, a dominant enzyme of the Trichoderma lulose. For soluble cellulose, Nidetzky etal. showed that
system, was shown to be affected by cellulose crystal- the initial velocity of cello-oligosaccharides degraded by
linity. A rough estimate of processivity, determined by CBHI increased with DP up to cellohexose and then
the ratio of cellobiose to glucose, was reported to be 23 remained constant [82] . Similar effects of DP of soluble
and 14 cellobiose units for bacterial microcrystalline cel- cellodextrins on CBHII and EGI activity were also
lulose (BMCC, CrI ~>85%) and amorphous cellulose, reported [51] . Furthermore, b-glucosidase activity was
respectively [72] . In another study, processivity values reported to decrease as DP was reduced [83,84] . However,
for CBHI from T.reesei were reported to be 8810, no information is available on the effect of insoluble
4210 and 342.0 cellobiose units for bacterial cel- cellulose DP on the catalytic efficiency of cellulases
lulose CrI ~88), BMCC (CrI ~92) and endoglucanase- except that higher DP could result in higher synergy
pretreated bacterial cellulose (unknown CrI), respec- between CBHI and EGI [65,81,85,86] . Furthermore, cel-
tively [73] . Further studies are needed to confirm and lulose DP may affect the processivity index, and full

future science group www.future-science.com 425


Review Yang, Dai, Ding & Wyman

processivity of CBHI may not be realized with short Nevertheless, because some bacterial cellulases, such as
cellulose chains [61] . Limited information is available cellulosome, a multienzyme complex with a size approx-
in the literature on the effect of cellulose DP on cellu- imately 100 times that of individual fungal cellulases,
lase adsorption. Kaplan etal. showed a significant drop hydrolyze cellulose at a higher rate than fungal cellu-
in cellulase adsorption resulting in reduced hydrolysis, lases, micropores appear to be less important [50,109] .
with a change in DP along with some ring openings Furthermore, it was observed that cellulase components
of cotton cellulose due to withering; however, crys- did not penetrate into the pores [110] , and no relation was
tallinity was not affected much [87] . Given the typi- observed between pore volume and digestibility [111] . A
cally large amount of CBHI in cellulase (>65%) and limited to negligible effect of particle size on cellulose
its preferences [82,8891] , one could quickly conclude adsorption [112114] and cellulose hydrolysis [47,115] was
that DP reduction should improve hydrolysis effective- reported, but the possibility of an increase in the rate of
ness by making more ends available to CBHI and be a cellulose fragmentation with smaller particles cannot be
promising target to enhance hydrolysis rates and yields, ruled out. In contrast, it was shown that larger particles
provided the enzyme formulation is adjusted to take could be inhibitory to effective hydrolysis [116] . On the
advantage of the lower DP. other hand, various studies on the effect of DP and
crystallinity on enzymatic digestibility demonstrated
Accessible surface for cellulase that susceptibility of pretreated substrates to enzymatic
Cellulose accessibility to cellulases is limited by the hydrolysis could not be easily predicted from differences
structure of cellulose microfibrils that are believed to be in cellulose DP or crystallinity [37,117] , possibly due to
nanometer-sized (Figure2) . Crosslinking among chains the complexity of real cellulosic substrates. However,
of cellulose fibers, coupled with their being imbedded accessible surface area can provide a useful perspective
in a matrix of polysaccharides involving lignin and on these features and help identify characteristics that
other polymers, provides extra rigidity in native plant can be changed by pretreatment.
cell walls but complexity for enzymatic digestion [92] .
Although extensive modification may occur during cel- Change in cellulose reactivity & enzyme functionality
lulose purification, the diameter of cellulose microfibrils with conversion
may remain approximately 35 nm in plant cell walls, The dramatic decline in overall enzymatic hydroly-
the same as in the original source, but the length of sis rates and rates per amount of adsorbed enzyme as
these microfibrils may be significantly reduced to several hydrolysis progresses is responsible for low yields, and
hundred nanometer (Figure2) . The accessibility of cel- long processing times cannot be attributed to just prod-
lulose to cellulases may refer to two levels of limitations, uct inhibitory effects. However, the mechanism still
with one being the face of crystalline cellulose available remains unclear [118,119] . In addition to enzyme-related
to cellulases binding, with the carbohydrate-binding factors, such as thermal instability of cellulases [120123] ,
module of CBH I attaching to only the hydrophilic face products inhibition [120,124128] , enzyme inactivation
[9395] . The second limitation is the anatomical structure [125,129135] , enzyme slowing down/stopping [136] , sub-
of the plant cell wall, which may also affect accessibility strate-related factors, including substrate transformation
for cellulases, specifically the pores existing in the plant into a less digestible form [137] , and the heterogeneous
cell walls that allow cellulases to enter into the boxes of structure of the substrate [137,138] , have been proposed
plant tissue to access the surface of cellulose microfibrils. to account for such phenomena. At one time, the drop
One of the impacts of pretreatment could be to enlarge in rate was explained by declining substrate reactivity as
pore sizes to enhance cellulase penetration into biomass. the more easily reacted material was thought to be con-
Based on the premise that enzymatic hydrolysis of sumed preferentially [137] , but other reports concluded
cellulose is a surface reaction, available surface area that substrate reactivity was not the principal cause
of cellulose for cellulase attack should be one of the of the long residence time required for good cellulose
most influential structural features of biomass that conversion [136] .
influences cellulase adsorption on the cellulose sur- Interrupt and restart experiments were conducted
face and subsequent enzymatic breakdown [16,96101] . to identify factors that control cellulose hydroly-
Many papers have discussed available pore volume and sis[136141] . A new restart approach, involving protein-
specific surface area in this context [51,44] . Accessibility ase treatment to remove cellulases followed by pro-
can be also correlated to other substrate-related factors, teinase inhibitors to deactivate the proteinase before
such as cellulose crystallinity or depolymerization. restarting cellulose hydrolysis at the original conditions,
However, some studies offered evidence of other sub- was developed to understand reactivity loss during
strate features, including pore volume [44,101105] and the dynamic process of enzymatic hydrolysis of cel-
particle size [37,103,106108] affecting cellulose hydrolysis. lulose [142] . The resulting hydrolysis rate and the rate

