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Bioresource Technology 135 (2013) 513522

Contents lists available at SciVerse ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

A review of enzymes and microbes for lignocellulosic biorenery


and the possibility of their application to consolidated bioprocessing technology
Tomohisa Hasunuma a, Fumiyoshi Okazaki a, Naoko Okai a, Kiyotaka Y. Hara a, Jun Ishii a, Akihiko Kondo b,
a
b

Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan

h i g h l i g h t s
" Biorenery approaches produce fuels and chemicals through biomass conversion.
" Consolidated bioprocessing of lignocellulose is desired for effective biorenery.
" This review focuses on the development of microbes for consolidated bioprocessing.
" The production of bio-based chemicals and advances fuels is emphasized.

a r t i c l e

i n f o

Article history:
Available online 23 October 2012
Keywords:
Biomass
Biorenery
Cellulase
Consolidated bioprocessing
Lignocellulose

a b s t r a c t
The biorenery manufacturing process for producing chemicals and liquid fuels from biomass is a promising approach for securing energy and resources. To establish cost-effective fermentation of lignocellulosic biomass, the consolidation of sacccharication and fermentation processes is a desirable strategy,
but requires the development of microorganisms capable of cellulose/hemicellulose hydrolysis and target
chemical production. Such an endeavor requires a large number of prerequisites to be realized, including
engineering microbial strains with high cellulolytic activity, high product yield, productivities, and titers,
ability to use many carbon sources, and resistance to toxic compounds released during the pretreatment
of lignocellulosic biomass. Researchers have focused on either engineering naturally cellulolytic microorganisms to improve product-related properties or modifying non-cellulolytic organisms with high product yields to become cellulolytic. This article reviews recent advances in the development of
microorganisms for the production of renewable chemicals and advanced biofuels, as well as ethanol,
from lignocellulosic materials through consolidated bioprocessing.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
To ensure a reliable future source of energy and raw materials,
the utilization of sustainable biomass has considerable advantages
over petroleum-based energy sources. A biorenery is a concept
that integrates biomass conversion processes and equipment to
produce fuels, power, and chemicals from biomass, instead of conventional oil renery processes. Lignocellulosic biomass obtained
as agriculture byproducts and industrial residues is an abundant,
inexpensive, and renewable source of sugars, and is a desirable
feedstock for the sustainable production of liquid fuels and chemical products through the biorenery processes (Menon and Rao,
2012). After pretreatment of the biomass, cellulosic and hemicellulosic materials are enzymatically decomposed into simple sugars
that can be metabolized by microorganisms and converted to
Corresponding author. Tel./fax: +81 78 803 6196.
E-mail address: akondo@kobe-u.ac.jp (A. Kondo).
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.10.047

desired chemical products, including alcohols, fatty acids, organic


acids and amino acids in microbial fermentation. Bio-ethanol is
currently one of the most promising alternatives to conventional
petroleum-based transport fuels. However, the recalcitrant structure of lignocellulosic biomass makes the process involved in its
bioconversion more complicated than that of starchy and sugary
materials, because current sugar-platform technologies require
enzymatic conversion of the substrate to fermentable sugars prior
to initiating microbial fermentation (Mussatto et al., 2010).
Lignocellulosic materials are mainly composed of cellulose,
hemicellulose, and lignin. In plant biomass, cellulose forms highly
crystalline microbrils consisting of homopolymers of b-1,4-linked
glucose units embedded in a hemicellulose, pectin and lignin matrix, a conrmation that makes the structure resistant to saccharication by hydrolytic enzymes. In general, the chemical and
physicochemical pretreatment of lignocellulose causes cellulose
to swell, thereby increasing its accessibility to saccharication enzymes (Chandel et al., 2012; Hong et al., 2012; Menon and Rao,

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2012). However, the hydrolysis of cellulose remains a major limiting factor for the efcient utilization of lignocellulosic materials
(Matano et al., 2012; Olson et al., 2011).
To release soluble sugars from cellulose, the activities of multiple enzymes, including endoglucanase, exoglucanase, and b-glucosidase, are required (Chandel et al., 2012). As cellulase reactions are
inhibited by their intermediary and nal products, such as cellooligosaccharides and glucose, microbial fermentation processes that
combine enzymatic hydrolysis with sugar consumption are preferential for the alleviation of cellulase activity inhibition (van Zyl
et al., 2007). However, the large difference in optimum temperatures between saccharication and fermentation during simultaneous saccharication and fermentation (SSF) is a drawback of
bio-ethanol production (Hasunuma and Kondo, 2012). To overcome this limitation, large amounts of saccharication enzymes
by fungi and bacteria are required, which severely impacts the cost
effectiveness of biorenery from lignocellulosic materials.
The recent development of microorganisms capable of efcient
cellulose hydrolysis and fermentation represents a signicant step
in reducing the requirement for enzyme addition into the SSF processes (Matano et al., 2012). Such consolidation of enzyme production, saccharication, and fermentation into a single process is
increasingly recognized as having potential for the low-cost production of biofuels and bio-based chemicals, as the high costs of
capital investment, raw materials, and equipment associated with
microbial enzyme production can be avoided (Menon and Rao,
2012; Mussatto et al., 2010; Olson et al., 2011). To develop such
organisms, researchers have focused on either engineering naturally cellulolytic microorganisms to improve product-related properties or modifying non-cellulolytic organisms with high product
yields to become cellulolytic (Hasunuma and Kondo, 2011; Olson
et al., 2011), as there is still no ideal organism to use in one-step
biomass conversion.
Although early efforts toward achieving efcient biorenery
processes from plant biomass have typically focused on ethanol,
recent advances in microbial metabolic engineering have enabled
to perform a challenge for producing renewable chemicals and advanced bio-fuels that are compatible with existing engines and fuel
distribution infrastructure (Zhang et al., 2011a). This review will
focus on a discussion of microbial strains for use in consolidated
bioprocessing (CBP) technologies. In particular, approaches for
the conversion of lignocellulosic materials into bio-based chemicals and fuels, as well as bio-ethanol, through microbial fermentation are emphasized.

