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Corresponding author at: Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and
Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel.: +81 92 642 3019; fax: +81 92 642 3019.
E-mail address: sonomoto@agr.kyushu-u.ac.jp
(K. Sonomoto).
0168-1656/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2011.06.017
M.A. Abdel-Rahman et al. / Journal of Biotechnology 156 (2011) 286301 287
3. Fermentative lactic acid production by LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
3.1. Improvement of lactic acid production by LAB in the eld of microbial technology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
3.2. Lactic acid production by LAB using lignocellulosic biomass and lignocellulose-derived sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
4. Metabolism of lignocellulose-derived sugars by LAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
5. Designed biomass study and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
1. Introduction
Lactic acid (2-hydroxypropanoic acid, CH
3
CH(OH)COOH) is
a natural organic acid with a long history of use in the food and
non-food industries, including the cosmetic and pharmaceutical
industries, and for the production of oxygenated chemicals, plant
growth regulators, and special chemical intermediates (Oshiro
et al., 2009; Singhvi et al., 2010; Tashiro et al., 2011). Currently,
there is an increased demand for lactic acid as a feedstock for the
production of biopolymer poly-lactic acid (PLA), which is a promis-
ing biodegradable, biocompatible, and environmentally friendly
alternative to plastics derived frompetrochemicals. PLA has many
uses in surgical sutures, orthopedic implants, drug delivery sys-
tems, and disposable consumer products (Adnan and Tan, 2007),
and its use would signicantly alleviate waste disposal problems.
The physical properties of PLAdepend on the isomeric composition
of lactic acid. Pure isomers, l- and d-lactic acid, are more valuable
than the racemic dl form because each isomer has its own spe-
cic industrial application. l-Lactic acid is used for the synthesis
of poly l-lactic acid (PLLA), a semi-crystalline biodegradable and
thermostable polymer that has a potentially large market in goods
packaging. PLLA has high tensile strength and lowelongation with
a high modulus that makes it suitable for medical products used in
orthopedic xation (e.g., pins, rods, ligaments, etc.), cardiovascular
applications (e.g., stents, grafts, etc.), dental applications, intestinal
applications, and sutures (John et al., 2006a). d-Lactic acid is used
for the production of poly d-lactic acid (PDLA) (John et al., 2009).
These pure polymers are relativelyheat sensitive, while stereocom-
plexes of PLLA and PDLA have a melting point 50
C higher than
their respective pure polymers (Ikeda et al., 1987; Tsuji and Fukui,
2003) and are more biodegradable (de Jong et al., 2001; Tashiro
et al., 2011). The ratio of l- and d-lactic acids inuences the prop-
erties and the degradability of PLA (Kharras et al., 1993).
Lactic acid can be produced either by chemical synthesis or
by microbial fermentation. Chemical synthesis from petrochemi-
cal resources always results in racemic mixture of dl-lactic acid,
which is a major disadvantage of this approach (Hofvendahl and
Hahn-Hgerdal, 2000). Conversely, microbial lactic acid fermenta-
tion offers an advantage in terms of the utilization of renewable
carbohydrate biomass, low production temperature, low energy
consumption, and the production of optically high pure lactic acid
by selecting an appropriate strain (Ilmen et al., 2007; Pandey et al.,
2001). Presently, almost all lactic acid produced globally is man-
ufactured by fermentation routes. In particular, there have been
numerous studies of lactic acid production by lactic acid bacteria
(LAB) in comparison with other microorganisms.
The demand for lactic acid has increased considerably due to its
wide range of applications; however, the highcost of the rawmate-
rials, e.g., starch and rened sugars, which accounts for the highest
portion of the production cost, represents one of the most serious
obstacles for the fermentative production of lactic acid to compete
with chemical synthesis (Datta et al., 1995). Cheap raw materials
are essential for the feasibility of the biotechnological production
of lactic acid because polymer producers and other industrial users
usually require large quantities of lactic acid at a relatively low
cost. The use of low-cost non-food materials for lactic acid pro-
duction appears to be more attractive because they do not have
any impact on the human food chain. Nowadays, lignocellulosic
materials from agricultural, agro-industrial, and forestry sources
represent a potentially inexpensive and renewable carbohydrate
feedstock for the large-scale fermentation of lactic acid due to their
abundance, low price, high polysaccharide content, and renewa-
bility (Duff and Murray, 1996; Parajo et al., 1996; Taniguchi et al.,
2005; Wyman, 1999). However, the cellulose and hemicellulose in
lignocellulose are not directly available for bioconversion to lac-
tic acid because of their intimate association with lignin (Schmidt
and Thomsen, 1998) and the lack of hydrolytic enzymes in LAB
(Tokuhiro et al., 2008).