426 Biofuels (2011) 2(4) future science group


Enzymatic hydrolysis of cellulosic biomass Review

per adsorbed enzyme of Avicel were nearly constant acetylated in many types of biomass, and deacetylation
with changing conversion for these restart experi- was reported to triple cellulose digestibility, with some
ments, but declined in continual hydrolysis. Thus, the differences reported in the degree of removal needed to
drop in hydrolysis rate for continual cellulose diges- be effective [163,164] . One study showed that this effect
tion could not be attributed to changes in substrate appeared to become less important beyond removal of
reactivity, while other enzyme-related effects such as 75% of the acetyl groups, while another study revealed
enzymes slowing down by getting stuck or jamming continued improvements up to full removal of hemi-
could be responsible [142,143] . For this restart approach, cellulose [156,165] . Grohmann and co-workers showed
the cellulose CrI increased slightly with cellulose con- that removing acetyl esters from aspen wood and wheat
version to approximately 80% within 5h [144] . AFM straw made them five to seven times more digestible.
images of the interrupted hydrolyzed Avicel showed Kong etal. observed a major effect on cellulose digest-
that the somewhat rough surface of the original Avicel ibility by the removal of acetyl content of aspen wood
became smoother and flatter as enzymatic hydrolysis while preserving lignin and polysaccharides [165] . Chang
progressed (Figure 3) . The new restart approach allows and Holtzapple applied similar methods as above but
one to revisit many aspects of enzymatic hydrolysis of showed that removal of acetyl bonds is less important
cellulose needed to advance the understanding of the than crystallinity reduction and/or lignin removal[166] .
dynamic interactions between enzyme and cellulose. In addition, a study by Weimer etal. suggested that inti-
mate association of xylan and cellulose does not inhibit
Derived insoluble matter distribution biodegradability of polysaccharides [167] . Removing
Cellulose, hemicelluloses and lignin are the major hemicellulose also removes acetyl groups and usually
polymers in the plant cell walls, and any change in or alters the form of lignin left, making it difficult to
removal of these components would be expected to isolate the factors most influential in improving per-
consequently affect enzymatic digestibility. However, formance. Unfortunately, it is still debatable whether
experimental results have been rather inconsistent. hemicellulose removal or the breakdown of the cross-
Grohmann etal. and others showed direct relationships linked network of polysaccharides and bonds among
between hemicellulose removal and glucose yields from them is responsible for enhanced digestion of cellulose
cellulose [111,145150] , but other reports do not support in pretreatedbiomass.
a role for hemicellulose removal in changing cellulose Studies such as these lead us to believe that even if
digestibility [151154] . Similarly, conflicting conclu- cellulose is made completely accessible to enzymes, they
sions have been reached on the importance of lignin would not be able to hydrolyze cellulose unless the net-
removal in enhancing cellulose conversion [102,155157] . work of biomass components including hemicellulose
All plant cell wall constituents are modified to different and acetylation is disrupted [168170] . Jeoh and co-work-
extents by pretreatments, depending on the technolo- ers observed increased cellulose accessibility, as mea-
gies and conditions applied, making it challenging to sured by the adsorption of fluorescent labeled CBHI,
deduce whether altering cellulose microfibrils, removing and an increase in hydrolysis with the extent of xylan
hemicelluloses, modifying or relocating lignin, or other removal [55,171] . Pan etal. suggested that acetyl groups in
effects on the substrate are responsible for improving pulp might restrict cellulase accessibility to cellulose by
enzyme effectiveness. inhibiting productive binding, which might be caused
by increased diameter of cellulose and/or changed
Hemicellulose hydrophobicity [168] . However, there is not much evi-
The enzymatic digestion of cellulose has been shown dence as to whether selective hemicellulose removal or
to significantly improve with hemicellulose removal, deacetylation impact cellulase adsorption. In addition,
thereby suggesting that hemicellulose provides the because recent results showed xylooligomers are strong
key barrier to cellulose breakdown by enzymes [157] . inhibitors of cellulase, their release during enzymatic
However, simultaneous lignin alteration during pre- hydrolysis could substantially slow hydrolysis, mak-
treatment can confound the role of hemicellulose ing their removal in pretreatment important [172,173] .
solubilization and modification [102,155,158,159] . From Deacetylation may indirectly affect cellulase effective-
a more applied perspective, some pretreatments such ness in enzymatic hydrolysis of lignocellulosics because
as ammonia fiber expansion (AFEX) produce highly the removal of acetyl and other substituents from xylan
digestible cellulose without removing any significant could increase xylan digestibility by xylanase [156,174178]
amounts of hemicellulose [160162] , although AFEX and result in increased cellulose digestibility [179182] .
may modify the chemistry of hemicelluloses. Less Thus, although its role in enhancing cellulose digestion
attention has been given to the degree of acetylation is ambiguous, xylan removal during pretreatment may
of the substrate. Hemicellulose chains are extensively be desirable for a number of economic and technical

future science group www.future-science.com 427


Review Yang, Dai, Ding & Wyman

reasons such as higher recovery of xylose, less inhibi- hemicellulose and lignin removal were very limited,
tion by xylooligomers and less need for hemicellulose needed less cellulase to achieve similar hydrolysis per-
degrading and accessory enzymes [183185] . formance than dilute acid pretreated solids, which had
little hemicellulose left and most of the original lignin
Lignin content [160] . Overall, lignin modification seems more
Lignin binds cellulosic fibers together in a composite important than hemicellulose dissolution, with the lat-
structure with excellent properties, but also reduces the ter perhaps just providing a convenient marker of lignin
accessibility of cellulose to enzymes [21] . Various studies alterations that improve cellulose digestibility [194] .
reported cellulose hydrolysis was improved with increas- Such information leads us to believe that lignin
ing lignin removal, although differences were reported modification is vital to enhance cellulose digestibility
in the degree of lignin removal needed [44,102,157,186] . and that lignin removal provides even greater benefits
The ratio of syringyl to guaiacyl moieties in lignin was [157] . Removing lignin enhances cellulose accessibil-
also considered to have important effects on digest- ity and reduces nonproductive binding of enzymes,
ibility [187] , yet the importance of lignin in limiting thus improving enzymatic hydrolysis performance [16] .
hydrolysis has been difficult to determine. One of the Interestingly, recent lignin-blocking technology brings
most significant limitations is the effect of lignin on new insight into disrupting the original celluloselig-
fiber swelling and its resulting influence on cellulose ninhemicellulose structure and fully liberating highly
accessibility [116,188,189] . Lignin has been claimed to susceptible cellulose and hemicellulose for enzymes to
depolymerize and then repolymerize during hemicel- hydrolyze, rather than pursue complete removal of lig-
lulose hydrolysis by pretreatment, although no doubt in nin and its derived compounds [16,197] .
a different morphology that could change its impact on Although literature regarding the effect of lignin on
cellulose digestion[117,190192] . The removal of lignin not cellulose hydrolysis is abundant, the role of lignin in
only increased cellulose accessibility but also allowed enzymatic hydrolysis of heterogeneous cellulosic bio-
more cellulase action [157] . Lignin and its derivatives mass is still unclear. However, it may be advantageous to
were reported to precipitate and bond with protein [16] , remove lignin before hydrolysis to enhance the technical
and condensed lignin was reported to adsorb protein and economic prospects of cellulose saccharification,
from aqueous solutions [193] . Thus, it appears that lignin because lignin will lead to less available enzyme due to
could physically and chemically resist cellulose attack unproductive binding, may be inhibitory to fermenta-
by enzymes. Lignin not only plays a very important tion [50] and may cause mixing problems at higher solids
role in irreversible cellulase absorption but also acts as loadings [198,199] . It is not clear whether lignin removal
a barrier to cellulase, limiting hydrolysis efficacy [194] . or disruption of its tight association with carbohydrates
Thus, lignin removal may both open more space for is necessary. Grabber and co-workers suggested that
enzymes and reduce enzyme nonspecific absorption inhibition of fungal hydrolases is not affected by the
on lignin [157] . Low levels of lignin have been shown composition of lignin [200] . However, lignin concentra-
to enhance cellulose hydrolysis due to a physical sepa- tion and its crosslinking with feruloylated xylans greatly
ration of microcellulose fibrils enhancing cellulase affect degradability of cell walls [201,202] . The negative
access/activity [16,55,157] . Lignin modifications in trans- impact of lignin concentration on cell wall digestibility
genic biomass have resulted in decreased recalcitrance of tobacco stems was observed by Sewalt etal. in another
to saccharification with improved fermentable sugar study [203] .
yield[18] . Ooshima and co-workers observed that dilute acid
Our recent findings suggest that enzymatic digest- pretreatment at higher temperatures led to shrinking
ibility of cellulose is related to both hemicellulose and and agglomeration of lignin that increased cellulase
lignin removal. For example, similarly high enzymatic adsorption on cellulose [134] . Similar observations of
digestion was observed for corn stover pretreated at opti- lignin melting and its relocation are affirmed by others
mized conditions for several CAFI pretreatments, even as well [204,205] . In a recent study, Selig etal. explained
though hemicellulose and lignin removal varied con- that droplets of lignin, formed during high temperature
siderably among the solid residues from each pretreat- dilute acid or water only pretreatment, may migrate to
ment technology [21,195] . Relocation and/or modifica- the surface and impede cellulase adsorption on cellu-
tion of lignin on the solid substrates could lead to lower lose[206] . Yuldashev etal. observed that the amount of
degree of observed final lignin removal [16] . Although cellulase on the surface of cotton stalks (cellulose: 44%
some demonstrated a linear relationship between lignin and lignin: 26.4%) was lower than the milled cotton
removal and cellulose digestion, others showed little stalks (cellulose: 92% and lignin: 0.6%), leading to a
or no effect of lignin removal on cellulose digestibil- drop in conversion; however, lignin did not inactivate
ity[196] . For example, AFEX-pretreated solids, in which free or bound enzymes [207] . In another study, Ishihara

