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New application of Bacillus strains for optically

pure l-lactic acid production: general overview and
future prospects

Pramod Poudel, Yukihiro Tashiro & Kenji Sakai

To cite this article: Pramod Poudel, Yukihiro Tashiro & Kenji Sakai (2015): New application
of Bacillus strains for optically pure l-lactic acid production: general overview and future
prospects, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1095069

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 Bioscience, Biotechnology, and Biochemistry, 2015
Review
New application of Bacillus strains for optically pure L-lactic acid production:
general overview and future prospects
Pramod Poudel1, Yukihiro Tashiro1,2,* and Kenji Sakai1,2
1
Laboratory of Soil and Environmental Microbiology, Division of Systems Bioengineering, Department of Bioscience
and Biotechnology, Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University,
Fukuoka, Japan; 2Laboratory of Microbial Environmental Protection, Tropical Microbiology Unit, Center for
International Education and Research of Agriculture, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Received June 26, 2015; accepted September 6, 2015
http://dx.doi.org/10.1080/09168451.2015.1095069
Downloaded by [NUS National University of Singapore] at 14:55 26 November 2015
Members of the genus Bacillus are considered to
be both, among the best studied and most com-
monly used bacteria as well as the most still unex-
plored and the most wide-applicable potent bacteria
because novel Bacillus strains are continuously
being isolated and used in various areas. Production
of optically pure L-lactic acid (L-LA), a feedstock for
bioplastic synthesis, from renewable resources has
recently attracted attention as a valuable application
of Bacillus strains. L-LA fermentation by other pro-
ducers, including lactic acid bacteria and Rhizopus
strains (fungi) has already been addressed in several
reviews. However, despite the advantages of L-LA
fermentation by Bacillus strains, including its high
growth rate, utilization of various carbon sources,
tolerance to high temperature, and growth in simple
nutritional conditions, it has not been reviewed. This
review article discusses new findings on LA-produc-
ing Bacillus strains and compares them to other
producers. The future prospects for LA-producing
Bacillus strains are also discussed.
Key words: lactic acid producing Bacillus strains;
optical purity; high temperature;
biomass; metabolic engineering
Genus Bacillus is Gram staining-variable
(Gram-positive, Gram-positive in the early stages of
growth, or Gram-negative), endospore-forming, aerobic
or facultative anaerobic rods with a wide range of
nutritional requirements (simple to complex), growth
conditions (ranging from acidophilic to alkaliphilic,
psychrophilic to thermophilic, and halotolerant), DNA
base composition, and metabolic diversity.1) Many
Bacillus strains have been widely used as producers of
fermented foods (e.g. natto in Japan),2) enzymes
(e.g. amylases, pullulanases, and proteases),3) purine
nucleotides,4) biopesticides,5) poly-(γ-glutamic acid),6)
7)
L-lactic acid, , etc. including probiotics for animals
and humans,8) promoters of plant growth,9), and
*Corresponding author. Email: tashiro@agr.kyushu-u.ac.jp
© 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
biocontrollers of plant disease10) in the broad areas of
food, cosmetics, medicine, agriculture, and chemistry.