There have been numerous investigations on the development
of biotechnological processes for lactic acid production, with the
ultimate objective of making the process more effective and eco-
nomical. In this review, we focus on the conventional processes
for lactic acid fermentation by LAB from lignocellulosic biomass
and lignocellulose-derived sugars. Moreover, we describe the lim-
itations of lactic acid production using such materials. We also
describe fermentative processes and technologies with practical
examples, the metabolism of biomass-derived sugars, and the
promising prospects of lactic acid fermentation.
2. Overviewof lactic acid production fromlignocellulosic
biomass
2.1. Composition of lignocellulosic biomass
The global production of plant biomass, of which over 90%
is lignocellulose, amounts to 20010
9
tons per year, where
82010
9
tons of the primary biomass remains potentially
accessible (Lin and Tanaka, 2006). Lignocellulosic biomass is
organic material derived from a biological origin, and represents
the most abundant global source of biomass that has been largely
unutilized (Lin and Tanaka, 2006). It is mainly composed of cellu-
lose (insoluble bers of -1,4-glucan), hemicellulose (noncellulosic
polysaccharides including xylans, mannans, and glucans), and
lignin (a complex polyphenolic structure), which form90% of the
dry matter, plus lesser amounts of minerals, oils, and other com-
ponents (Balat, 2011; Molina-Sabio and Rodrguez-Reinoso, 2004;
Yang et al., 2009). This biomass includes forest and crop residues
(Chen and Lee, 1997; Melzoch et al., 1997), municipal solid waste
(John et al., 2007), waste paper (McCaskey et al., 1994), and wood
(Linko et al., 1984). The structural and chemical composition of lig-
nocellulosic material has varying amounts of these components
because of genetic and environmental inuences and their inter-
actions (Demirbas, 2005). The proportion of biomass constituents
varies between species, and there are distinct differences between
hardwoods and softwoods. The total content of cellulose and hemi-
cellulose is higher in hardwoods (78.8%) than in softwoods (70.3%),
but the total content of lignin is higher in softwoods (29.2%) than in
hardwoods (21.7%) (Balat, 2009). As shown in Table 1, the cellulose,
hemicellulose, andlignincontent depends onthetypeof lignocellu-
losic biomass, which indicates that an appropriate material should
be selected for the corresponding fermentation.
Cellulose, the major component of plant biomass (3060% of
total feedstock dry matter), is a homopolysaccharide composed
of -d-glucopyranose units, linked by -(14)-glycosidic bonds.
288 M.A. Abdel-Rahman et al. / Journal of Biotechnology 156 (2011) 286301
Table 1
The contents of cellulose, hemicellulose, andlignininvarious types of lignocellulosic
biomass (% dry weight).
a
Lignocellulosic materials Cellulose (%) Hemicellulose (%) Lignin (%)
Algae (green) 2040 2050 NA
b
Aspen hardwood 51 29 16
Birch Hardwood 40 39 21
Chemical pulps 6080 2030 210
Coastal Bermuda grass 25 35.7 6.4
Corn cobs 45 35 15
Cornstalks 3947 2631 35
Cotton seed hairs 8095 520 0
Cotton, ax, etc. 8095 520 NA
b
Grasses 2540 2550 1030
Hardwood 452 305 204
Hardwood barks 2240 2038 3055
Hardwood stems 4055 2440 1825
Leaves 1520 8085 0
Newspaper 4055 2540 1830
Nut shells 2530 2530 3040
Paper 8599 0 015
Pine softwood 44 26 29
Primary wastewater
solids
815 NA
b
2429
Softwood 422 272 283
Softwood barks 1838 1533 3060
Softwood stems 4550 2535 2535
Solid cattle manure 1.64.7 1.43.3 2.75.7
Sorted refuse 60 20 20
Spruce softwood 43 26 29
Swine waste 6.0 28 NA
b
Switch grass 45 31.4 12.0
Waste papers from
chemical pulps
6070 1020 510
Wheat straw 3741 2732 1315
WillowHardwood 37 23 21
, Not determined.
a
Source: Balat (2011), Olsson and Hahn-Hgerdal (1996) and Sun and Cheng
(2002).
b
NA not available.
The orientation of the linkages and additional hydrogen bond-
ing make the polymer rigid and difcult to break. Hemicellulose
(2040% of total feedstock dry matter) is a short, highly branched
heterogeneous polymer consisting of pentose (xylose and arabi-
nose), hexose (galactose, glucose, and mannose), and acid sugars
(Saha, 2000). Mannose is the dominant hemicellulose sugar in soft-
woods, while xylose is dominant in hardwoods and agricultural
residues (Taherzadeh and Karimi, 2008). Hemicellulose is more
readily hydrolyzed compared to cellulose because of its branched
and amorphous nature. Lignin (1525% of total feedstock dry mat-
ter) is an aromatic polymer synthesized from phenylpropanoid
precursors. The phenylpropane units of lignin (primarily syringyl,
guaiacyl, and phydroxy phenol) are bonded together by a set of
linkages toforma verycomplexmatrix(Demirbas, 2008). This com-
plex matrix consists of a variety of functional groups, e.g., hydroxyl,
methoxyl, and carbonyl groups, which impart a high polarity to
the lignin macromolecule (Feldman et al., 1991). Lignin is consid-
ered to be difcult to use as a fermentation substrate because it
makes the biomass resistant tochemical andbiological degradation
(Taherzadeh and Karimi, 2008).