428 Biofuels (2011) 2(4) future science group


Enzymatic hydrolysis of cellulosic biomass Review

and co-workers showed that lignin slows down enzyme ammonia-recycled percolation pretreatment of corn
adsorption but does not restrict the conversion of car- stover, containing xylooligomers, soluble lignin, sugar
bohydrates in steamed shirakamba wood [208] . Limited and lignin degradation products, inhibited cellulase and
delignification of wheat straw by sodium hydroxide microbial activity significantly [222] . However, little was
was shown to result in increased cellulase adsorption by known about exactly which soluble compounds from
Estrada etal. [209] . Conversely, Mooney etal. studied hemicellulose, lignin and other biomass components
the effect of amount of lignin in four different types affect enzymatic hydrolysis, or how they acted. However,
of pulp on cellulase adsorption and concluded that the recent work by Kumar and Wyman revealed that xylo-
proportion of lignin did not influence cellulase adsorp- oligomers strongly inhibit cellulase action [176] , and a
tion [210] . However, Selig and co-workers showed that follow-on study showed that inhibition by xylooligomers
lignin droplets deposited on cellulose may interact with was stronger than by glucose or cellobiose, with longer
water and form a boundary layer impeding cellulase chained xylooligomers having the greatest impacts [175] .
movement [205] . Furthermore, lignin linkages with cel- This research also showed that soluble xylooligomers
lulose may presumably impede the processive action had strong inhibitory effects on cellulases and such
of cellulase. Although lignin may reduce the amount effects increased with concentration [175] . It was also
of active enzyme available for cellulose hydrolysis, its reported that soluble products from xylan, including
impact on the effectiveness of adsorbed cellulase still xylooligosaccharides and xylose, are significantly more
requires clarification. inhibitory to glucan hydrolysis even though xylan and
pectin inhibited glucan hydrolysis [223] . Xylooligomers
Derived soluble matter distribution effects were found to significantly inhibit cellulase adsorption
Much attention has been paid to removing hemicellu- onto Avicel (Shi et al., Unpublished Data) . Other sugars
lose and lignin from biomass solids as obvious physical from hemicellulose, such as mannose and galactose,
barriers to cellulose access by enzymes, but little work were found to be inhibitory to cellulases and b-gluco-
has been devoted to understanding how soluble matter sidase[224] . Another study identified sugars in pretreat-
(e.g., sugar, sugar oligomers, sugar degradation prod- ment hydrolyzate as the primary inhibitor to enzymatic
ucts and lignin-derived compounds) released during hydrolysis of dilute acid pretreated whole slurry, while
pretreatment and enzymatic hydrolysis affect enzymatic other soluble compounds, including acetic acid, phe-
hydrolysis of cellulose. In addition, in most research, nolic compounds and furans, only slightly inhibited
pretreated cellulosic biomass solid was separated from enzymatic cellulose hydrolysis[225] .
the hydrolyzate and thoroughly washed to get a clear- Soluble lignin derivatives were reported to affect not
cut evaluation of the effect of pretreatment on cellulose only microorganisms but also enzymes such as cellu-
digestibility independent of dissolved inhibitors. On the lases and b-glucosidases [226238] . Mendels and Reese
other hand, enzymatic hydrolysis of pretreated whole found that substituted phenols had moderate inhibition
slurry, including both pretreated solids and liquor (at on cellulases [233] . Panagiotou and Olsson tested mul-
least partially if not all of the liquor), will likely be nec- tiple compounds (including furans, phenols and low
essary to lower capital and operating costs. Even with molecular weight acids) and reported formic acid as the
washed pretreated solids, the concentration of soluble strongest inhibitor to cellulases by complete inactivation
matter released from the pretreated solids during enzy- of enzymes [239] . Because the lignin preparation used in
matic hydrolysis becomes more significant as the solid the study partially dissolved, the observed inhibition of
loadings increase. However, it was reported that cellu- cellulases was believed to be due to not only cellulase
lose conversion by enzymatic hydrolysis was reduced adsorption on the major particulate lignin component
when pretreated solids were not washed [211] , pretreat- but also on solubilized small-molecular lignin com-
ment hydrolyzate was added back to the pretreated sol- pounds [226] . Soluble phenol compounds, including van-
ids [212] or the whole slurry (i.e., pretreated solids and illin, syringaldehyde, trans-cinnamic acid and hydroxy-
hydrolyzate) was enzymatically hydrolyzed [213217] . benzoic acid, were reported to inhibit cellulose hydrolysis
These results suggest that compounds in the pretreat- in wet cake by endo- and exo-cellulases, and cellobiose
ment hydrolyzate have inhibitory effects on enzymatic hydrolysis by b-glucosidase [223] . In this study, vanillin
hydrolysis of cellulose. showed strongest inhibition on the mixture of Spezyme
Research revealed that some compounds in the pre- CP and Novozyme 188, while hydroxybenzoic acid had
treatment hydrolyzate, which usually contains soluble the greatest inhibition of these individual commercial
lignin, oligomeric sugars primarily from hemicellulose, enzymes. On the other hand, soluble lignin degradation
sugars, and lignin degradation products, had profound aldehydes (vanillin, syringaldehyde and 4-hydroxybezal-
inhibitory effects on cellulase and microbial activi- dehyde) or corresponding carboxylic acids were reported
ties [175,218221] . Kim etal. showed that effluent from to have minor inhibitory effects on cellulases [221].