The first Bacillus strain was discovered in 18721), and
new Bacillus strains have been continuously isolated
from different sources.11) The number of Bacillus
strains is presently around 300 species (http://www.bac
terio.net/bacillus.html). This strongly encourages
researchers around the world to screen for novel Bacil-
lus isolates and to determine new functions or applica-
tions. Among the ca. 2277 total genera in the domain
bacteria (http://www.bacterio.net), those of the genus
Bacillus can be considered to not only be among the
best studied and most commonly used, but also the
most still unexplored and most wide-applicable potent
bacteria for human life. Recently, production of opti-
cally pure lactic acid (LA) has attracted attention as a
feasible application of Bacillus strains.7)
LA has applications in the agricultural, medicinal,
food, pharmaceutical, and chemical industries,12–14) in
particular, there is much interest in optically pure (L or
D-isomer) LA for the synthesis of high-quality polylac-
tic acid (PLA). PLA is used for the production of
biodegradable and environmentally friendly bio-plastics
as a substitute for petrochemical-based plastics.15) Due
to the numerous applications for this material, it has
been estimated that global LA production demand is
130,000–150,000 tons per year.16) Currently, LA is
mainly produced by microbial fermentation processes
rather than by traditional chemical synthesis. The tradi-
tional chemical synthesis of LA is based on the hydrol-
ysis of lactonitrile derived from acetaldehyde and
hydrogen cyanide, which leads to the formation of
racemic mixtures of DL-LA.12) Optically pure D-LA or
L-LA can only be produced by microbial fermentation
using specific microbes.17–19) Another significant
advantage of microbial fermentation over traditional
LA chemical synthesis is that various plant-derived
renewable biomass sources can be used as substrates
for LA production by microbial fermentation.
In general, fermentative LA production is mostly
accomplished by members of the domain bacteria. A
 2 P. Poudel et al.
few micro-organisms in the domains archaea (Scenedes-
mus sp. and Nannochlorum sp.) and eukaryota (fungi
such as Rhizopus oryzae) have been shown to produce
LA.7) Within the domain bacteria, all the wild-type LA
producers belong to the phylum Firmicutes and the class
Bacilli that includes two orders: the Lactobacillales and
Bacillales. The order Lactobacillales contains lactic acid
bacteria (LAB), which are known as LA producers,
including the genera Lactobacillus (L-, D- or DL-LA),
Enterococcus (L-LA), Lactococcus (L-LA), Carnobac-
terium (D-LA), Pediococcus (L- or DL-LA), Leuconostoc
(D-LA), Streptococcus (L-LA), Weissella (D- or DL-LA)
and so on. On the other hand, within the order Bacil-
lales and the family Bacillaceae (48 genera to date),
most bacteria in the genus Bacillus have been reported
as potent L-LA producers. Only two species of genus
Geobacillus (G. stearothermophilus)20) and Halo-
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lactibacillus (H. halophilus)21) have been reported as L-
LA producers. In the domain Eukaryota, several wild-
type fungi, especially R. oryzae22) and Rhizopus micro-
sporus,23) are known as potent L-LA producers. In addi-
tion, some genetically modified bacteria and yeast have
been developed for either D-LA or L-LA production
depending upon the genetic manipulation.24) In the
future, approaches to screen for new wild-type LA-
producers and to genetically modify LA producers for
targeted processes will be performed actively and
continuously.
Fermentative LA production by LAB25,26) and fungi22)
have been already reviewed and their recent issues have
been addressed. In contrast, despite their interesting fea-
tures such as high growth rates, tolerance to high temper-
atures, growth in simple nutritional conditions, and the
ability to ferment a wide range of sugars including pen-
toses and hexoses to produce optically pure L-lactic acid
(up to 100%), the recent developments in LA fermenta-
tion by Bacillus strains have not yet been reported.27)
Therefore, in this review article, we will provide an over-
view of the special features of LA-producing Bacillus
strains in comparison to other LA producers, report the
efficient utilization of various biomass resources by
Bacillus strains, elucidate the major metabolic pathways
involved and the genetic approaches for improving LA
production, as well as make concluding remarks and
summarize the future prospects.
I. Special features of LA-producing
Bacillus strains and comparison to other
LA producers
Here, we compared special features (nutritional
requirement, tolerance to high temperature, tolerance to
pH, ability to produce optical pure LA, oxygen demand,
and sugar metabolism) among Bacillus, LAB, and Rhi-
zopus sp. (Table 1), and described them in each section.