2.2. Conventional processes for lactic acid production by LAB
fromlignocellulosic biomass
Despite the advantages in its sustainability and availability,
the commercial use of lignocellulose in lactic acid production is
still problematic due to its complexity. The biochemical conver-
sion of lignocellulosic biomass requires several processing steps
designed to convert structural carbohydrates to monomeric sug-
ars, e.g., glucose, xylose, arabinose, and mannose. These sugars
Lignocellulosic
biomass
Chemical/physical
Pretreatment
Cellulose
Hydrolyzed
Hemicellulose
Enzymatic
hydrolysis
Lignin
Mainly
Glucose
Mainly
Pentoses
Fermentation
Lactic acid
Separation
Fig. 1. A general owchart of the conventional process for lactic acid production
fromlignocellulosic biomass materials.
can be fermented to lactic acid by wild-type and breeding strains,
with varying degrees of effectiveness. Once the technologies are
established and commercialized, a wide range of valuable products
could be produced fromlignocellulosic biomass. The conventional
processes for producing lactic acid from lignocellulosic biomass
include the following 4 main steps (Fig. 1):
(1) Pretreatmentbreaking down the structure of the lignocellu-
losic matrix.
(2) Enzymatic hydrolysisdepolymerizing lignocellulose to fer-
mentative sugars, such as glucose and xylose, by means of
hydrolytic enzymes.
(3) Fermentationmetabolizing the sugars to lactic acid, generally
by LAB.
(4) Separation and purication of lactic acidpurication of lactic
acid to meet the standards of commercial applications.
2.2.1. Pretreatment methods
The enzymatic susceptibility of native lignocellulose is difcult
and slow due to the association of cellulose and hemicellulose
with lignin (Schmidt and Thomsen, 1998). The main goals of
pretreatment are to remove lignin, separate cellulose and hemicel-
lulose, increase the accessible surface area, partially depolymerize
cellulose, and increase the porosity of the materials to aid in
the subsequent access of the hydrolytic enzymes (Chandel et al.,
2007; Hendriks and Zeeman, 2009; Kumar et al., 2009; Sun and
M.A. Abdel-Rahman et al. / Journal of Biotechnology 156 (2011) 286301 289
Cheng, 2002; Taherzadeh and Karimi, 2007; Zhang et al., 2009).
The hemicellulose should be removed or altered without degra-
dation for a high ultimate yield of sugars (Mosier et al., 2005).
Pretreatment includes physical (milling and grinding), chemical
(alkali, dilute acid, oxidizing agents, andorganic solvents), physico-
chemical (steamexplosion/autohydrolysis, hydrothermolysis, and
wet oxidation), and biological methods. Some methods disrupt
the lignincarbohydrate complex, and other disrupts the highly
ordered crystalline cellulose structure (Sun et al., 1995).
Different pretreatment methods have been extensively devel-
oped, including ammonia ber explosion and ammonia recycle
percolation (Jorgensen et al., 2007), lime (Kaar and Holtzaple,
2000), organosolv (Pan et al., 2006), liquid hot water (Antal, 1996),
ionic liquid (Dadi et al., 2006), alkaline pretreatment (Lau et al.,
2008), dilute acid and steam explosion (Laser et al., 2002; Mosier
et al., 2005; Parisi, 1989; Yang and Wyman, 2008), and enzymatic
treatment (Anderson et al., 2005; Converse, 1993; Hayn et al.,
1993; Ladisch et al., 1983). Among these methods, dilute acid pre-
treatment is still the method of choice in several model processes
(Wyman et al., 2005). The initial pretreatment reaction involves a
mild acid-catalyzing hydrolysis of the glycosidic bonds of hemi-
cellulose and the ether linkages in lignin (Fengel and Wegener,
1989), in which the organic acids, formed by the cleavage of labile
ester groups, catalyze the hydrolysis of hemicellulose. Fractiona-
tion is achieved by the enlargement of the inner surface. The effects
of different pretreatment methods upon different lignocellulosic
materials, e.g., corn stover (Chen et al., 2009), wheat straw (Sun
and Chen, 2008), switchgrass (Esteghlalian et al., 1997), rice straw
(Zhang and Cai, 2008), and sugarcane bagasse (Rabelo et al., 2009),
have been investigated.