future science group www.future-science.com 429


Review Yang, Dai, Ding & Wyman

Enzyme-related factors properties such as thermal, acid or alkaline tolerance


Enzymatic hydrolysis of cellulose, typically character- under unusual culture environments. Based on their
ized by an insoluble reactant (cellulosic substrate) and protein structures, the glycosyl hydrolases are further
a soluble catalyst (enzymes), is not only influenced by classified into four groups: multienzyme complex (cel-
structural features of the solid substrate but also by lulosome) systems, noncomplex cellulase systems, and
enzyme-related factors, such as enzyme source, prod- hemicellulase and ligninase systems. Since the cellu-
uct inhibition, thermal inactivation, activity balance losome system in the anaerobic thermophilic bacte-
for synergism, specific activity, nonspecific binding, rium Clostridiumthermocellum was first identified in
enzyme processibility and enzyme compatibility. Due the early 1980s by Bayer, Lamed and their colleagues
to the complexity of both the cellulose substrate and [246,247] , substantial progress has been realized in under-
the cellulase system, the mechanism of cellulose hydro- standing the protein complex, characteristics, genes
lysis is still not completely understood, although some governing formation of protein complexes, diversity
knowledge of enzyme structure, enzyme molecular and their interaction with plant cell walls. So far,
properties, fibers and cellulose ultrastructure has been the cellulosome system is found only in anaerobic
obtained through extensive study over the decades. microbes. Many elegant reviews have discussed these
Since many enzyme-related factors have been exten- complex cellulase systems [248253] . Cellulosomes have
sively reviewed [240243] , we will focus more on the several unique features: efficient nanomachines to
enzyme source, enzyme-specific interaction with cellu- deconstruct plant cell wall polysaccharides, molecular
losic substrates, synergistic effects of glycosyl hydrolases integration of cellulases and hemicellulases into cellu-
and strategies to improve enzyme effectiveness. losomal multienzyme complexes resulting from high-
affinity interaction between type I dockerin domains
Features of glycosyl hydrolases from of the modular enzymes and type I cohesion modules
differentmicrobes of a noncatalytic scaffoldin, and a scaffoldin-borne car-
In order to significantly improve the efficiency of bohydrate binding module (CBM) to attach to plant
enzymatic hydrolysis of cellulosic biomass and lower cell walls [252] .
costs, approaches have been taken to find more robust In contrast, noncomplex glycosyl hydrolases are
enzymes and advance the understanding of enzyme found in all microbes, even those with cellulosomal
interactions with cellulosic biomass. Different sets of systems. In this review, properties of the noncomplex
hydrolytic enzymes, such as cellulases, hemicellulases, glycosyl hydrolases and their interaction with cellu-
accessory enzymes to attack hemicellulose debranch- loses will be discussed in more detail. Noncomplex gly-
ing, phenolic acid esterases and ligninases for lignin cosyl hydrolases have been extensively studied in the
degradation/modification are required for complete several filamentous fungi. Among these, Trichoderma
deconstruction of the various components of lignocel- and Aspergillus strains are well developed for industrial
lulosic biomass [244] . However, it is not well known glycosyl hydrolase production with enzyme production
how the glycosyl hydrolases and their associated conditions extensively optimized. Generally, T.reesei
enzymes/proteins function together to breakdown lig- secreted at least two cellobiohydrolases (CBHI and
nocellulosic biomass. Diverse microorganisms, includ- CBHII), five to six endoglucanases (EGI, EGII, EGIII,
ing bacteria and fungi, can produce various glycosyl EGIV, EGV, and EGVI), b-glucosidase (BGL I and II),
hydrolases for biomass conversion and deconstruc- two xylanases and various accessory hemicellulases[254] .
tion. In nature, lignocellulosic biomass is completely The effectiveness of cellulase components acting on
deconstructed by a mixture of glycosyl hydrolases from insoluble substrates, and especially crystalline cellulose,
various microbes in specific communities, such as the is affected by the proportion of these components, with
hindgut of termite, the rumen of cows, various ligno- some ratios being particularly effective due to their
cellulosic biomass composts and the extreme environ- synergistic action [60,66] . Although the CBHI:EGI ratio
mental niches. Those anaerobic or aerobic microbial of commercial cellulase preparations from T.reesei is
communities may consist of only bacteria, only fungi, typically approximately 45:1, recent studies suggested
or bacteria and fungi together [245] . Selected microbial that the optimal enzyme ratio is affected by both pre-
strains that have been explored for various glycosyl treatment conditions and feedstock sources [255,256] .
hydrolase applications and their characteristics are Although several studies validate that CBHI and EGI
listed in Table1. share common sites on cellulose, CBHI has higher
T
hese microbes were isolated from different environ- binding capacity and affinity than EGI, and CBHII
mental niches and grouped into aerobic or anaerobic has separate binding sites than CBHI [56,257] . However,
bacteria or fungi on the basis of their growth condi- the influence of the molar ratio of these components
tions. The glycosyl hydrolases have evolved different on binding and/or of bound enzyme on synergism has

430 Biofuels (2011) 2(4) future science group


Enzymatic hydrolysis of cellulosic biomass Review

Table1. Selected bacterial and fungal strains for glycosyl hydrase production.
Name Enzymes types Ref.
Bacteria (aerobic)
Acidothermus cellulolyticus NC/HC T [375]
Bacillus sp. NC/HC M/AT [376]
Bacillus pumilus NC/HC M/AT [377,378]
Bacillus substilis NC/HC M/T [379]
Bacillus agaradhaerens JAM-KU023 NC/HC T/A [380]
Brevibacillus sp. strain JXL NC/HC T [381]
Cellulomonas flavigena NC/HC T/AT [382]
Cellulomonas fimi NC/HC M [273,383]
Geobacillus thermoleovorans NC/HC T/AT [384]
Paenibacillus campinasensis BL11 NC/HC T [385]
Paenibacillus strain B39 NC T [386]
Streptomyces sp. NC/HC M/T [387]
Thermoactinomyces sp. NC/HC T [388]
Thermomonospora curvata NC/HC T [389]
Thermomonospora fusca NC/HC T [390]
Bacteria (anaerobic)
Acetivibrio cellulolyticus Cellulosome/NC M [391]
Bacteroides cellulosolvens Cellulosome M [267]
Clostridium acetobutylicum Cellulosome M [392]
Clostrium cellulolyticum Cellulosome/NC M [393]
Clostrium cellulovorans Cellulosome/NC M [394]
Clostrium josui Cellulosome M [395]
Clostrium papyrosolvens Cellulosome M [396]
Clostrium thermocellum Cellulosome/NC T [246]
Ruminococcus albus Cellulosome M [397]
Ruminococcus flavefaciens Cellulosome M [398]
Filamentous fungi (aerobic)
Acremonium cellulolyticus NC/HC M [399]
Acrophialophora nainiana HC/HC M [400]
Aspergillus acculeatus NC/HC M [401,402]
Aspergillus fumigatus NC/HC M/T [403]
Aspergillus niger NC/HC M [404]
Aspergillus oryzae NC/HC M [405]
Fusarium solani NC/HC M [406]
Humicola grisea var. thermoidea NC/HC T [407]
Irpex lacteus NC/HC/LN M [408]
Penicillium funmiculosum NC/HC M [409]
Penicillium atrovenetum NC/HC T [410]
Penicillium citrinum NC/HC M [411]
Phanerochaete chrysosporium NC/HC/LN M [412]
Schizophyllum commune NC/HC M [413]
Sclerotium rolfsii NC/HC M [414,415]
Sporotrichum cellulophilum NC/HC T [501]
Talaromyces emersonii NC/HC T [416]
Thielavia terrestris NC/HC T [502]
Trichoderma koningii NC/HC M [406]
Trichoderma.reesei NC/HC M [406,417]
Trichoderma viride NC/HC M [418]
AT: Alkali tolerant; HC: Hemicellulase; LN: Ligninase; M: Mesophilic; NC: Noncomplexed cellulase; T: Thermophilic.

future science group www.future-science.com 431


Review Yang, Dai, Ding & Wyman

Table1. Selected bacterial and fungal strains for glycosyl hydrase production (cont.).
Name Enzymes types Ref.
Anaerobic fungi
Anaeromyces elegans NC/HC M [419]
Anaeromyces mucronatus NC/HC M [420]
Caecomyces CR4 NC/HC M [421]
Neocallimastic frontalis Cellulosome M [422]
Neocallimastic hurleyensis Cellulosome M [423]
Neocallimastic patriciarum Cellulosome M [424]
Orpinomyces joyonii Cellulosome M [425]
Orpinomyces PC-2 Cellulosome M [426,427]
Piromyces communis Cellulosome M [428]
Piromyces equi Cellulosome M [429]
Piromyces E2 Cellulosome M [430]
AT: Alkali tolerant; HC: Hemicellulase; LN: Ligninase; M: Mesophilic; NC: Noncomplexed cellulase; T: Thermophilic.