I.i. Nutritional requirements
Bacillus strains are commonly isolated from nutri-
tionally poor environments such as soil under various
climatic conditions.28) In contrast, LAB are only iso-
lated from nutritionally rich environments that contain
high amounts of amino acids, vitamins, and nucleotides
in addition to a carbon source,29,30) while Rhizopus sp.
requires fewer nutrients for the growth compared to
LAB and have the ability to grow in simple inorganic
salt containing media.22) Yeast extract and peptone are
rich in nitrogen, vitamins, and other nitrogenous
growth-stimulating factors. Culture media for LAB
require supplementation with organic nitrogen sources
such as yeast extract, peptone, and other proteinous
substances, which ultimately increases the cost of the
medium formulation.15,28) Altaf et al.31) reported that
yeast extract accounted for ca. 38% of the total med-
ium cost for LA production by LAB. Therefore, it is
necessary to use cheap substitutes for these expensive
substances or to employ LA producers that can grow
well at low concentrations of organic nitrogen sources.
Interestingly, Bacillus strains profusely grow and
produce L-LA in a simple mineral salt medium contain-
ing a very small amount of yeast extract.32,33) Many
researchers have already described the minimal nutri-
tional requirements for growth and LA production by
Bacillus strains.33,34) For Bacillus strains, ammonium
sulfate,35) NH4Cl,36) low grade peanut meal,37) corn
steep liquor,38) excess sludge hydrolysate,39) and yeast
autolysate32) have been reported as cheap alternative
nitrogen supplements for LA fermentation. In addition,
Meng et al.37) reported that Bacillus sp. strain WL-S20
efficiently produced L-LA using inexpensive peanut
meal as a nitrogen source and yields 20% higher LA
concentration than that using yeast extract. Further-
more, among the several sugars (glucose, fructose,
mannose, arabinose, xylose, lactose, sucrose, maltose,
and starch), it is reported that glucose and sucrose
exhibited the highest LA concentration of ca. 18 g/L
by Bacillus sp. strain WL-S20.37) Meantime, LA pro-
duction by Bacillus sp. WL-S20 LA using various glu-
cose concentrations (40, 60, 80, 100, 110, and 120 g/L)
results in different LA concentrations (16, 17, 19, 17,
17, and 16 g/L, respectively) and LA yield (0.73, 0.85,
0.95, 0.74, 0.78, and 0.67 g/g, respectively) using the
same strains.37) These findings reveal that types of car-
bon sources affect LA production as well as sugar con-
centrations. On the other hand, Ma et al.39) reported
that LA production (98.1 g/L of L-LA, 0.98 g/g yield,
99.6% optical purity, 3.63 g/L h productivity) using
10 g/L excess sludge hydrolysate was comparable to
LA production (98.9 g/L L-LA, 0.98 g/g yield, 99.8%
optical purity, 3.92 g/L h productivity) using 2 g/L
yeast extract by B. coagulans NBRC12583T. Therefore,
it is better to develop microbial strains that can produce
LA in a simple and nutritionally low medium.
I.ii. Tolerance to high temperature
LA-producing Bacillus strains show optimum growth
temperatures between 45 and 60 °C and limited growth
up to 70 °C, whereas the optimum growth temperatures
for most LAB range from 30 to 43 °C.27) As excep-
tions, the thermotolerant D-LA producer Lactobacillus
delbrueckii subsp. lactis QU 4140) and the L-LA pro-
ducer Enterococcus faecium QU 5041) are reported to
be tolerant to temperatures up to 55 °C. In case of
Rhizopus sp., optimum temperatures for LA production
range from 27 to 35 °C, and the maximum temperature
at 45 °C for cell growth is reported for Rhizopus
 Bacillus strains for optically pure L-lactic acid production
Table 1. Special features of LA-producing Bacillus strains and comparison to other LA producers.