The pretreatment process is a very critical stage in lignocellu-
lose bioconversion. If pretreatment is not sufciently efcient, the
resultant residue is not easily saccharied by hydrolytic enzymes
and, if it is too severe, toxic compounds can be produced that
inhibit microbial metabolism and growth (Kodali and Pogaku,
2006). Therefore, pretreatment has a great potential to inuence
the downstreamcosts by determining fermentation toxicity, enzy-
matic hydrolysis rates, enzyme loading, mixing power, product
concentrations, product purication, waste treatment demands,
power generation, and other process variables. An effective pre-
treatment process should meet the following requirements: (1)
highly digestible pretreated solid; (2) no signicant degradation
of sugars; (3) good recovery of high sugar concentrations; (4) sugar
formation by subsequent enzymatic hydrolysis; (5) effective at low
moisture contents; (6) form minimal or no microbial inhibitory
by-products; (7) require minimal energy input; (8) high degree of
simplicity; (9) not require biomass size reduction; (10) low cost
materials for the construction of pretreatment reactors and to be
easily managed at large volumes; (11) produce less residues; (12)
consume fewand cheap chemicals; and (13) have environmentally
acceptable features (Galbe and Zacchi, 2007; Lynd, 1996; Sun and
Cheng, 2002; Wu et al., 2011; Yang and Wyman, 2008).
2.2.2. Enzymatic hydrolysis
Enzymatic hydrolysis is the most promising means to yield fer-
mentable sugars from pretreated lignocellulosic biomass, and is
necessary to allowLABto utilize polysaccharides as a carbonsource
(Hinman et al., 1992; Lin and Tanaka, 2006; Lynd et al., 1996; Ogier
et al., 1999; Taniguchi et al., 2005; Yu and Zhang, 2004). The goal
of enzymatic hydrolysis is to depolymerize the polysaccharides in
the water-insoluble solid fraction that remains after pretreatment.
There are 2 general categories of enzymes necessary to convert
cellulose and hemicellulose into soluble sugars: cellulases and
hemicellulases, respectively. To maximize enzymatic hydrolysis,
mixtures of these enzymes are needed to increase hemicellulose
hydrolysis and thus increase the access of cellulase, leading to a
decreased hydrolysis time and process cost (hgren et al., 2007; Tu
and Saddler, 2010; Zhang et al., 2010a).
The rate of the enzymatic hydrolysis of cellulose is greatly
affected by its degree of polymerization (Chang and Holtzapple,
2000; Cohen et al., 2005; Kumar et al., 2008). Efcient degradation
and saccharication of cellulose require a synergistic reaction of
the following 3 classes of cellulolytic enzymes:
(a) Endo--1,4-glucanases (EG; EC 3.2.1.3): randomly hydrolyze
accessible intramolecular -1,4-glucosidic bonds of cellulose
chains and insert a water molecule in the -(1,4) bond, creating
a newreducing and non-reducing chain end pair.
(b) Exo--1,4-glucanases or cellobiohydrolases (CBH; EC 3.2.1.91):
cleave cellulose chains at the ends of the polymer, releasing
soluble cellobiose or glucose.
(c) -Glucosidases (-G; EC 3.2.1.21) (cellobiases): complete the
hydrolysis bycleavingcellobiose into2glucose molecules (Lynd
et al., 2002) and thus relieve the system from end-product
inhibition (Fujii et al., 1995). They are also active on cello-
oligosaccharides (Kumar et al., 2008).
Individual cellulases have very limited hydrolytic activity, while
a mixture of cellulases can exhibit a synergistic effect (Nidetzky
et al., 1994; Zhang et al., 2007). Extensive research has been per-
formedtoimprove the hydrolytic efciencyof suchenzymes (Baker
et al., 1998; Mais et al., 2002; Selig et al., 2008). In addition to these
3 major groups of cellulases, accessory or helper enzymes that
attack hemicellulose (Berlin et al., 2005) and lignin (Palonen and
Viikari, 2004) may also play a role in hydrolysis by clearing the
access of the main enzymes to cellulose.
Unlike cellulose, xylans are chemically quite complex, and
their degradationrequires multiple enzymes. Enzymatic hydrolysis
of hemicellulose requires endo-1,4--xylanase, -xylosidase, -
glucuronidase, -l-arabinofuranosidase, and acetylxylan esterase,
which act on xylan degradation and saccharication (Carvalheiro
et al., 2008; Saha, 2004), and -mannanase and -mannosidase,
which cleave the glucomannan polymer backbone (Kumar et al.,
2008). Although more enzymes are required for xylan hydrolysis
than for cellulose hydrolysis, the substrate is more easily accessible
because xylan does not formtight crystalline structures (Keshwani
and Cheng, 2009).
The hydrolytic efciency of a multi-enzyme mixture in the
process of lignocellulose hydrolysis depends on the properties of
the individual enzymes and their ratio in the multi-enzyme cock-
tail (Irwin et al., 1993; Zhou et al., 2009). Recently, Lin et al.