received little attention. Nidetzky etal. showed that exoglucanase with exoglucanase, endoglucanase with
competitive rather than synergistic binding is observed endoglucanase [266] , exoglucanase or endoglucanase
for cellulase components [258] . On the other hand, Jeoh with b-glucosidase [267,268] , catalytic domain with
and co-workers concluded that the presence of Cel5A CBM [269] or two catalytic domains [270] , cellulose-
(endocellulase) of Thermomonospora fusca increased enzyme-microbe synergism [271] and spatial synergism
binding of an exocellulase and an endocellulase [259,260] . for cellulase complexes (i.e., the cellulosome of C.ther-
The rationale for both strongly and weakly bind- mocellum) [241] . Such synergisms depend on cellulase
ing enzymes is still unclear. Typical cellulases contain sources or even substrate features. For example, syn-
carbohydrate-binding modules (CBMs) [261] that are ergism between the catalytic domain and CBM was
beneficial for enzyme efficiency by adhering to and reported for CenA of Cellulomonasfimi on cotton fibers
sometimes possibly disrupting the substrate. CBMs but was not observed on
bacterial microcrystalline cel-
from different enzymes and different taxonomic origins lulose (BMCC) [269] . Endoendo type synergism was
have been classified into families with similar amino only reported in fungal cellulases of Gloeophyllumsepia-
acid sequences and 3D structures. CBMs of T.reesei rium and Gloeophyllumtrabeum [266] . Cellcellulase
CBH1, CBHII and EGI have aromatic residues that cellulose synergism has been shown for some cellulo-
are critical for the binding of a CBM onto crystalline lytic microorganisms such as C.thermocellum that have
cellulose. Structural studies indicate that the spacing tightly cell-associated cellulase systems.
of the three aromatic residues coincides with the spac- Extensive study of synergisms in noncomplex cellu-
ing of every second glucose ring on a glucan chain. lases showed that they act in a synergistic or coopera-
Therefore, it has been postulated that the aromatic tive manner. The synergism among different cellulases
amino acids of the CBMs form van der Waals interac- depends on several factors including the nature of the
tions and aromatic ring polarization interactions with substrate, enzyme compositions and concentration, cel-
the pyranose rings exposed on the surface of cellulose lulase affinity for substrate, component stereospecific-
[262] . It was reported that the CBHI-CBM was capable ity and the enzyme to substrate ratio. The synergistic
of interacting with approximately ten cellobiose units interaction between cellulolytic components of T.reesei
(20 glucose units), and its catalytic core with approxi- was reported high on crystalline cellulose but decreased
mately 3654cellobiose units [263] . Cellulases proces- as substrate concentration increased [272] . Endoexo and
sivity, which involves CBM and catalytic domains of exoexo synergism was reported to be influenced by
cellulases, was studied in some recent reports [264,265] . the nature of the substrate such as DP [272] . Studies of
It is vital to understand how features of individual cel- the size distribution during hydrolysis of BMCC and
lulase components and their synergism dynamically acid-swollen cellulose also showed that the behavior
change as enzymatic hydrolysis of cellulose progresses. of endoglucanases and cellobiohydrolases (e.g., puri-
fied CenA, CenC and CbhA, and CbhB from C.fimi)
Synergistic enzyme effects on overall varied with different substrates [273] .
degradation processes Complex cellulosomes consist of many cellulases
Synergistic phenomena are widely observed in cellu- and hemicellulases that function synergistically to
lose hydrolysis, with many forms reported and pro- degrade celluloses. For example, the cellulosomal sub-
posed, including endoglucanase with exoglucanase, units of C.thermocellum include 12 endoglucanases,

432 Biofuels (2011) 2(4) future science group


Enzymatic hydrolysis of cellulosic biomass Review

two cellobiohydrases, two exoglucanases, six xylanases, with examples being cellulolytic Key term
one chitinase, one lichenase and one mannanase [265] . systems in insect hindguts [289291] , Metagenomics: Study of genetic
Besides cellulosomes, noncellulosomal cellulases have rumen microbial communities[292] material recovered directly from
been found in anaerobic microbes. However, atten- and various environmental com- environmental samples, where the
microorganisms are not easily cultured
tion to these noncellulosome cellulases is limited even posts. Those synergetic systems
in laboratory or simply studied in their
though many noncellulosomal cellulases have been can consist of just bacterial or fun- natural environment. In addition,
identified in cellulosomal microbes. Noncellulosomal gal communities or communities metagenomics allows researchers to
cellulases may act synergistically with cellulosomes for of bacteria with fungi, and may look at the genomes of all of the
microbes in an environment at once,
cellulose degradation [240] . include some contributions by the providing a meta view of the whole
One of the major challenges for cellulase research- host. Metagenomic and functional microbial community and the complex
ers is to elucidate the synergistic interactions between analysis of hindgut microbiota of interactions within it, such as
individual components [274] . In order to determine the a wood-feeding higher termites lignocellulosic biomass degradation
and conversion in certain
degree of synergism between cellulase components, it is showed the presence of a large, environmental composites.
imperative that each component be purified to homo- diverse sets of bacterial genes for
geneity, but aggregates and enzymeenzyme com- cellulose and xylan hydrolysis[293] .
plexes between cellobiohydrolases and endoglucanases Brulc etal. examined the gene-centric metagenomics of
are extremely difficult to break into their constituent the complex fiber-adherent bovine rumen microbiome
parts. Such complexity has been shown in cellulase and compared it with termite hindgut microbiota[294] .
from T.reesei [275] . On the other hand, some cellulase The study indicated fundamental differences in the
components, such as endoglucanases, are quite diffi- GH content that appeared to be diet driven for either
cult to purify to homogeneity. More detailed models bovine rumen (forages and legumes) or termite hind-
of cellulose degradation depend on firm knowledge gut (wood). Both studies suggested that Clostridium
of the kinetics and substrate specificities of individual cellulosomes are rarely present in both synergetic
cellulases. This area has been dramatically improved communities. In contrast, Clostridium species were
by introduction of a low molecular mass chromo- major players in microbial communities in cellulolytic
genic 4-methylumbelliferyl-b-glycosides and by the enrichment cultures from thermophilic compost [295] .
expression of cloned cellulases genes in heterologous Furthermore, fungal species found in enriched cultures
expression systems, such as Saccharomyces cerevisiae, were Piromyces species that produced cellulosomes as
which eliminate the problem of cross-contamination well. Interestingly, celY, encoding the noncellulosomal
of enzyme purified to homogeneity [276278] . We also glycosyl hydrolase family 48 along with its cellulo-
need to consider the potential impacts of glycosylation somal systems, previously observed in Clostridium
on functions of glycosyl hydrolases in the heterologous stercorarium was found in Clostridium straminisolvens
expression systems. Furthermore, because synergistic and Clostridium clariflavum [296,297] , and C.thermo-
effects between cellulases are influenced by the nature cellum [298] . Clostridiumstercoraium produced cellulo-
of the substrate, such as chemical composition, degree somes with a large number of hemicellulases but only
of crystallinity, DP and solubility, it is often challeng- two noncellulosomal cellulases, GH9 endoglucanase
ing to compare research results in the literature using CelZ and GH48 exoglucanase celY, that synergistically
different substrates. Therefore, establishing a set of degrade crystalline cellulose in biomass. This suggests
cellulose model substrates and widely employing such that a stand-alone cellulosome may not be sufficient
model substrates in the research community would cer- to degrade complex lignocellulosic biomass, and addi-
tainly facilitate comparisons and help in understand- tional glycosyl hydrolases may be required for complete
ing mechanisms and kinetics of cellulose hydrolysis by degradation. This hypothesis is supported by results
cellulases. In addition, results from such studies would from metagenome studies of cellulolytic enrichment
provide experimental evidence to validate computa- cultures from different composts [295,299,300] , in which
tional simulations of synergetic interactions among a series of noncellulosomal bacteria coexisted with cel-
enzymes/proteins and lignocellulosic biomass, a new lulosomal Clostridium species. Therefore, the study of
research area to determine molecular dynamics of those synergetic microbial communities may lead to potential
complex interactions [279284] . breakthroughs in lignocellulosic conversion. Besides
Besides synergism among cellulase components, the glycosyl hydrolases, associated proteins/enzymes and
synergetic effects of various glycosyl hydrolases (e.g., reagents may also play important roles in lignocellulosic
core cellulases and enzymes involved in hemicellulose biomass conversion. Thus far, tool-kits are insufficient
and lignin degradation) on lignocellulosic degradation to quantify the contribution of individual components
have been evaluated [192,250,268,285288] . Natural syner- to synergisms, further impeding improvements in
gism for lignocellulose degradation is very common, enzyme characteristics.