2. Hydrolysis of cellulosic materials by cellulases


2.1. Cellulase enzymes
The hydrolysis of insoluble cellulose by microorganisms requires the production of either free or cell-associated extracellular
cellulases. The biochemical analyses of cellulase systems from aerobic and anaerobic bacteria and fungi performed during the past
two decades have revealed that multiple enzymatic activities are
needed to hydrolyze cellulose into soluble sugar monomers that
can be metabolized by microorganisms (van Zyl et al., 2007; Zhang
and Lynd, 2004). At least three major types of enzymes are
required for hydrolyzing cellulose: (i) endoglucanase (EG) or 1,4b-D-glucan-4-glucanohydrolase; (ii) exoglucanase, including 1,4b-D-glucan glucanohydrolase (also known as cellodextrinase) and
1,4-b-D-glucan cellobiohydrolase (cellobiohydrolase; CBH); and
(iii) b-glucosidase (BGL) or b-glucoside glucohydrolase.
EG randomly hydrolyzes the b-glycoside linkages of internal
amorphous regions in cellulose to produce oligosaccharides of various degrees of polymerization and generate new chain ends. Exo-

glucanases hydrolyze cellulose in a processive manner from the


reducing or non-reducing ends of cellulose chains to generate
either glucose or cellobiose as major products. Exoglucanases can
also hydrolyze microcrystalline cellulose, conceivably by peeling
cellulose chains from the microcrystalline structure. BGL cleaves
soluble cellodextrins and cellobiose into glucose. The correct combination of the activities and production level of each cellulase enzyme is critical for efcient lignocellulosic biomass utilization
(Chandel et al., 2012).
2.2. Non-complexed cellulase systems
Fungi belonging to the genus Trichoderma have received intensive attention due to their high level production of secreted cellulases. In particular, the cellulase system of Trichoderma reesei has
been the focus of research for more than 50 years. The high-level
production of complex mixture of cellulases produced by Trichoderma species, which often exceeds 100 g L 1, is the current gold
standard for commercialized cellulase production (Cherry and
Fidantsef, 2003). T. reesei produces at least two exoglucanases
(CBHI and CBHII), ve endoglucanases (EGI, EGII, EGIII, EGIV, and
EGV), and two b-glucosidases (BGLI and BGLII) (Zhang and Lynd,
2004). The cellulase activity of T. reesei is predominantly attributed
to CBHI, CBHII, and EGII (Nidetzky and Claeyssens, 1994). Although
T. reesei secrets BGL, the production level of this enzyme is significantly lower than that found in other fungi, such as Aspergillus
species. The expression levels of the T. reesei BGL gene are presumably sufcient to permit cell growth on cellulose, but not to allow
the industrial use of BGL as a cellulase reagent. Furthermore, BGL
from T. reesei displays product (glucose) inhibition (van Zyl et al.,
2007), whereas those of Aspergillus species are more glucose tolerant. Sakamoto et al. (1985) isolated BGL1 and BGL2 from Aspergillus
aculeatus and found that the two enzymes were potently active not
only on soluble cellooligosaccharide substrates, from cellobiose to
cellohexaose, but also on insoluble cellooligosaccharides with an
average degree of polymerization of 20. Nakazawa et al. (2011)
constructed a recombinant T. reesei strain expressing A. aculeatus
BGL1. The supernatant from the recombinant strain grown on Avicel and xylan efciently hydrolyzed NaOH-pretreated rice straw at
a low enzyme dose. Based on these ndings, cellulase cocktails
from T. reesei engineered to express BGL from the genus Aspergillus
appear most suitable for cellulose saccharication. Acremonium
cellulolyticus, which efciently produces both cellulase and
b-glucosidase in addition to carboxymethyl cellulose-hydrolyzing
enzyme and small amounts of xylanase, b-1,3-glucanase and amylase because of its high accessibility to cellulose, is an alternative
cellulase producer (Park et al., 2011). Park et al. (2011) noted that
the level of cellulase produced from pretreated waste milk pack in
cultures of A. cellulolyticus is similar to those obtained with pure
cellulose.
2.3. Complexed cellulase systems (cellulosome)
Cellulosome systems are multi-enzymatic complexes produced
by anaerobic bacteria that efciently degrade plant biomass (Fontes and Gilbert, 2010). In these systems, different types of cellulose-degrading enzymes are assembled on the structural
scaffoldin subunits through strong non-covalent proteinprotein
interactions between the docking modules (dockerin) and complementary modules (cohesins). In addition, scaffoldin contains a carbohydrate-binding module, which binds the entire enzymatic
complex to the cellulose surface. As the scaffoldin subunits are
covalently bound to the cell walls of microbes by their anchoring
proteins, microbes expressing cellulosomes can utilize cellulose
as a source of carbon and energy. The efcient synergistic degradation of plant biomass results from the combination of targeting the

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T. Hasunuma et al. / Bioresource Technology 135 (2013) 513522

Francisco et al. (1993) used an immobilization technique to prepare whole-cell biocatalysts of an E. coli strain that specically
binds to cellulosic materials. Genes of the Cellulomonas mi exoglucanase, Cex, and its cellulose-binding domain, CBDCex, were expressed in E. coli as fusion proteins with Lpp-OmpA, consisting of
the major outer membrane lipoprotein, Lpp, and amino acids
46159 of the outer membrane protein, OmpA, resulting in the
localization of the both Cex and CBDCex on the E. coli cell surface.
Cell-surface anchored CBDCex tightly bound to cellulose microbrils, while Cex successfully hydrolyzed the chromogenic substrate
p-nitrophenyl-b-D-cellobioside. Recently, Thermobida fusca BGL
was displayed on the E. coli cell surface using the Blc anchor
protein (Tanaka et al., 2011). The recombinant strain demonstrated
direct growth on cellobiose and cellooligosacchride. As the binding
of recombinant E. coli to cellulosic materials enables the recovery
of cells by separating the biomass from the fermentation supernatant, cellulase-immobilized cell biocatalysts have clear advantages
for CBP.
To achieve the CBP of lignocellulosic materials, genetically engineered E. coli strains were used for the production of ethanol from
sugar beet pulp (SBP) composed of pectin, cellulose, and hemicellulose. Edwards et al. (2011) successfully produced ethanol from
SBP by conferring cellobiose-fermenting and pectin-degrading
abilities to recombinant E. coli strain KO11, which stably expresses
chromosomally integrated pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) genes from Zymomonas mobilis. To allow strain KO11 to utilize both cellobiose and pectin, which
contains partially methylesteried 1,4-linked a-D-galacturonic
acids, as carbon sources, the Klebsiella oxytoca cellobiose phosphotransferase operon (casAB), encoding a cellobiose permease and
phospho-b-glucosidase, Erwinia chrysanthemi pectate lyase (PelE)
gene, E. chrysanthemi out operon, which is involved in the Secdependent pathway for the secretion of PelE, and E. chrysanthemi
ogl gene, encoding an oligogalacturonide lyase, were integrated
into the KO11 genome. Following the addition of commercially
available cellulase and pectin methylesterase, the engineered
E. coli strain showed improved ethanol production from SBP due
to the effective hydrolysis of the methylester moiety of pectin by
pectin methylesterase (Edwards et al., 2011). Although additional