(2011) constructed a cellulase cocktail for a more efcient enzy-
matic hydrolysis of lignocellulose and a more rational utilization of
enzymes by using combinations of the 3 enzymes, 2 cellulases, and
1 xylanase.
2.2.3. Fermentation process with LAB
The hydrolysate of a lignocellulosic biomass is a mixture of
hexoses (e.g., glucose) and pentoses (e.g., xylose and arabinose).
Lignincannot be used for lactic acid fermentation. The effective uti-
lization of cellulose- and hemicellulose-derived sugars can reduce
the production cost of biomaterials by as much as 25% (Hinman
et al., 1989). Fermentation technologies must be cost competitive
withchemical synthesis to validate the use of biotechnological pro-
cesses on an industrial scale (Bustos et al., 2007). The key economic
drivers inthe fermentationprocess are highproduct yields, produc-
tivity, and the concentration of products formed, which strongly
inuences the product recovery costs. In order to achieve maxi-
mum lactic acid yield and productivity, a large number of studies
have investigated lactic acid fermentation by LAB from lignocel-
lulosic biomass in the eld of microbial technology, as described
290 M.A. Abdel-Rahman et al. / Journal of Biotechnology 156 (2011) 286301
in detail in Section 3.2. Biomass materials have been used as
substrates for lactic acid production by LAB, including lignocel-
lulose/hemicellulose hydrolysates (Karel et al., 1997), cottonseed
hulls (Vickroy, 1985), corncob (Guo et al., 2010; Moldes et al., 2006;
Shen and Xia, 2006; Wang et al., 2010), corn ber hydrolysates and
stalks (Saha and Nakamura, 2003; Vickroy, 1985), apple pomace
(Gullon et al., 2008), wood hydrolysate (Wee et al., 2004), beet
molasses (Gksungur andGvenc , 1999; Kotzamanidis et al., 2002),
sugar cane press mud and bagasse (Xavier and Lonsane, 1994;
Laopaiboonet al., 2010), cassava bagasse (Johnet al., 2006a,b; Rojan
et al., 2005), cellulose (Venkatesh, 1997; Singhvi et al., 2010), paper
sludge(Marques et al., 2008), carrot processingwaste(Pandeyet al.,
2001), molasses spent wash (Sharma et al., 2003), and wheat bran
(John et al., 2006c; Naveena et al., 2005a,b).
2.2.4. Separation and purication of lactic acid
In the traditional chemical separation process, the fermentation
brothis rst neutralized by calciumcarbonate. The calciumlactate-
containing broth is then ltered to remove cells, carbon treated,
decolored, evaporated, and acidied with sulfuric acid to produce
lactic acid and insoluble calcium sulfate (Datta and Henry, 2006).
Pure lactic acid is further obtained by hydrolysis, esterication, and
distillation. The disadvantages of this process include the produc-
tion of a large amount of calciumsulfate (gypsum) as a by-product
and high sulfuric acid consumption (Qin et al., 2010). Other alter-
native lactic acid separation technologies such as adsorption (Chen
and Ju, 1998), reactive distillation (Kumar et al., 2006), ultraltra-
tion and electrodialysis (Choi et al., 2002; Datta and Henry, 2006;
Hbov et al., 2004; Kim and Moon, 2001; Madzingaidzo et al.,
2002), and nanoltration (Gonzalez et al., 2008; Li and Shahbazi,
2006) have also been studied as lactic acid separation and puri-
cation processes that do not yield salt waste. These separation
processes are more cost and energy efcient when compared with
traditional chemical separation processes. In addition, they have
several advantages including the lack of energy-intensive phase
changes or potentially expensive solvents or adsorbents as well as
the potential for the simultaneous separation and concentration of
lactic acid (Li et al., 2008).
2.3. Difculties in using lignocellulosic biomass for efcient lactic
acid production by LAB
The effective utilization of lignocellulosic biomass has some
inherent limitations due to its seasonal availability, scattered dis-
tributions, and the high costs of storage and transportation (Lin
and Tanaka, 2006; Polman, 1994). In addition, the main problems
encountered in the efcient conversion of lignocellulosic biomass
to lactic acid are: (a) the resistant nature of biomass and pre-
treatment problems; (b) high cost enzymes and their feedback
inhibition; (c) formation of by-products due to the heterofermen-
tation of pentose sugars; and (d) carbon catabolite repression
caused by the heterogeneity of hydrolysate-sugar composition.