future science group www.future-science.com 433


Review Yang, Dai, Ding & Wyman

Advanced technologies for discovery, 27, 31, 32, 36, 37, 38, 43, 47, 51, 53, 54, 55, 61, 63, 67,
characterization & over-expression of 72, 75, 79, 81, 92, 105 and 114. The exocellobiohydro-
glycosylhydrolases lase I and II and EG I, II, III and IV belong to the GH
DNA sequence technology for whole genome sequence families GH5, 6, 7, 12 and 61, while the hemicellulose-
of biocatalystic microbes degrading enzymes belong to the GH families GH10,
The next-generation of DNA sequencing has the poten- 11, 26, 29, 43, 51, 53, 54, 62, 67, 74 and 95. Sizes
tial to dramatically accelerate lignocellulosic biomass of CAZyme families for the 23 fungal genomes with
conversion research by enabling inexpensive, routine complete sequences available, are summarized by class
and widespread comprehensive analyses of genomes, in Table2 .
transcriptiomes and interactomes. To date, it has been P.chrysosporium, a white rot fungus that efficiently
applied to determine the whole genome sequence degrades lignin, has the lowest number of genes encod-
related to lignocellulosic biomass production and ing GHs, glycosyltransferases and carbohydrate ester-
microbial systems for lignocellulosic biomass conver- ases among the filamentous fungi. T.reesei, an efficient
sion. Recent progress in whole genome sequencing of plant polysaccharide degrader and an important model
cellulosic biofuel crops has been reported in several of biomass degradation systems, has surprisingly few
publications[301305] . Such genome sequence informa- genes encoding GHs compared with other filamentous
tion provides the foundation for improvements in plant fungi. Thorough inspection of the T. reesei genome
oil and lignocellulosic biomass production in selected revealed only seven genes encoding well-known cel-
biofuel crops and especially regulation of complex lig- lulases (endoglucanases and cellobiohydrolases), 16
nocellulosic biomass formation. Genome sequences also hemicellulase genes and five genes for pectin breakdown
provide a foundation to examine the potential of heter- enzymes [306] , the smallest set of genes for glycosyl
ologous expression of microbial glycosyl hydrolases in hydrolases among plant cell wall degrading fungi.
biofuel crops, as discussed in a latersection. Rapid improvements in DNA sequencing technol-
As noted above, both eukaryotic and prokaryotic ogy also provide powerful tools for metagenomics.
microbes can produce glycosyl hydrolases. The eukary- Metagenomics, a relatively new field of genetic research,
otic microbes mainly consist of various fungal species, enables studies of organisms that are not easily cul-
with the most common being filamentous fungi. The tured in a laboratory as well as studies of organisms
genome sequence of T. reesei, which is widely used in their natural environment. Functional metagenom-
for commercial production of cellulases and hemi- ics has been applied to examine cellulolytic systems in
cellulases, was recently determined [306] . In addition, insects[289291] , rumen microbial communities [292,293]
genome sequences have been determined for several and various environmental composts (e.g., a switch-
other filamentous fungi, such as Phanerochaete chryso- grass-adapted compost community) [317] , thermophilic
sporium [307] , Aspergillusniger [308] , Aspergillus
fumiga- biocompost [295] and agricultural soils [318] . All com-
tus[309] , Aspergillusnidulans [310] , Aspergillusoryzae[311] , munities contain different family sizes but all have the
Fusariumgraminearum [312] , Magnaporthegrisea [313] , families GH1, 2, 3, 5, 8, 10, 28, 35, 38, 42, 43 and 53.
Neurospora crassa [314] , Penicillium chrysogenum [315] With the significant interest in identification of novel
and Ustilago maydis [316] . Such genome sequence data enzymes for lignocellulosic biomass degradation and con-
allow examination of carbohydrate-active enzymes version, functional metagenomics studies are being rap-
(CAZymes) categorized into different classes and fami- idly extended to expand the CAZyme families [319323] .
lies that include GHs, glycosyltransferases, polysaccha- Although genes encoding the diversity of CAZyme fami-
ride lyases, carbohydrate esterases and carbohydrate- lies have been identified via whole genome sequencing
binding modules [601] . Those enzymes cleave, build and metagenomics, detection of an open reading frame
and rearrange oligo- and poly-saccharides and play a alone does not warrant actual production of protein, nor
central role in fungal metabolisms, which are crucial for does it readily indicate the spatial and temporal expres-
biomass degradation. Each enzyme class has different sion of the gene in the biosystem. Therefore, new tech-
families. Gylcosyl hydrolases, such as cellulolytic and nologies in protein identification and characterization
hemicellulose-degrading enzymes, are classified with will be crucial for improvements in enzyme production,
GHs and key enzymes for lignocellulosic biomass deg- pathway optimization and biofuels crops.
radation. So far, 118 families of GHs have been identi-
fied in all biological systems, a number that will grow as Mass spectrometry technology for secretomes
more genes are sequenced. More than 60 of them have & subcellular organelle proteomics of
been found in the filamentous fungi mentioned above. biocatalysticmicrobes
The 36 families of GHs found in all those filamentous Mass spectrometry is a promising tool to assess rela-
fungi are GH1, 2, 3, 5, 6, 7, 10, 11, 13, 15, 16, 17, 18, tively abundant proteins, provided that the biosystem

434 Biofuels (2011) 2(4) future science group


Enzymatic hydrolysis of cellulosic biomass Review

Table2. Sizes of CAZyme families by class identified in genome databases of 23 fungi.


Name Glycoside Glycosyl- Polysacch- Carbohydrate Carbohydrate-
hydrolase transferase aridelyase esterase binding module
Filamentous fungi
Aspergillus fumigatus 263 103 13 29 55
Aspergillus niger 248 114 8 25 38
Aspergillus oryzae 303 119 23 30 34
Aspergillus nidulans 252 90 21 33 41
Fusarium graminearum 243 110 20 42 61
Magnaporthe grisea 232 94 5 47 65
Neurospora crassa 174 78 4 22 42
Penicillium chrysogenum 219 102 9 22 49
Phanerochaete chrysosporium 166 57 14 14 N/A
Podospora anserine 230 89 7 49 97
Trichoderma reesei 200 103 3 16 36
Single cell fungi
Candida albicans 58 69 0 3 4
Candida dubliniensis 49 69 0 3 10
Candida glabrata 39 79 0 3 12
Cryptococcus neoformans 77 66 3 7 12
Saccharomyces cerevisiae 50 68 0 3 12
Schizosaccharomyces pombe 51 61 0 5 8
Debaryomyces hansenii 74 74 0 3 11
Eremothecium gossypii 44 57 0 2 9
Kluyveromyces lactis 46 62 0 2 11
Lachancea thermotolerans 45 59 1 2 11
Pichia pastoris 39 60 1 2 16
Pichia stipitis 53 67 0 4 7

or community is not too complex and has been sam- fungus Phanerochaete carnosa grown on spruce and
pled deeply enough at the molecular level. In the last microcrystalline cellulose [327] and the cellulosome
decade, mass spectrometry for proteomics has pro- composition of C.thermocellum grown on the diluted-
gressed radically and is now on par with most genomic acid pretreated switchgrass [328] . Proteins identified in
technologies in high throughput and comprehensive- the extracellular filtrates of P.carnosa included GH2,
ness [324,325] . So far, only a few proteomic studies have 3, 5, 6, 7, 10, 11, 13, 15, 16, 18, 31, 35, 43, 47, 55, 61,
directly examined proteins and enzymes in the bio- 92, glucuronoyl esterase (CE1), pectin esterase (CE8),
systems or communities involved in lignocellulosic polysaccharide lyase (PL14) and proteins corresponding
biomass degradation under realistic environmental to glyoxal oxidases, monooxygenase P450, peroxidases
conditions. However, proteomics have been applied and multicopper oxidase. Results from C.thermocellum
to examine production and dynamics of complex and suggested that a coordinated substrate-specific regula-
noncomplex glycosyl hydrolases in different microbes tion of the cellulosomal subunit composition occurred
rapidly. Recently, proteome analysis of fungal and to better suit the organisms need for growth under spe-
bacterial involvement in leaf litter decomposition was cific carbon source conditions. Furthermore, the devel-
conducted for a co-culture of two model organisms, opment of methods for effective sample preparation for
Pectobacterium carotovorum (Gram-negative bacte- the extracellular proteome of the extreme thermophile
rium) and A.nidulans (filamentous fungus), in culture Caldicellulosiruptorsaccharolytucus suggested that two
with beech litter [326] . Proteome analysis revealed that levels of sample purification were necessary to effec-
most of the extracellular biodegradative enzymes (pro- tively desalt the sample and provide sufficient protein
teases, cellulases and pectinases) in the culture were identification [329] . More recently, mass spectrometry
secreted by the fungus, while the bacterium produced proteomics in combination with transcriptomics and
only low levels of pectinases. Proteomic studies were genomics provided a powerful system to examine regu-
also employed to examine lignocelluloses-degrading lation of glycosyl hydrolase production under different
enzymes secreted by the white-rot softwood degrading culture conditions [330332] .