enzymatic complex to the substrate (target effect) and the spatial


proximity of the different types of cellulases to each other (proximity effect) (Fierobe et al., 2002).
3. Fermentative production of chemicals with recombinant
cellulose-utilizing microbes
3.1. Recombinant cellulase expression
A number of microorganisms used in industry, including Escherichia coli, Bacillus sp., lactic acid bacteria, Corynebacterium glutamicum, and Saccharomyces cerevisiae, have been engineered to
tolerate toxic compounds and metabolize a range of carbon
sources such as arabinose and xylose present in hemicellulose as
well as glucose. The availability of extensive genetic manipulation
tools has enabled the engineering of metabolic pathways to not
only improve production titers and yields, but also to expand the
utilization of carbon sources during fermentation. Thus, the heterologous expression of cellulase enzymes in industrial strains would
be an effective method for one-step fuel and chemical production.
Expressing recombinant cellulases in target strains represents
the development of microorganisms as biocatalysts (Table 1). By
displaying cellulases on microbial cell surface, efcient whole cell
biocatalysts for SSF have been constructed. To date, cellulases
derived from fungi and bacteria have been successfully
heterologously expressed as a multiple enzyme complex in noncellulolytic microorganisms. One of the most common sources
for cellulase genes expressed in yeast strains are fungi, particularly
T. reesei, while mesophilic and thermophilic bacteria have been
used as sources of cellulase genes for conferring cellulolytic ability
to both bacterial and yeast species.
3.2. E. coli
E. coli is arguably the most common host organism for the production of recombinant proteins. The use of E. coli as an expression
host has also inuenced approaches towards CBP, which have been
shaped by the generalized idea of immobilized cell biocatalysts.

Table 1
Production of chemicals from cellulosic materials with non-cellulolytic bacteria in SSF and CBP processes.
Strain
B. coagulans 36D1

Relevant genes
None

Substrate
96 g L

Enzyme added

cellulose

A. aculeatus bglucosidase
None

55 g L 1 IL-treated
switchgrass

None

71 mg L

55 g L 1 IL-treated
switchgrass

None

28 mg L 1
n-butanol

55 g L 1 IL-treated
switchgrass

None

1.7 mg L
pinene

A. aculeatus bglucosidase
A. aculeatus bglucosidase

1.27 g L 1 L(+)lactic acid


1.47 g L 1 L(+)lactic acid

B. coagulans DSM2314

None

B. coagulans P4-102B

Dldh, DalsS, GlyDH (D121N/F245S)

B. subtilis168

B. subtilis EG (BsCel5), DalsS

C. glutamicum pCCT-engD

C. cellulovorans engD

7.0 g L 1 amorphous
cellulose
15 g L 1 barley b-glucan

C. glutamicum ATCC 13032

C. cellulovorans celE, cbpA

Carboxymethyl cellulose

E. coli MG1655 DfadE

C. japonicas cel3A and gly43F, Bacillus sp. cel,


C. stercoranium xyn10B, FAEE biosynthesis
pathway (fadD, atfA, pdc, adhB, LtesA, atfA)
C. japonicas cel3A and gly43F, Bacillus sp. cel,
C. stercoranium xyn10B, butanol biosynthesis
pathway (crt, bcd, etfB, etfA, hbd, atoB, adhE2)
C. japonicas cel3A and gly43F, Bacillus sp. cel,
C. stercoranium xyn10B, pinene biosynthesis
pathway (atoB, HMGS, MK, PMK, PMD, idi,
PINE, GPPS)
C. thermocellum celA Dldh1

E. coli DH1 DadhE

E. coli MG1655

L. plantarum NCIMB 8826


pCU-CelA/DldhL1
L. plantarum NCIMB 8826
pCU-CelA/DldhL1
N.D., not determined.

C. thermocellum celA Dldh1

Reference

80 g L L(+)lactic acid
39.6 g L 1 L(+)lactic acid
2.0 g L 1 h 1 D(-)
lactic acid
3.1 g L 1 L(+)lactic acid
0.17 g L 1 Lglutamic acid
N.D.

154.9 g L limepretreated wheat straw


40 g L 1 lignocellulose

2gL

cellohexaose

2gL

barley b-glucan

15 FPU g-cellulose
biocellulase W
98 mg g-biomass 1
cellulase GC220
7.5 FPU g-cellulose
cellulase
None

Product
1

FAEE

Ou et al.
(2011)
Maas et al.
(2008)
Wang et al.
(2011)
Zhang et al.
(2011b)
Tsuchidate
et al. (2011)
Hyeon et al.
(2011)
Bokinsky et al.
(2011)
Bokinsky et al.
(2011)
Bokinsky et al.
(2011)

Okano et al.
(2010)
Okano et al.
(2010)

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enzymes were added into the fermentation medium, this study


provided useful information for establishing the CBP of lignocellulosic biomass.
Innovative CBP research with engineered microorganismas and
ionic liquid (IL) has been reported (Bokinsky et al., 2011). ILs are
salts in the liquid state at ambient temperature have been used
to develop a promising new class of cellulose-dissolving solvents
and bio-catalytic reaction media. Bokinsky et al. (2011) has demonstrated not only cell growth, but also biofuel production, from
IL-pretreated biomass using recombinant E. coli strains that were
engineered to express cellulase, xylanase, glucosidase, and xylobiosidase. The expression of endocellulase, Cel from Bacillus sp. D04
and xylanase, Xyn10B from Clostridium stercoranium xylanase,
Xyn10B, which were fused with the protein OsmY for the targeted
secretion from E. coli, enabled the recombinant strain to hydrolyze
phosphoric acid swollen cellulose (PASC) and beechwood xylan. To
utilize soluble oligosaccharides released by cellulolytic and hemicellulolytic enzymes, the glucosidase gene, cel3A, and xylobiosidase gene, gly43F, from Cellvibrio japonicas were expressed under
the control of wrbA and cstA promoters, respectively. Using this approach, good growth was observed on fractions of IL-treated
switchgrass, eucalyptus and yard waste without exogenously
added enzymes. Furthermore, the biosynthesis of three types of
biofuel (fatty acid ethyl esters (FAEE), n-butanol, and pinene) was
achieved by constructing biofuel biosynthesis pathways in cellulolytic and hemicellulolytic E. coli strains (Bokinsky et al., 2011).
E. coli has also been engineered to biosynthesize a wide variety
of chemicals, including isopropanol, butanol, fatty acids, alkanes,
and isoprenoids (Zhang et al., 2011a). Despite the obvious requirements for increasing yields and lowering costs, this signicant
proof-of-concept will allow the development of engineered E. coli
strains for advancing CBP technologies.
3.3. Bacillus sp.
3.3.1. Bacillus subtilis
Under anaerobic conditions, B. subtilis produces mainly L(+)-lactic acid and 2,3-butanediol, in addition to acetic acid. Homo-lactic
acid fermentation based on glucose and cellobiose has been demonstrated using a metabolically engineered B. subtilis strain 168
(Romero-Garcia et al., 2009). B. subtilis strain 168 possesses one
secretory glycoside hydrolase family 5 endoglucanases (BsCel5),
and one intracellular BGL, but cannot grow on cellulose due to
the very low expression level of endoglucanase and lack of exoglucanase. The heterologous expression of cellulase genes has enabled
Bacillus sp. to utilize various cellulosic materials. Zhang et al.
(2011b) demonstrated that overexpression of an endogenous EG,
BsCel5, enabled the growth of non-cellulose-utilizing B. subtilis
on amorphous cellulose and well-pretreated lignocellulosic biomass without the requirement for other added organic nutrients.
The authors also improved the specic activity and expression/
secretion level of BsCel5 by two-round directed evolution. Moreover, to enhance lactate yields, a-acetolactate synthase (alsS) involved in the 2,3-butanediol biosynthesis pathway was
inactivated in the recombinant cellulolytic B. subtilis strain, thereby
effectively streamlining lactate production from cellulose (Table 1).
3.3.2. Bacillus coagulans
B. coagulans, a thermophilic bacterium, has many desirable
properties for the fermentation of lignocellulosic sugars into lactic
acid. B. coagulans strains capable of converting both hexoses and
pentoses to homofermentative L(+)-lactic acid have a unique pentose phosphate (PP) pathway that efciently functions at 5055 C
and pH 5.0, which are optimal condition of commercial fungal
cellulases. Therefore, B. coagulans have the potential to function
as lignocellulose-fermentation biocatalysts. Maas et al. (2008)