Characterization Bacillus strains
Nutritional Requirement of less nutrients to grow
requirements than LAB
for growth Ability to grow in mineral salt
medium containing low-organic
nitrogen sources (e.g. yeast extract)
Tolerance to Optimum temperature for LA
high production: 45–60 °C
temperature Maximum temperature for cell growth:
70 °C with B. coagulans NBRC
12583
Tolerance to Optimum pH for LA production:
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pH 5.0–9.0
Sensitive to acidic pH (<5.0) and
alkaline pH (>9.0) except for tolerant
to pH at 10 with Bacillus sp. WL-S20
Ability to L-LA producers (Major strains):
produce 97–100% of optical purity
optically D-LA producer (B. laevolacticus NCIB
pure LA 10269): >99% of optical purity
Sugar Hexoses: ferment to LA and by-
metabolism products (acetic acid, formic acid,
2,3-butanediol) via EMP (max. LA
yield >0.90 g/g)
Pentoses: ferment to LA via PPP and
EMP (max. LA yield >0.90 g/g)
Oxygen Facultatively anaerobes
demand In the absence of O2: grow
oligosporus TISTR 3518.22,23) Many thermotolerant
LA-producing Bacillus strains have been isolated from
various environments. For instance, Poudel et al.33) iso-
lated thermotolerant Bacillus sp. MC-07 from compost
and produced L-LA directly from starch at 50 °C. High
temperature LA fermentation has several advantages
compared to mesophilic fermentation.
First, high-temperature fermentation would signifi-
cantly reduce the cooling cost compared to that
incurred using mesophiles, which require temperature
control, facilitated by the inclusion of additional cool-
ing systems to maintain growth during fermentation.7)
Second, high-temperature fermentation minimizes con-
tamination; B. coagulans JI12 produced 137.5 g/L
L-LA with a high yield of 0.98 g/g, productivity of
4.4 g/L h, and optical purity of 99.6% from D-xylose at
50 °C even under non-sterile conditions.42) Third, the
3
Lactic acid producers
Lactic acid bacteria (LAB) Rhizopus sp.
Requirement of nutritionally rich Requirement of less nutrients to grow than
medium (amino acids, vitamins, and LAB
nucleotides) Ability to grow in simple inorganic salt
containing media
Optimum temperature for LA Optimum temperature for LA production:
production: 30–43 °C with general 27–35 °C
LAB, 50 °C with E. faecium QU50 Maximum temperature for cell growth: 45 °C
Maximum temperature for cell with Rhizopus oligosporus TISTR 3518
growth: 55 °C with E. faecium
QU50
Optimum pH for LA production: Optimum pH for LA production: 5.0–6.0
4.0–7.0 Tolerant to low pH ~3.5 (R. oryzae ATCC
Tolerant to low pH ~4.0 (Lb. 52311)
plantarum ATCC 21028) and to
high pH ~10.5 (Alkalibacterium
olivoapovliticus)
L-LA producers (genera of L-LA producers (Major strains): 98.5–100% of
Lactococcus, some Lactobacillus, optical purity L-LA
Enterococcus, etc.) : >~95%
D-LA producers (genus
Leuconostoc, Lactobacillus
delbrueckii subsp. lactis, etc.) :
>~99%
Hexoses: ferment to LA and by- Hexoses: ferment to LA and by-products
products (acetic acid, formic acid, (ethanol, CO2, fumaric acid, malic acid, citric
ethanol) via EMP (max. LA yield acid,) via EMP and tricarboxylic acid pathway
>0.90 g/g) (max. LA yield ≈ 0.71–0.79 g/g)
Pentoses: ferment to LA via PPP Pentoses: ferment to via PPP (max. LA yield ≈
(max. LA yield >0.90 g/g) and PKP 054–0.65 g/g)
(max. LA yield < ca. 0.60 g/g)
Facultatively anaerobes Obligatory aerobes
In the presence of O2: grow and In the presence of O2: grow very well using O2
tolerant to O2, but cannot utilize O2 as the electron acceptor in metabolism
as the electron acceptor in In the absence of O2: cannot grow
metabolism
In the absence of O2: grow very
well
fermentation temperature affects microbial activity and
substrate conversion rate.43) Heriban et al.32) reported
that a high temperature is beneficial for the synthesis of
LA, as it improves the rate of the biochemical reactions
and results in the higher activity of micro-organisms.