future science group www.future-science.com 435


Review Yang, Dai, Ding & Wyman

All glycosyl hydrolases are secreted into the external physical (UV light or x-ray) induction [360363] , have
environment for lignocellulosic biomass degradation. been applied, but w
ith advances in gene transfer tech-
Most of those glycosyl hydrolase proteins undergo nology, the potential regulation of glycosyl hydrolase
post-translation modification with glycosylation production can be determined by gene deletion or
playing important roles in enzyme function, stability over-expression. For example, deletion of the glucose
and interaction with lignocellulose. During the last repressor gene Cre1 from T.reesei improved cellulase
decade, mass spectrometry methods have also been production [364,365] and similar responses have been
developed to analyze glycoproteins in different organ- observed for deletion of the ACE1 gene, which also
isms [333338] . The methods were mainly employed to affects xylanase expression in T.reesei [366] . Besides the
analyze N- and O-glycosylation sites and glycan struc- negative regulation of transcriptional factors ACEI and
tures in several glycosyl hydrolases (endoglucanase I, II CREI, expression of cellulases and xylanases was also
and exoglucanase I, II), mostly from T.reesei[339342] . positively regulated by transcriptional factors XYR1,
Some evidence indicated that glycosylation also occurs ACE2 and HAP2/3/5. Recent completion of the
in bacterial cellulosomes [343,344] . More and more genome sequences of several filamentous fungal species
studies indicate that glycosylation plays an impor- including two glycosyl hydrolase-producing strains
tant role in glycosyl hydrolase function and biomass of T.reesei [306] and A.niger [308] , and broad studies
conversion and is influenced by different organisms of glycosyl hydrolase production in those hosts can
and culture conditions [345347] . However, thus far, no enhance strain improvements for glycosyl hydrolases
studies have globally examined glycosylation in the production by genetic engineering.
large GH families involved in lignocellulosic biomass In heterologous systems, over-expression of glycosyl
degradation. Complete genome sequences of several hydrolases has been evaluated in different fermenta-
glycosyl hyrodrases producing filamentous fungi will tive microbes such as bacteria, yeast and filamentous
lay an important foundation to globally study the fungi [367369] , and in higher plants with or without
glycosylation of glycosyl hydrolases and its effects on tissue-specific targeting [370372] . Several recent pub-
lignocellulosicdegradation. lications have reviewed detailed strategies for heter-
ologous expression of glycosyl hydrolases in higher
Gene transfer technologies for improvement of plants[373,374] . The ultimate goal is to realize economic
glycosyl hydrolase production in both homologous & production of glycosylhydrolases.
heterologous organisms
Genetic engineering technology has been drastically Future perspective
improved for both microbial systems and higher plants, For lignocellulosics, cellulase adsorption and efficacy
especially with the development of Agrobacterium- cannot be simply related to a few substrate features.
mediated transformation of higher plants 30 years Thus, hemicellulose and lignin removal, deacety-
ago [348] . This method has been widely applied to lation, decrystallization, accessible surface area and
functional genomics in higher plants for large-scale the nature of different cellulase components could all
targeted and random mutagenesis to provide one of affect access of enzymes to substrate and their effec-
the most effective strategies to understanding gene tiveness once they attach. Yet, some of these factors are
functions and improve productivity and quality of likely more influential than others, and a concerted
various lignocellulosic biomasses [349354] . Later, this effort is needed to understand fundamental physical
method was adapted to fungal transformations and and chemical features of lignocellulosic biomass that
as a tool for functional genomics in fungi [355,356] . impede glycosyl hydrolase access to carbohydrates
Concurrently, other transformation methods have and slow the rate of biomass deconstruction into
been developed and adapted to both plant and fungal fermentable sugars. Understanding factors that con-
transformations, such as chemical-mediated protoplast trol interactions between lignocellulosic biomass and
transformation [357] and biolistic nuclear transforma- glycosyl hydrolases as well as inhibitory compounds
tion [358,359] . These effective gene transfer systems that are either natural biomass compounds released
allow us to examine the potential for improvement during deconstruction or formed by degradation of
of glycosyl hydrolase production in both homologous sugars and other biomass constituents in up-stream
and heterologous systems. processing would be invaluable in identifying better
Research on homologous systems is needed to opti- pretreatments and enzyme systems to lower the cost
mize glycosyl hydrolase production in known produc- of biomass conversion to meet industrial needs. For
tion hosts, where the endogenous glycosyl hydrolases example, understanding how pretreated cellulosic bio-
have been identified and characterized. Traditionally, mass reactivity changes with conversion and structure
various mutagenesis methods, such as chemical or and the effects of enzymesubstrate interactions on

436 Biofuels (2011) 2(4) future science group


Enzymatic hydrolysis of cellulosic biomass Review

sugar release could suggest advanced technologies with in both homologous and heterologous systems. Further
lower costs. Improved analytical methods are needed advanced biotechnologies are crucial for discovery and
to better characterize biomass composition and struc- characterization of new enzymes and improvement of
ture and interactions between biomass, enzymes and the enzyme characteristics and production in homolo-
other compounds, and to follow the details of biomass gous or heterologous systems and ultimately lead to
deconstruction. Results from such research can guide low-cost conversion of lignocellulosic biomasses into
further optimization of glycosyl hydrolases production fuels and chemicals.

Executive summary
Advancements in pretreatment and cellulase technologies have contributed significantly to historical cost reductions for biological
conversion of cellulosic biomass to ethanol and other products.
A key to lower cellulosic ethanol cost is to reduce enzyme usage and costs.
Emerging biotechnology tools offer great promise in discovery of new enzyme sources with desirable features.
Improving the understanding of the structure and function of both lignocellulosic materials and their degrading enzymes will be
invaluable in determining the roles of biomass pretreatment, hydrolysis and enzymes in influencing cellulose conversion and in targeting
appropriate strategies to enhance rates and yields.
Substrate-related factors that affect enzymatic hydrolysis of cellulosics
Characteristics of cellulose:
Advanced imaging techniques provide new insights into plant cell wall structure and changes during hydrolysis, as well as new
understanding of enzymesubstrate and enzymeenzyme interactions;
Cellulose characteristics (e.g., size, structure, crystallinity, degree of polymerization and accessible surface area) were shown to affect
cellulase adsorption, synergism and processivity;
The mechanism of the rapid decline in hydrolysis rate with cellulose conversion during enzymatic hydrolysis remains unclear, and
innovative research is needed to shed new light on what slows cellulase action.
Derived insoluble matter distribution effects
Deacetylation was reported to improve cellulose digestibility.
Hemicellulose (e.g., xylan) and lignin removal appeared to improve cellulose digestibility, but some pretreatment methods are effective
without removing either (e.g., ammonia fiber expansion).
Lignin modification rather than complete removal by thermal pretreatment could result in highly accessible cellulose while the remaining
solid lignin could cause nonspecific binding of cellulases.
Hemicellulose removal is favorable in regards to reducing inhibitory effects of hemicellulose sugars (e.g., xylan mono/oligomers)
oncellulases.
Derived soluble matter distribution effects
The inhibitory effects of soluble compounds released in thermochemical pretreatments are not well understood.
Some xylan and lignin derivatives, especially xylan oligomers, were reported to show different degrees of inhibition of enzymatic
hydrolysis of cellulose.
Identification of soluble compounds derived from thermal pretreatment that account for the greatest inhibition effects and strategies to
reduce or eliminate their deleterious effects needs further investigation.
Enzyme-related factors that affect enzymatic hydrolysis of cellulosics
Features of glycosyl hydrolases from different microbes:
Lignocellulosic biomass is naturally deconstructed by glycosyl hydrolase mixtures from various microbes in specific communities.
Glycosyl hydrolases from different microbes have different functional features that potentially can become new sources of
effectiveenzymes.
Enzyme synergy effects on overall degradation process
Many forms of enzyme synergism have been observed among cellulases.
Enzyme synergism depends on enzyme sources and substrate features.
Producing purified enzymes in heterologous systems and establishing standard substrates would improve the understanding of
enzymesynergism.
Study of synergetic microbial communities may lead to potential breakthroughs in lignocellulosic biomass conversion.
Advanced technologies for discovery, characterization, & over-expression of glycosyl hydrolases
DNA sequence technology for whole genome sequencing of biomass feedstocks, biocatalystic microbes, metagenomics, and functional
genomics and metagenomics, can build a foundation for advances in biomass production, enzyme categorization and applications.
Mass spectrometry technology has been applied to examine secretomes and subcellular organelle proteomics of biocatalystic microbes
and protein glycosylation.
Gene transfer technologies improve glycosyl hydrolase production in both homologous and heterologous organisms.
Cellulase engineering through directed evolution, rational design, post-translational modifications, and their combination may greatly
increase cellulase performance and dramatically decrease enzyme use.