produced L(+)-lactic acid from lime-pretreated wheat straw with


the commercial hydrolytic enzyme GC220 and B. coagulans DSM
2314. Dairy manure compost, which is rich in lignocellulosic
material, is a good reservoir of thermophilic biocatalysts that
can efciently utilize sugars derived from biomass, because it
typically reaches elevated temperature due to high microbial
activity. Bischoff et al. (2010) described the enrichment of
xylose-fermenting thermophilic bacteria from dairy manure
compost, demonstrating that B. coagulans can be applied for the
production of lactic acid from hydrolysates of corn ber.
Native stains of B. coagulans produce optically pure L(+)-lactic
acid from both glucose and xylose (Ou et al., 2011). Although B.
coagulans also has the potential to function as a biocatalyst for
the conversion of cellulose to optically pure L(+)-lactic acid, no
strains capable of generating the D( )-isomer of lactic acid had
been identied until recently. Wang et al. (2011) developed a general method for the genetic engineering of B. coagulans to overcome the genetic recalcitrance of this species, which is a major
limiting factor for the industrial use of this bacterium. In their
study, a derivative of B. coagulans strain P4-102B obtained by
mutagenesis and adaptive evolution successfully produced D( )lactic acid at 50 C and pH 5.0 due to expression of a mutated form
of glycerol dehydrogenase.

3.4. C. glutamicum
C. glutamicum is a non-pathogenic, non-sporulating, non-motile,
Gram-positive soil bacterium belonging to the order Actinomycetales, which includes species of Corynebacteria, Nocardia, and Rhodococci. C. glutamicum is an important industrial microorganism due
to its high production of amino acids, which are widely used in
medicine, fodder, and as food supplements. Genetically engineered
strains of C. glutamicum are also superior to other microorganisms
for producing various kinds of organic compounds, including ethanol, 1,5-diaminopentane (cadaverine) as a component of bio-based
nylons, and succinic acid as a polymer building block (Verts et al.,
2012). Current processes for the industrial production of amino
acids and building blocks for organic polymers with C. glutamicum
predominantly focus on media supplemented with glucose, fructose, and sucrose, because wild-type C. glutamicum lacks the ability
to utilize pentose sugars, including xylose and arabinose, and cellobiose. As most C. glutamicum strains also lack the EG and BGL
activities necessary to hydrolyze plant cellulose, many attempts
have been made to construct C. glutamicum strains capable of utilizing biomass-derived sugars.
To identify suitable strains for cellobiose fermentation, the
screening for spontaneous mutant strains of C. glutamicum R capable of growing on cellobiose as the sole carbon source was performed (Kotrba et al., 2003). One mutant was identied with a
single mutation in codon 317 of bglF, which is part of the b-glucoside utilization operon consisting of the genes bglF, bglA, and bglG,
encoding a b-glucoside-specic IIBCA component of the
phosphoenolpyruvate: sugar phosphotransferase system, phospho-b-glucosidase, and anti-terminator protein from the BglG/SacY
family of transcription regulators, respectively. To allow simultaneous utilization of the sugars glucose, xylose, and cellobiose, ve
copies of clusters of xylA and xylB, which encode xylose isomerase
and xylulokinase (XK), respectively, from E. coli and one copy of a
variant bglF317A-bglA cluster from the spontaneous C. glutamicum
mutant were integrated into the genome of C. glutamicum R (Sasaki
et al., 2008). The resulting strain, X5C1, completely consumed
40 g L 1 glucose, 20 g L 1 xylose, and 10 g L 1 cellobiose within
12 h and predominantly produced lactic and succinic acids under
growth-arrested conditions. Recently, the expression of xylA and
xylB from E. coli in C. glutamicum yielded cadaverine from