Fourth, a high temperature is beneficial for simultane-
ous saccharification and fermentation (SSF) because
several hydrolytic enzymes (cellulase, amylase) exhibit
optimum temperatures higher than ca. 50 °C.39,44)
According to Ou et al.44), thermophilic B. coagulans
36D1 (ca. 26.99 g/L LA and 0.903 g/g LA yield)
showed better LA production performance than did
mesophilic LAB (Lactococcus lactis subsp. lactis
NRRL B-4449; ca. 20.34 g/L LA and 0.709 g/g LA
yield) by SSF of 40 g/L crystalline cellulose carried
out using 20 FPU-cellulase/g-cellulose at 50 and 40 °C,
respectively. Therefore, high-temperature fermentation
 4 P. Poudel et al.
minimizes cooling cost, has no need for sterilization,
and improves the efficiency of enzymatic hydrolysis in
SSF.
I.iii. Tolerance to pH
One of the key parameters in LA fermentation is pH.
Similar to LAB,7) Bacillus strains also show an opti-
mum pH for growth or LA production at neutral, but
are sensitive to acidic pH (under pH 5.0). The pH for
LA production by Bacillus strains is 5.0–9.0, whereas
the pHs for LA production by most of LAB and Rhizo-
pus species are 4.0–7.016,26,45) and 5.0–6.0,22) respec-
tively. The ability of some Bacillus (tolerance up to pH
10.0 with Bacillus sp. WL-S20) and LAB (tolerance up
to pH 10.8 with Alkalibacterium olivoapovliticus)46)
strains to tolerate alkaline pH can minimize contamina-
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tion.21) At pH 9.0, Bacillus sp. WL-S2037) and H. halo-
philus21) efficiently produced L-LA (225 g/L, optical
purity >99%, yield 0.99 g/g, productivity 1.04 g/L h;
65.8 g/L, optical purity of 98.3%, yield 0.76 g/g,
productivity 0.83 g/L h, respectively) from D-glucose.
Fermentation at acidic pH values close to the pKa of
LA (3.86) can reduce the amount of agents added to
neutralize the LA produced; however, most LA-produc-
ing Bacillus strains are more sensitive to acidic condi-
tions than LAB (tolerance up to pH ~4.0 with
Lactobacillus plantarum ATCC 21028)45) and fungi
(tolerance up to pH ~3.5 with R. oryzae ATCC
52311).47) Comparison of one species in the genus
Bacillus, Bacillus acidicola, was reported to tolerate
pH values up to 3.5 lower than the pKa value of LA;
however, the efficiency of LA fermentation by this
strain under acidic conditions has not been well stud-
ied.48) Therefore, additional studies are required to
improve L-LA fermentation by Bacillus strains under
acidic conditions, using techniques such as genome
shuffling, which has enhanced the pH tolerance of Lac-
tobacillus rhamnosus ATCC 11443 by up to 3.6.49)
Under non-controlled pH conditions, Bacillus strains
are not able to accumulate high concentrations of LA
because of their intolerance to acidic pH values. Under
controlled pH, the LA concentration and productivity
in batch fermentation by Bacillus strains were found to
be improved. The pH of the fermentation broth is con-
trolled by various neutralizing agents such as
CaCO3,50) Ca(OH)2,50) NaOH,42) KOH,51) and NH3.18)
Among these neutralizing agents, CaCO3 and
Ca(OH)242,50) have been widely used. However, one
disadvantage of using them is the formation of calcium
salts such as calcium lactate in the fermentation broth,
which requires an additional acidification procedure
using H2SO4 during the recovery process to obtain free
LA.52) This disadvantage can be minimized using NH3
as neutralizing agent because calcium lactate is not
formed and NH3 can be recovered in the supernatant of
culture broth. Sakai and Ezaki18) reported L-LA fermen-
tation (concentration, 86 g/L; productivity, 0.72 g/L h,
optical purity, 97%, yield, 0.97 g/g) by B. coagulans
NBRC 12583T using NH3 as a neutralizing agent to
maintain the pH at 7.0 from food waste. Furthermore, a
total recycled PLLA production process has been pro-
posed, in which NH3 is recycled and utilized as the
neutralizing agent for the subsequent LA fermentation
after recovery of the free L-LA.53)
I.iv. Ability to produce optical pure LA
Optical purity of LA produced in fermentation is
mainly dependent on gene-expression levels and the
enzyme activities of L-lactate dehydrogenase (L-LDH),
D-lactate dehydrogenase (D-LDH), and lactate racemase.