These obstacles are briey discussed as follows:
2.3.1. Resistant nature of biomass and pretreatment problems
Crystalline nonreactivity and, inparticular, resistance to hydrol-
ysis are the major problems for efcient lignocellulose utilization
(Kumar et al., 2008). Although various pretreatment procedures
have been evaluated, its utilization is a major drawback and affects
the total economy of the bioconversion of lignocellulosic biomass
(Zhang et al., 2009). Pretreatment is an expensive step and has a
major inuence on the cost of the enzymatic hydrolysis and fer-
mentation processes (Lynd et al., 1996; Wooley et al., 1999). Also,
lignin limits the rate of cellulose hydrolysis by acting as a physi-
cal barrier that prevents the digestible parts of the substrate from
being hydrolyzed (Chang and Holtzapple, 2000; Esteghlalian et al.,
2001). Different strategies have been examined to overcome the
nonproductive adsorption of lignin to cellulase by alkali extraction
and the addition of proteins or other additives, e.g., polyethylene
glycol and Tween (Brjesson et al., 2007; Pan et al., 2005).
Another obstacle is the release of inhibitory compounds dur-
ing the pretreatment process. Although the composition of the
released compounds depends not only on the type of lignocellu-
losic material and the chemistry but also on the characteristics of
the pretreatment process. These inhibitory compounds interfere
with the hydrolysis of cellulosic substrates by cellulase (Mes-
Hartree et al., 1987). In addition, many potentially inhibitory
compounds released by pretreatment processes have been iden-
tied for the microorganisms used for fermentation (Mussatto
and Roberto, 2004; Palmqvist and Hahn-Hgerdal, 2000). Although
detoxication methods such as bioabatement (Lopez et al., 2004;
Nichols et al., 2005) and overliming (Nilvebrant et al., 2003) have
been proposed, the efciency of fermentation is still in need of
improvement. The isolation of superior LAB strains or genetically
engineered strains that are resistant to inhibitors, and advances
and improvements in the pretreatment of lignocellulose are still
needed to reduce the overall cost. Recently, wild-type Lactobacillus
brevis S3F4 was shown to have strong resistance to fermentation
inhibitors such as ferulic acid and furfural (Guo et al., 2010). Biolog-
ical delignication with white rot fungi, which selectively degrade
lignin and leave cellulosic materials, also has potential advantages
such as low-capital cost, low-energy input, no chemical require-
ments, mild environmental conditions, and high yields without
generating polluting by-products (Chaudhary et al., 1994; Kuhad
and Johri, 1992; Kumar et al., 2009). However, drawbacks of this
process include its long treatment period and low hydrolysis rate
(Kumar et al., 2008; Sun and Cheng, 2002).
2.3.2. High-cost enzymes and their feedback inhibition
The high costs of enzyme production, feedback inhibition, and
the excessive enzymatic dosages necessary to hydrolyze pre-
treatedbiomass aresomeof thedrawbacks limitingthecommercial
application of lignocellulose hydrolysis as a lignocellulosic-lactate
industry (Himmel et al., 1999; Wooley et al., 1999). During cellu-
lose saccharication, the sugar end products of hydrolysis do not
accumulate quickly because the sacchariedglucose andcellobiose
inhibit the EGandCBHactivities byfeedbackinhibition(Adsul et al.,
2007b; Ghosh and Das, 1971; Holtzapple et al., 1990; Lee and Fan,
1983). Several methods have been developed to reduce this inhi-
bition, including the improvement of -G activity in the cellulase
system (Shen and Xia, 2004), removing the released sugars dur-
ing hydrolysis by ultraltration or simultaneous saccharication
and fermentation (SSF) (Rezaei et al., 2008), optimizing cellulase
enzyme conditions (i.e., temperature, pH, and enzyme loading
amounts) (Sun and Cheng, 2002; Ou et al., 2009), or supplying -G
during hydrolysis to avoid cellobiose accumulation (Caminal et al.,
1985; Moldes et al., 2001; Ramos and Saddler, 1994). Studies to
remove such forms of inhibition are still in progress.
2.3.3. Formation of by-products due to the heterofermentation of
pentose sugars
Lignocellulosic hydrolysates contain not only hexoses but also
pentoses. Hexoses can easily be fermented by LAB, while pen-
tose sugars cannot be fermented by most LAB (Hofvendahl and
Hahn-Hgerdal, 2000; Tanaka et al., 2002). In general, a few LAB
metabolize pentose sugars via the phosphoketolase (PK) pathway,
whichexhibits heterofermentationwithequimolar amounts of lac-
tic acidandacetic acidproducedandreaches only0.60C-mol/C-mol
in terms of the theoretical yield of lactic acid to pentose sugars
(Abdel-Rahman et al., 2011b; Oshiro et al., 2009; Patel et al., 2006;
Tanaka et al., 2002). This co-productionof acetic acid and lactic acid
also results in an increase of the lactic acid purication cost (Garde
M.A. Abdel-Rahman et al. / Journal of Biotechnology 156 (2011) 286301 291
et al., 2002; Patel et al., 2006); therefore, this approach is not con-
ducive for the industrial fermentation of pentoses to lactic acid. To
overcome this obstacle, we previously isolated a LAB strain, Lac-
tococcus lactis IO-l, which could utilize xylose with a high lactate
yield and lowacetate production (Tanaka et al., 2002). Okano et al.