T. Hasunuma et al. / Bioresource Technology 135 (2013) 513522

enzymatic hydrolysates of hemicellulose derived from oat spelts


(Buschke et al., 2011).
To promote the utilization of arabinose, the E. coli genes araA,
araB, and araD, encoding L-arabinose isomerase, L-ribulokinase,
and L-ribulose-5-phosphate 4-epimerase, respectively, were expressed in the recombinant C. glutamicum strain CRA1 under control of a constitutive lac promoter (Kawaguchi et al., 2008). Strain
CRA1 was able to grow on minimal medium containing arabinose
as the sole carbon source and produced several organic acids,
including succinic, lactic, and acetic acids. Heterologous expression
of the araBAD operon from E. coli in the wild-type and lysine-producing strains of C. glutamicum enabled production of L-glutamate
and L-lysine, respectively, from arabinose (Schneider et al., 2011).
L-ornithine and L-arginine were also generated from arabinose
when this operon was expressed in L-ornithine- and L-arginineproducing strains of C. glutamicum.
One of the major requirements for C. glutamicum to utilize cellulose is the extracellular production of cellulase. To promote the
secretion of foreign proteins in this species, the Tat-dependent signal sequence torA from E. coli, joined to the Clostridium thermocellum CelA, encoding EG, was introduced into wild-type C.
glutamicum, leading to the successful detection of cellulase activity
in the fermentation medium (Tsuchidate et al., 2011). Furthermore,
a recombinant C. glutamicum strain harboring a chimeric gene consisting of the E. coli torA and Clostridium cellulovorans EG genes
(engD) was able to grow in medium containing the cellulosic material barley b-glucan as the sole carbon source and produce glutamate in the presence of A. aculeatus BGL1 (Tsuchidate et al.,
2011). Tateno et al. (2007) reported a cell surface display system
in C. glutamicum using B. subtilis PgsA as an anchor protein. PgsA,
which is one of the poly-c-glutamic acid synthetase complexes,
has a transmembrane region at its N terminus. Recently, Streptococcus bovis a-amylase (AmyA) was successfully displayed on the
C. glutamicum cell surface by fusing AmyA with the C terminus of
the porins, PorB, PorC and PorH (Tateno et al., 2009). As the next
strategy, cellulolytic enzymes would be displayed on the cell surface to develop whole cell biocatalysts. A C. glutamicum strain
expressing functional mini-cellulosomes composed of chimeric
EG (CelE) bound to the scaffolding protein miniCbpA from C. cellulovorans was constructed using the cg0955 signal peptide (Hyeon
et al., 2011). The mini-cellulosome-expressing C. glutamicum strain
hydrolyzed carboxymethyl cellulose (CMC). Further engineering of
C. glutamicum will help realize one-step production processes for
not only various amino acids, but also plastic precursors such as
cadaverine, lactate and succinate from lignocellulosic materials
through CBP.
3.5. Lactic acid bacteria
Lactic acid bacteria (LAB) have been used for the industrial production of lactic acid that is applicable for use as a polymerization
substrate to generate poly-lactic acid (PLA), which has several
applications. Poly-L-lactic acid is commercially used as a biodegradable plastic, while stereocomplex PLA, consisting of L- and
D-lactic acid, is a promising material due to its high melting point
and physiological stability. For both materials, the isomeric purity
of lactic acid is important, because isomer separation is a costly
process.
The industrial production of lactic acid has typically involves
the bacterial fermentation of glucose or maltose derived from starchy biomass as carbon sources. Although the use of lignocellulosic
biomass as carbon sources for lactic acid production has many
advantages, including low cost and environmental friendliness,
most LAB strains lack cellulase activity. Several attempts have been
made to apply LAB strains for cellobiose utilization. Lactobacillus
delbrueckii mutant Uc-3 strain with intercellular BGL activity pro-

517

duced 90 g L 1 L(+)-lactic acid from 100 g L 1 cellobiose at a productivity rate of 2.25 g L 1 h 1 (Adsul et al., 2007). More recently,
UV mutagenesis of Lactobacillus lactis NCIM 2368 led to the isolation of mutant strain RM2-24, which was able to produce high
amounts of D-lactic acid (110 g L 1) from 150 g L 1 acid hydrolysate of sugar cane containing cellobiose (Joshi et al., 2010).
For the efcient fermentation of lignocellulosic hydrolysates,
microorganisms must utilize not only cellobiose, but also pentoses,
particularly xylose and arabinose, present in the material. In some
LAB, arabinose and xylose are converted to xylulose-5-phosphate
(X5P) by a number of reaction steps (Okano et al., 2010). X5P is
converted to equimolar amounts of lactic acid and acetic acid via
the phosphoketolase (PK) pathway, which is an obstacle to highyield lactic acid production. In Lactococcus lactis, X5P is assimilated
by the PP pathway that produces only lactic acid as a nal product
in addition to the PK pathway. Thus, purity of D-lactic acid was improved by the redirection of the PK pathway to the PP pathway in a
recombinant Lactobacillus plantarum DldhL1 strain by substituting
an endogenous PK gene with L. lactis transketolase gene involved
in the PP pathway (Okano et al., 2010). Okano et al. (2010)
successfully produced homo-D-lactic acid from several components of lignocellulosic hydrolysate, including xylose, and
cellooligosaccharide, using metabolically engineered strains of L.
plantarum. To more effectively ferment cellulosic material, EG from
C. thermocellum has been expressed in L. plantarum (Bates et al.,
1989) and the probiotic lactobacilli Lactbacillus gasseri and
Lactbacillus johnsonii (Cho et al., 2000). Secretion of CelA by the
CelA-secreting DldhL1 strain led to the production of 1.27 g L 1
1
D(-)-lactic acid with an optical density of 99.5% from 2 g L
of
cellohexaose. Moreover, by adding BGL from A. aculeatus to the
medium of the modied strain, D(-)-lactic acid was directly produced from b-glucan (Okano et al., 2010). Narita et al. (2006) developed a cell surface display system in Lactobacillus casei using the B.
subtilis PgsA anchor protein. The display of cellulolytic enzymes on
the cell surface of LAB would enable efcient lactic acid production
from cellulosic materials.
Expression of cellulosomes in LAB strains would be a signicant
step towards the realization of the CBP of plant biomass. Recently,
a prototypic cellulosome-displaying LAB strain was constructed by
functionally binding BGL fused with the type 1 dockerin motif of
the cellulosomal enzyme, CelS, to the scaffolding protein CipA from
C. thermocellum (Wieczorek and Martin, 2010). The authors conrmed that the cellulosome-inspired enzyme complexes were displayed on the cell surface of Lactococcus lactis. BGL from Fibrobacter
succinogenes was also displayed on the cell surface of a probiotic
Lactobacillus reuteri strain (Huang et al., 2011). The construction
of mini-cellulosomes on L(+)- or D(-)-lactic acid-producing LAB
strains represents a promising platform for the production of bioplastics from cellulosic materials, and will likely be achieved in the
near future.
3.6. S. cerevisiae
The yeast S. cerevisiae is one of the most well-studied organisms
for ethanol fermentation from cellulosic and hemicellulosic materials. In addition, S. cerevisiae displays several advantageous characteristics for use in industrial applications, including its inherent
resistance to low pH, high temperature, and various inhibitors
(Hasunuma and Kondo, 2011). Accordingly, numerous studies have
described CBP using cellulase-expressing S. cerevisiae. Notably,
yeast cell-surface display technology has been widely used to
introduce cellulose-degrading abilities to S. cerevisiae. Hasunuma
and Kondo (2011) have recently reviewed lignocellulolytic ethanol
production by cell surface-engineered S. cerevisiae; therefore, here,
we have only briey described a few notable examples of CBP
using engineered S. cerevisiae.