future science group www.future-science.com 437


Review Yang, Dai, Ding & Wyman

Financial & competing interests 2 Reese ET, Mandels M. Degradation of saccharification-and-fermentation-based


cellulose and its derivatives. Enzymic bioethanol process. Technical and economic
disclosure
degradation. High Polymers 5(5), 10791094 evaluation. Appl. Biochem. Biotechnol.,
Bin Yangs research is sponsored by the Center for
(1971). 121124, 485499 (2005).
Bioproducts and Bioenergy and Department of
3 Wright JD. Ethanol from biomass by 14 Himmel ME, Ruth MF, Wyman CE.
Biological Systems Engineering at Washington State
enzymatic-hydrolysis. Chem. Eng. Prog. 84(8), Cellulase for commodity products from
University. Ziyu Dais research was funded by the 6274 (1988). cellulosic biomass. Curr. Opin. Biotechnol,
Biomass Program of the US Department of Energy. 10(4), 358364 (1999).
4 Wright JD. Ethanol from lignocellulose: an
Pacific Northwest National Laboratory is operated overview. Energ. Prog. 8(2), 7178 (1988). n
Very comprehensive review, covering the
by Battelle Memorial Institute, Pacific Northwest fundamental knowledge to key cost drivers
5 Montencourt BS, Kelleher TJ, Eveleigh DE.
Division, for US Department of Energy under con- Biochemical nature of cellulases from mutants for celluase production issues.
tract No: DE-AC05-76RL01830. Shi-You Ding is of Trichoderma reesei. Biotechnol. Bioeng. Symp. 15 Himmel ME. biomass recalcitrance:
supported by the US Department of Energy, the 10, 1526 (1980). engineering plants and enzymes for biofuels
Office of Science, Office of Biological and 6 Spindler DD, Wyman CE, Grohmann K, production. Science 316(5827), 982 (2007).
Environmental Research through the BioEnergy Mohagheghi A. Simultaneous saccharification 16 Yang B, Wyman CE. BSA treatment to
Science Center, a DOE Bioenergy Research Center. and fermentation of pretreated wheat straw to enhance enzymatic hydrolysis of cellulose in
Charles Wyman is supported by the Ford Motor ethanol with selected yeast strains and lignin containing substrates. Biotechnol.
b-glucosidase supplementation. Appl. Biochem. Bioeng. 94(4), 611617 (2006).
Company Chair in Environmental Engineering at
Biotechnol. 21, 529540 (1988).
the Center for Environmental Research and 17 Pan X, Kadla JF, Ehara K, Gilkes N, Saddler
7 American Chemical Society. Novozymes, JN. Organosolv ethanol lignin from hybrid
Technology of the Bourns College of Engineering at
DOE claim cost cut. Chemical and Engineering poplar as a radical scavenger: relationship
UCR; the BioEnergy Science Center, a DOE
News, 10 (2005). between lignin structure, extraction
Bioenergy Research Center, supported by the
8 American Institute of Chemical Engineering. conditions, and antioxidant activity. J.Agric.
Biological and Environmental Research Office in the Food Chem. 54(16), 58065813 (2006).
Genencor makes strides in the conversion of
DOE Office of Science, contract DE-AC05- biomass to ethanol. Chem. Eng. Prog. 15 18 Chen F, Dixon R A. Lignin modification
00OR22725, and subcontract 4000063616; the (2004). improves fermentable sugar yields for biofuel
University of California at Riverside; the USDA 9 Wooley R, Ruth M, Glassner D, production. Nat. Biotechnol. 25(7), 759761
National Research Initiative Competitive Grants SheehanJ.Process design and costing of (2007).
Program, contract 2008-35504-04596; the Defense bioethanol technology: a tool for determining 19 Chen F, Reddy MSS, Temple S, Jackson L,
Advanced Research Projects Agency through subcon- the status and direction of research and Shadle G, Dixon R A. Multi-site genetic
tract SUB-226-UCR1 from Logos Technologies sup- development. Biotechnol. Prog. 15, 794803 modulation of monolignol biosynthesis
(1999). suggests new routes for formation of syringyl
ported by contract HR0011-09-C-0075; the
10 Wooley R, Ruth M, Sheehan J, Ibsen K, lignin and wall-bound ferulic acid in alfalfa
Defense Advanced Research Projects Agency and
Majdeski H, Galvez A. Lignocellulosic Biomass (Medicago sativa L.). Plant J. 48(1), 113124
Army Research Lab through Defense Science Office
to Ethanol Process Design and Economics (2006).
Cooperative Agreement W911NF-09-2-0010 and
Utilizing Co-Current Dilute Acid Prehydrolysis 20 Argyropoulos DS, Liu Y. The role and fate
subcontract 09-005334-000 to the University of and Enzymatic Hydrolysis: Current and of lignins condensed structures during
Massachusetts, Amherst; and the DOE Office of the Futuristic Scenarios. National Renewable oxygen delignification. J.Pulp Paper Sci.
Biomass Program, contract DE-FG36-07GO17102. Energy Laboratory, Golden, CO, USA (1999). 26(3), 107113 (2000).
Dr Wyman is also a co-founder, SAB chair, and n
Important information on cellulase 21 Wyman CE, Dale BE, Elander RT,
Chief Development Officer of Mascoma Corporation. production costs for cellulosic ethanol. Holtzapple M, Ladisch MR, Lee YY.
The authors have no other relevant affiliations or 11 Aden A, Ruth M, Ibsen K etal. Lignocellulosic Coordinated development of leading
financial involvement with any organization or biomass to ethanol process design and biomass pretreatment technologies.
entity with a financial interest in or financial conflict economics utilizing co-current dilute acid Bioresource Technol. 96(18), 19591966
prehydrolysis and enzymatic hydrolysis for (2005).
with the subject matter or materials discussed in the
manuscript apart from those disclosed. corn stover. In: National Renewable Energy n
Very comprehensive review covering
Laboratory Technical Report # NREL/ leading pretreatment technologies.
No writing assistance was utilized in the produc-
TP-51032438. National Renewable Energy
tion of this manuscript. 22 Morris VJ, Kirby AR, Gunning AP. Atomic
Laboratory, Golden, CO, USA (2002)
Force Microscopy for Biologists. Imperial
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