(2009a,b) engineered the LAB Lb. plantarum strain to produce lac-
tic acid fromxylose and arabinose via the pentose phosphate (PP)
pathway, leadingtohomolactateproduction. Recently, we reported
that a novel LAB, Enterococcus mundtii QU 25, consumed xylose
homofermenatively without acetate production (Abdel-Rahman
et al., 2010a, 2011b); therefore, the purication cost will be sig-
nicantly decreased with this strain.
2.3.4. Carbon catabolite repression caused by the heterogeneity
of the hydrolysate-sugar composition
One of the major obstacles in using lignocellulosic biomass as
a feedstock is the inherent heterogeneity of its sugar composition
(Kim et al., 2010a). To achieve maximum product yield and pro-
ductivity, the complete utilization of these sugars is essential (Kim
et al., 2010b; Saha, 2003). In many LAB, fermentation of mixed
carbohydrates is achieved sequentially, whereby the utilization of
glucose represses the consumption of alternative sugars due to car-
bon catabolite repression (Saier, 1998; Stulke and Hillen, 1999;
Titgemeyer and Hillen, 2002). This sequential utilization of mixed
sugars makes the fermentation process complex and often reduces
the yield and productivity (Bothast et al., 1999). Carbon catabolite
repression by LAB has been reported in many strains, e.g., Lb. casei
(Gosalbes et al., 1999; Veyrat et al., 1994; Viana et al., 2000), Lb.
pentosus (Chaillou et al., 1999, 2001; Mahr et al., 2000), Lb. plan-
tarum (Marasco et al., 1998), Lb. sakei (Zuniga et al., 1998), and Lb.
delbrueckii (Morel et al., 1999; Schick et al., 1999). AfewLAB strains
have been reported to simultaneously consume lignocellulose-
derived sugars, e.g., Lb. brevis (Guo et al., 2010; Kim et al., 2009),
Lb. plantrum (Guo et al., 2010), and our newly isolated strain E.
mundtii QU 25 (Abdel-Rahman et al., 2010a,b, 2011a). Mixed LAB
cultures havealsobeenusedtomaximizetheyieldandproductivity
frommixed sugars (Cui et al., 2011; Taniguchi et al., 2004). There-
fore, it is essential to isolate LAB strains or to establish genetically
engineered strains for efcient lignocellulose utilization.
3. Fermentative lactic acid production by LAB
3.1. Improvement of lactic acid production by LAB in the eld of
microbial technology
It has generally been observed that pH, nutrient concentration,
substrate concentration, end products concentration, and tem-
perature signicantly affect the growth of LAB and lactic acid
production. These factors may decrease cell density and the lac-
tic acid titer, yield, and productivity in some cases. Researchers in
the eldof microbial technology have conductednumerous studies
to establish an efcient method of lactic acid production by LAB.
In lactic acid fermentation, low pH has an inhibitory effect on
cellular metabolismand lactic acid production. The majority of LAB
cannot grow below pH 4, although the pK
a
of lactic acid is 3.78
(Adachi et al., 1998); therefore, neutralizing agents such as cal-
ciumcarbonate, sodiumhydroxide, or ammoniumhydroxide must
be added to keep the pH at a constant value in order to reduce
the inhibitory effects of lowpH. pH-controlled batch fermentation
signicantly improves lactic acid production, yield, and produc-
tivity by different LAB strains, e.g., Lb. delbrueckii (Tashiro et al.,
2011), E. mundtii QU 25 (Abdel-Rahman et al., 2011a,b), and E. fae-
cium (Shibata et al., 2007). Batch fermentation is a simple closed
culture systemthat contains an initial and limited amount of nutri-
ent, and nothing is added during fermentation except possibly acid
or alkali for pH control. The low levels of nutrient limit the cell
concentration, nal lactic acid concentration, and productivity. It
was reported that the addition of nutrients to a culture broth of E.
mundtii QU 25 during batch fermentation increased the cell mass
and lactic acid production and productivity (Abdel-Rahman et al.,
2011a). In addition, batch fermentation was superior to contin-
uous fermentation in some respects, particularly lactic acid titer
(Buyukgungor and Bueschelberger, 1986; Hofvendahl and Hahn-
Hgerdal, 2000; Nomura et al., 1987). High lactic acid production
has been obtained with batch fermentation of LAB with the pro-
duction of 150g/L l-lactate fromglucose (Bai et al., 2004), 87.2g/L
d-lactate from glucose (Tashiro et al., 2011), and 119g/L l-lactate
(Abdel-Rahmanet al., 2011a) and80g/L d-lactate (Joshi et al., 2010)
fromcellobiose.