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Fujita et al. (2004) described the direct fermentation of PASC to


ethanol by the synergistic saccharication activity of T. reesei
CBHII, EGII, and A. aculeatus BGL1 on the cell surface of S. cerevisiae,
resulting in the production of 2.9 g L 1 ethanol from 10 g L 1 PASC.
Yamada et al. (2011) constructed a recombinant S. cerevisiae strain
whose cellulase activity was optimized by controlling the expression levels of three types of cellulases (EGII, CBHII, and BGL1) using
the newly developed cocktail d-integration method, which allows
the simultaneous integration of multiple gene expression cassettes
into yeast chromosomes at desired ratios. The constructed recombinant strain, which possessed 16 copies of EGII, 6 copies of CBHII,
and 1 copy of BGL1, produced 3.1 g L 1 ethanol from 20 g L 1 PASC
(Yamada et al., 2011).
Although S. cerevisiae has the potential to ferment ethanol from
various types of cellulosic biomass, it cannot naturally utilize the
xylose contained in the hemicellulosic fraction. Therefore, the xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from
Scheffersomyces stipitis are typically introduced into S. cerevisiae
strains targeted for ethanol production from hemicellulose. Additionally, the endogenous XK gene from S. cerevisiae is also often
overexpressed to increase the efciency of xylose utilization
(Hasunuma and Kondo, 2011). Sakamoto et al. (2011) developed
a recombinant hemicellulose-fermenting S. cerevisiae strain that
co-displayed xylanase II (XynII) from T. reesei and b-xylosidase
(XylA) from Aspergillus oryzae and BGL1 from A. aculeatus on the
cell surface of a xylose-fermenting strain harboring chromosomally encoded genes for XR, XDH and XK. The recombinant strain
successfully produced 8.2 g L 1 ethanol from a rice-straw hemicellulosic hydrolysate obtained by liquid hot water treatment that did
not involve the addition of cellulase reagents or detoxication of
the hydrolysate. Based on these ndings, S. cerevisiae clearly appears suitable for lignocellulosic bio-ethanol production in CBP.
A few recent works have demonstrated that metabolically engineered S. cerevisiae can also produce useful compounds other than
ethanol. In particular, the construction of a lactate-producing
S. cerevisiae has provided impressive evidence for the versatility
of this yeast (Tokuhiro et al., 2009). However, as S. cerevisiae favors
the production of ethanol as a metabolic waste product during fermentation to obtain cellular energy, the metabolic engineering of
S. cerevisiae remains challenging. For example, isobutanol production from glucose by S. cerevisiae has remained at levels only
slightly higher than 100 mg L 1, despite signicant efforts to
improve yields (Kondo et al., 2012b). In addition, the yields of
n-butanol typically only reach 2.5 mg L 1 (Steen et al., 2008). Thus,
the development of novel metabolic engineering strategies are
needed for improving the production of bio-based chemicals by
S. cerevisiae, because the CBP strategies being developed for ethanol production may be applicable for these chemicals in the near
future.
4. Advances in cellulase-expressing microbes used for the
production of chemicals
4.1. Cellulase-expressing strains
Microorganisms capable of utilizing diverse substrates would
provide a clear competitive advantage in biorenery processes.
Natural plant-degrading microorganisms producing extracellular
multiple enzyme systems with different substrate specicities,
such as cellulase and xylanase, would be able to utilize cellulosic
and hemicellulosic materials as carbon sources. Thus, CBP of lignocellulosic materials has been developed with native cellulolytic
microorganisms, including Clostridia sp., fungi, and several thermophilic microorganisms. Notably, anaerobic Firmicutes, including
the class Clostridia, possess several desirable traits, such as the
ability to utilize a broad spectrum of carbon sources, diverse

pathways for the production of useful metabolites, and tolerance


to many toxic compounds present in fermentation medium (Tracy
et al., 2011). Although a lack of advanced genetic tools has long
hindered applied research in clostridial hosts, signicant improvements in genetic engineering technologies have been reported over
the past four years (Tracy et al., 2011). Cellulolytic microorganisms
have been traditionally modied by random breeding techniques,
such as mutation and crossbreeding, for the alteration of microbial
features that lead to the increased production of useful compounds
during fermentation; however, recent advances in genetic engineering have rapidly accelerated molecular breeding techniques
(Table 2).
4.2. Clostridium cellulolyticum
Cellulolytic microorganisms play an important role in the recycling of carbon xed in the form of cellulose. In particular, clostridial species that degrade cellulose in anaerobic environments play a
key role in the regeneration of low-molecular-weight carbon compounds from plant biomass and waste matter. Olson et al. (2011)
reviewed the recent progress in the conversion of lignocellulosic
materials to ethanol, isobutanol, and n-butanol by native celluloseand hemicellulose-utilizing microorganisms. C. cellulolyticum,
which is the best-understood mesophilic clostridial bacterium,
uses cellulosomes to efciently degrade the crystalline cellulose
and hemicellulose present in lignocellulosic biomass. This species
displays high carbon ux through glycolysis that leads to pyruvate
accumulation and the early cessation of cell growth. Guedon et al.
(2002) expressed pyruvate decarboxylase and alcohol dehydrogenase from Z. mobilis in a recombinant C. cellulolyticum strain to decrease pyruvate accumulation and found that the fermentation
patterns were markedly altered. Specically, the concentrations
of acetate and ethanol in the fermentation medium increased by
93% and 53%, respectively, while that of lactate decreased by 48%
as a result of the increased cellulose consumption. Although C. cellulolyticum utilizes both pentoses and hexoses to produce ethanol,
acetate, lactate, hydrogen, and CO2, the proportion of ethanol
among these compounds is relatively low. In a pioneering study
by Li et al. (2012), the successful disruption of both the paralogous
L-lactate and L-malate dehydrogenases in C. cellulolyticum to improve ethanol yields was reported. During growth on acidpretreated switchgrass, the double mutant exhibited 4-fold higher
ethanol yields than wild-type cells. This study was the rst to describe the targeted mutagenesis for C. cellulolyticum through genetic engineering.
The genetic engineering of C. cellulolyticum will allow better
understanding of the genetic and metabolic processes that are required to enhance biofuel production. Although most research concerning biofuel production by CBP microorganisms has focused on
ethanol production, higher alcohols are better candidates for gasoline replacement because of their high energy density, octane value, and Reid vapor pressure. To this end, Higashide et al. (2011)
expressed ve heterologous genes (alsS from B. subtilis, ilvCD and
yqhD from E. coli, and kivd from Lactococcus lactis) in C. cellulolyticum and successfully converted pyruvate to isobutanol from crystalline cellulose using the recombinant strain.
4.3. C. thermocellum
C. thermocellum, a Gram-positive, anaerobic, and thermophilic
bacterium, is also a candidate microorganism for CBP because it
can rapidly hydrolyze cellulose at rates approaching 2.5 g L 1 h 1
(Argyros et al., 2011) due to the expression of a highly efcient
cellulosome. C. thermocellum also produces ethanol, acetate, lactate, formate, hydrogen, and CO2, similar to C. cellulolyticum. The
metabolic engineering of C. thermocellum is required to attain

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T. Hasunuma et al. / Bioresource Technology 135 (2013) 513522


Table 2
Recent data on alcohol production from cellulosic materials with metabolically-engineered native-cellulolytic microorganisms.
Strain
C. cellulolyticum CC-pMG8
C. cellulolyticum pLyc1217/Er0137,
pLyc1217/Er2485

C. cellulolyticum pWH320

Relevant genes
Z. mobilis adhII, pdc

Dldh, Dmdh

C. japonicas MSB280

B. subtilis alsS, E. coli ilvCD and yqhD, L.


lactis kivd
Z. mobilis adhB, pdc

C. thermocellum M1570

Dhpt, Dldh, Dpta

high-yield production of target compounds by CBP, but the construction of stable transgenic strains has been limited by the lack
of effective genetic tools. Tripathi et al. (2010) developed positive
and negative selectable markers to allow for the selection of strains
that undergo allele replacement in the absence of replicating plasmids. As C. thermocellum is sensitive to the anti-metabolite 5-uoroorotic acid, the gene encoding PyrF, which participates in de novo
pyrimidine biosynthesis, was used as a marker for selection or
counter selection. This technology was applied to delete a gene involved in acetic acid formation (pta), which improved ethanol
yields (Tripathi et al., 2010). Argyros et al. (2011) created a counter-selection system based on endogenous hpt and Thermoanaerobacterium saccharolyticum tdk genes. Combining these selection
markers enabled the use of replicating plasmids to insert and remove markers from the host chromosome. Following the deletion
of the L-lactate dehydrogenase (ldh) and pta genes from C. thermocellum with the counter-selection system and a 2000-h adaptation
period, the ethanol yield of the mutant increased 4.2-fold compared to wild-type cells. Co-culture of the C. thermocellum mutant
and T. saccharolyticum for 146 h yielded 38.1 g L 1 ethanol from
92.2 g L 1 Avicel microcrystalline cellulose and concentrations of
acetic and lactic acids that were below detection limits.
One of the challenges for the industrial application of Clostridium species is increasing its low ethanol tolerance, which typically
ranges from 1020 g L 1 in wild-type strains, as this would have a
signicant effect on lowering production costs due to the reduced
size of the necessary equipment, such as fermentation tanks and
distillation columns (Mussatto et al., 2010). Using an evolutionary
engineering approach, Shao et al. (2011) isolated ethanol-tolerant
strains of C. thermocellum that were capable of growing in medium
containing up to 50 g L 1 ethanol. Analysis of the genome sequences of the ethanol-tolerant strains revealed six common
mutations, which were found in genes involved in ethanol, arginine, and pyrimidine biosynthesis pathways. Brown et al. (2011)
revealed that a shift of cofactor specicity from NADH to NADPH
for the bi-functional acetaldehyde-CoA/alcohol dehydrogenase enzyme affords ethanol tolerance to C. thermocellum, likely by affecting electron ow.
4.4. Clostridium phytofermentans and other cellulolytic bacteria
C. phytofermentans, which has the highest number of genes
associated with the degradation of lignocellulosic material among
sequenced clostridial species (Weber et al., 2010), is one of the
promising native cellulolytic microbes for CBP. Similar to fungi,
C. phytofermentans secretes non-complexed enzymes for the
hydrolysis of lignocellulosic materials, including cellulose, hemicellulose, and pectin, to fermentable sugars. In addition, C. phytofermentans can consume nearly all of the sugars present in

Substrate
50 g L

cellulose

10 g L Avicel microcrystalline
cellulose
10 g L 1 acid-pretreated
switchgrass
10 g L 1 crystalline cellulose (Sigma
type 50)
10 g L 1 Avicel microcrystalline
cellulose
20 g L 1 Avicel microcrystalline
cellulose

Product

Reference
1

0.83 g L
ethanol
2.7 g L 1
ethanol
1.3 g L 1
ethanol
0.42 g L 1
isobutanol
0.0035 g L
ethanol
5.6 g L 1
ethanol

Guedon et al. (2002)


Li et al. (2012)
Li et al. (2012)
Higashide et al. (2011)
1

Gardner and Keating,


(2010)
Argyros et al. (2011)

lignocellulosic biomass, such as arabinose, cellobiose, fructose, galactose, gentiobiose, glucose, lactose, maltose, mannose, ribose, and
xylose, to produce ethanol and acetate as the major products
(Weber et al., 2010). In 10-day fermentation experiments with
AFEX-treated corn stover containing 0.5% (w/w) glucan,
C. phytofermentans hydrolyzed 76.0% and 88.6% of available glucan
and xylan, respectively, and produced 2.8 g L 1 ethanol. The ethanol yield achieved by the CBP with C. phytofermentans was 71.8%
of those obtained by SSCF using commercially available cellulase
and a xylose-fermenting S. cerevisiae strain (Jin et al., 2011).
Numerous microorganisms, including hyperthermophilic marine archaea, which can tolerate elevated temperatures are able to
utilize carbohydrates such as a- and b-linked glucan, xylan, and
mannan for growth, whereas the utilization of crystalline cellulose
is limited to a number of terrestrial bacteria with upper temperature limits for growth of below 75 C (Blumer-Schuette et al.,
2008). Anaerocellum thermophilum is a Gram-positive thermophilic
anaerobe that grows optimally at 75 C. A. thermophilum strain
DSM 6725 (Caldicellulosiruptor bescii) efciently utilizes various
types of untreated plant biomass, including hardwoods, grasses,
crystalline cellulose, and xylan, with the predominant end-products being hydrogen, acetate, and lactate (Lochner et al., 2011).
Caldicellulosiruptor saccharolyticus, which is a close relative of A.
thermophilum, can degrade crystalline cellulose (Blumer-Schuette
et al., 2008). Interestingly, C. saccharolyticus strain DSM 8903 can
grow on switchgrass, but not on poplar. Yang et al. (2009) demonstrated a signicant difference in the biomass hydrolysis activity
between A. thermophilum DSM6725 and C. saccharolyticus
DSM8903, which would be due to the recalcitrance of poplar for
the DSM8903 strain.
C. thermocellum exhibits a high growth rate on crystalline cellulose, but does not utilize xylan and cannot grow on xylose or other
pentoses. However, hemicellulolytic thermophiles could be used in
conjunction with C. thermocellum to hydrolyze hemicellulose and
ferment all of the sugars present in the biomass substrate (Shaw
et al., 2008). The thermophilic anaerobe T. saccharolyticum is able
to utilize xylan and biomass-derived sugars, including arabinose,
mannose, and xylose, as well as glucose, as carbon sources to produce ethanol and organic acids. In order to improve ethanol yields,
Shaw et al. (2008) knocked out the genes involved in organic acid
formation, such as acetate kinase, phosphate transacetylase, and Llactate dehydrogenase, which resulted in a T. saccharolyticum
strain capable of producing ethanol as the only detectable product.
Using the homoethanologenic strain for the SSF of Avicel at 50 C
resulted in a 2.5-fold reduction in cellulase loading compared to
that required for S. cerevisiae cultured at 37 C (Shaw et al.,
2008). Recently, cellulose- and xylan-degrading thermophilic
anaerobes isolated from self-heated biocompost were able to utilize xylan with roughly the same efciency as that for cellulose