Substrate inhibition always occurs at high sugar concentra-
tions (Ding and Tan, 2006; Gatje and Gottschalk, 1991; Oshiro
et al., 2009). To overcome or reduce substrate inhibition, fed-batch
cultures were a better fermentation systemthan batch and contin-
uous cultures because they allowfor an increased maximumviable
cell concentration and prolonged culture lifetime, which result in
product accumulation to a higher concentration. Bai et al. (2004)
developed a process for the production of ammoniumlactate with
Lb. lactis in pH-controlled fed-batch fermentation with 161.2g/L
of lactic acid. Ding and Tan (2006) developed a high lactic acid
concentration process using 4 different fed-batch feeding strate-
gies with Lb. casei: pulse fed-batch, constant feed rate fed-batch,
constant residual glucose concentrationfed-batch, andexponential
feed rate fed-batch fermentations. They generated up to 210g/L of
lactic acid and a 97% yield with an exponential rate of feeding glu-
cose solution and yeast extract (Ding and Tan, 2006). However, in
all fed-batch technologies, the substrate concentration in the fer-
mentation broth is unstable, thereby generating more stress on the
producing strain. Recently, a method was developed to control the
concentration of the substrate through the automatic adjustment
of pH. Using this method, 96.3g/L and 170g/L of lactic acid were
obtained with Lb. lactis-11 (Zhang et al., 2010b) and Lb. rhamnosus
LA-04-1 (Li et al., 2010), respectively. Chang et al. (2011) suggested
multi-stage continuous high cell density culture as a new produc-
tion platform for obtaining high lactic acid titers (212.9g/L) and
productivity (10.6gL
1
h
1
) with Lb. rhamnosus. In addition to fed-
batchfermentation, continuous andsemi-continuous fermentation
processes have been used for lactic acid production by reducing
substrate inhibition (Amrane and Prigent, 1996; Nolasco-Hipolito
et al., 2002; Tashiro et al., 2011). The choice of the most suitable fer-
mentation process will depend upon the kinetic properties of the
LAB species used, the type of substrates, and the economic aspects
of the process.
High productivity was achieved with a high LAB cell density
without reducing the yield (Ohleyer et al., 1985). Many reports
showed that high cell density by cell recycling through ltration
drastically increased lactic acid productivity with Lb. helveticus
(Kulozik and Wilde, 1999), E. faecium(Shibata et al., 2007), and Lb.
delbrueckii (Tashiro et al., 2011). The cell recycling system, along
with repeated batch and continuous processes, generated a high
cell concentration and productivity in these processes (Kwon et al.,
2001; Oh et al., 2003). The immobilization of cells has been one
of the means used for high cell retention in bioreactors; however,
manystudies were not verysuccessful interms of yieldandproduc-
tivity (Cotton et al., 2001; Gksungur and Gvenc , 1999; Senthuran
et al., 1999; Zhang et al., 2011).
One of the major problems associated with lactic acid pro-
duction by fermentation is end-product inhibition; therefore, to
decrease the inhibitory effect of lactic acid during fermentation, it
must be selectively removed from the fermentation broth. Many
attempts have been directed to develop processes that remove
lactic acid from the fermentation broth, e.g., extraction from the
292 M.A. Abdel-Rahman et al. / Journal of Biotechnology 156 (2011) 286301
fermentation broth (Hano et al., 1993; Honda et al., 1995; Iyer and
Lee, 1999a,b; Yabannavar and Wang, 1991; Ye et al., 1996), elec-
trodialysis (Boyaval et al., 1987; Hongo et al., 1986; Kimand Moon,
2001; Min-Tian et al., 2005; Nomura et al., 1998; van Nispen and
Jonker, 1991; Vonktaveesuk et al., 1994), and nanoltration mem-
branes and ion exchange resins (Jeantet et al., 1996; Monteagudo
andAldavero, 1999; Srivastava et al., 1992; Vaccari et al., 1993). The
continuous removal of lactic acid with extraction or electrodialysis
results in even higher lactic acid concentrations and yields com-
pared to conventional batch fermentation processes. Li et al. (2004)
developed a bioreactor combining conventional electrodialysis and
bipolar membrane electrodialysis for product removal and pHcon-
trol inlactic acidfermentation. Min-Tianet al. (2005) achievedhigh
lactic acid productivity and yields with a continuous electrodial-
ysis fermentation system. Even though these methods lower the
purication cost, the price of the membranes and decreases in the
permeate owrate make the process less cost efcient and are still
considerable drawbacks. Other methods, e.g., two phase systems,
have been developed; however, the extracting material must be
biocompatible and not harm the organism in order to be efcient
(Planes, 1998).
The inuence of temperature on lactic acid fermentation is
related to the growth kinetic parameters of LAB, lactic acid pro-
duction, and substrate consumption. Among LAB, most lactic acid
productivity studies have been conducted at temperatures of
3043
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