520

T. Hasunuma et al. / Bioresource Technology 135 (2013) 513522

(Sizova et al., 2011). Phylogenetic analysis of 16S rRNA and glycosyl hydrolase family 48 gene sequences suggested that the isolates
were related to a strain of Clostridium clarifravum.

pathways for the synthesis of target products would have profound


signicance for increasing the yield and productivity of products.
6. Perspectives for future CBP microorganisms

4.5. Fungus for CBP


The capabilities of various lamentous fungi, including members of the genera Aspergillus, Rhizopus, Monilia, Neurospora, Fusarium, Tricoderma, and Mucor, have been explored for the improved
production of ethanol from biomass (reviewed in Xu et al., 2009).
Recently, the white rot fungus Trametes hirsute was shown to be
capable of directly fermenting starch, wheat bran, and rice straw
to ethanol without prior acid or enzymatic hydrolysis (Okamoto
et al., 2011). The methophilic fungus Fusarium oxysporum is among
the few microbial species that possess the enzymatic system to
break down cellulose and hemicellulose while simultaneously fermenting the generated hexoses and pentoses to ethanol. This capability allows for single-step ethanol production from agricultural
and forestry residues. A yield of 109 g ethanol per kg of dry
Brewers spent grain (BG) was obtained from alkali-pretreated BG
using F. oxysporum cultivated under microaerobic conditions
(0.01 vvm), corresponding to 60% of the theoretical yield based
on the total glucose and xylose content of BG (Xiros and Christakopoulos, 2009).
The mycelium fungus Rhizopus oryzae has been well studied in
regard to the production of fumaric acid, a four carbon unsaturated
dicarboxylic acid (Meussen et al., 2012). Because of its double bond
and two carboxylic acid groups, the potential industrial applications of fumaric acid range from the manufacture of synthetic resins and biodegradable polymers to the production of intermediates
for chemical synthesis. R. oryzae has several advantageous characteristics in the ability to utilize pentoses, low growth requirements,
tolerance to the inhibitors present in acid hydrolysates of lignocellulosic biomass, and ability to directly utilize non-pretreated cellulose and hemicellulose (Meussen et al., 2012).

Establishing economically feasible fermentation processes requires markedly increasing nal product titers due to the high energy demands of subsequent product recovery steps, as well as the
capital and production costs associated with biorenery equipment (Hasunuma and Kondo, 2011; Mussatto et al., 2010).
Although high-yield production of target compounds by metabolically optimized microbes is necessary, achieving higher titers inevitably requires increased loading of solid lignocellulose in the SSF
and CBP processes. Increase in the solid concentration results in
corresponding increases in chemical production (Matano et al.,
2012). However, by increasing the lignocellulose content in the
bioreactor, the concentration of fermentation inhibitors released
during the pretreatment of biomass would reach higher levels.
Accordingly, microorganisms that are resistant to inhibitors are a
prerequisite for the high-titer production of fuels and chemical
products (Hasunuma and Kondo, 2011).
To further engineer cellulolytic recombinant and native strains
for use in CBP, system-wide modications of intracellular metabolic pathways using advanced engineering tools such as minimal
hosts, vectors, genetic controllers, and characterized enzymes
(Keasling, 2012) are needed, which would improve the potential
of not only target productivities but cell growth and viability during the fermentation. In the near future, it may be possible to design optimized metabolic systems with the aid of computer
simulations based on metabolic proling data (Kondo et al.,
2012a). The integration of cellulolytic capabilities with metabolic
systems specied for targeted chemical production will allow customized CBP microorganisms to be developed using advanced gene
manipulation technologies.
7. Conclusions

5. Synthesis
Recent progress in the development of microorganisms through
evolutionary, metabolic, and synthetic engineering approaches
have paved the way for utilizing lignocellulosic biomass as a bioresource for fuel and chemical production. However, the one-step
fermentative conversion of cellulosic material to chemical products represents a major challenge to achieving this goal. When
using recombinant cellulose-utilizing microbes, low cellulolytic
activities of heterologous enzymes often result in unutilized cellulose, thereby decreasing yields. One major obstacle to modifying
non-cellulolytic microorganisms for the utilization of lignocellulose is the low inherent capacity of these strains for foreign cellulase secretion (Bokinsky et al., 2011). To overcome this limitation,
protein export systems that do not compromise the cell membrane
should be introduced into recombinant strains. In addition, as the
efcient degradation of crystalline cellulose requires the synergistic reaction of cellulases, appropriate combinations and quantitative ratios of cellulase subsets need to be expressed in strains
used for the fermentation of lignocellulosic biomass (Yamada
et al., 2011). As an alternative approach, cellulase engineering,
which has offered improvement in specic activity at a broad range
of pH and temperature by directed evolution and rational design
(Chandel et al., 2012), might be implemented. When using native
cellulose-utilizing microbes for industrial applications, these
microorganisms should exhibit high product yields, productivities,
and product titers, while not over-burdening cells energetically.
Although genetic manipulation tools for the microbes are under intense development, the ability to engineer complete metabolic

In this review, recent advances in the development of microorganisms for the fermentative production of bio-based fuels and
chemicals from cellulosic materials were highlighted. Direct conversion of cellulose to key commodities such as alcohols, fatty
acids, isoprenoid and organic acids has been achieved through
the engineering of metabolic pathways and cellulose hydrolysis
abilities of microorganisms. Despite of the obvious requirements
for increasing yields and lowering production costs, this signicant
proof-of-concept will allow the application of the engineered
microorganisms to advancing CBP technologies.
Acknowledgements
This work was supported by project P07015 of the New Energy
and Industrial Technology Development Organization (NEDO) under the sponsorship of the Ministry of Economy, Trade, and Industry (METI) of Japan. This work was also supported by Grants-in-Aid
for Young Scientists (B) to TH and KYH from the Ministry of Education, Culture, Sports and Technology (MEXT) of Japan and Special
Coordination Funds for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research
Areas (Innovative Bioproduction Kobe), MEXT, Japan.
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