L-LDH and D-LDH catalyze the conversions of pyruvic
acid to L-LA and D-LA, respectively, and almost all
LAB possess both D-LDH and L-LDH.54) In contrast, a
few LAB have been reported to possess lactate race-
mase, which transforms D-LA to L-LA.55) Therefore,
the optical purity of LA varies considerably among
LAB including L-LA producers (>95%; genera of Lac-
tococcus, some Lactobacillus, and Enterococcus, etc.),
DL-LA producers, and D-LA producers (>99%; Leu-
conostoc genus, and Lactobacillus delbrueckii subsp.
lactis, etc. Majority of Rhizopus sp. produce L-LA with
an optical purity 98.5–100%.22) In contrast, most
LA-producing Bacillus strains possess both L-LDH
(encoded by the ldhL gene) and D-LDH (encoded by
the ldhD gene), the enzymes responsible for the pro-
duction of L-LA and D-LA, respectively, but lack the
lactate racemase enzyme,54) even though they produce
L-LA with an optical purity of greater than 97%. Fur-
thermore, it has been reported that the optical purities
of L-LA produced by Bacillus strains are variable even
within same species, such as the potent L-LA-producing
B. coagulans strains, which ranges from 97–100%.50,56)
However, the mechanism underlying the synthesis of
high optical purity LA by Bacillus strains has not been
studied well.
I.v. Oxygen demand
LA-producing Bacillus strains and LAB are faculta-
tive anaerobes, whereas Rhizopus species are obligatory
aerobes (Table 1) and the demands of oxygen for cell
growth and LA production are different among three
LA-producers. LA-producing Bacillus strains aerobi-
cally grow very well using oxygen as the electron
acceptor with less LA production56), while anaerobic
culture exhibit less cell growth, but mainly produce
LA. Although aerobic culture by LAB show cell
growth due to their tolerance to oxygen, LAB cannot
utilize oxygen as the electron acceptor, which results in
lower LA production than anaerobic culture.12,57) On
the other hand, Rhizopus species can grow under only
aerobic condition.22)
One of the most interesting findings is that B. coagu-
lans can produce L-LA as a major fermentative product
either under strict anaerobic or aerobic (high oxygen)
conditions. In particular, B. coagulans strain M21,
accumulated 100% optically pure L-LA (12.5 g/L, yield
0.62 g/g, productivity 0.31 g/L h) from 20 g/L D-glu-
cose under high oxygen conditions.56) In contrast, it
has been reported that LAB (L. plantarum, L. rhamno-
sus) degrade LA, especially in presence of oxygen as
an electron accepter, and convert LA produced from
sugars (glucose and cellobiose) to acetic acid, H2O2,
and CO2.26,58)
 Bacillus strains for optically pure L-lactic acid production
I.vi. Sugar metabolism
LA-producing Bacillus strains metabolize pentose
and hexose sugars by the pentose phosphate pathway
(PPP) and the Embden-Meyerhof-Parnas pathway
(EMP), respectively, to produce LA as a major end
product homofermentatively (Fig. 1).59) In EMP, hexose
sugars such as glucose (six carbons) in the presence of
ATP are phosphorylated by hexokinase to form glucose
6-phosphate, which is then isomerized by phosphoglu-
cose isomerase to fructose 6-phosphate (F6P). F6P is
further phosphorylated to fructose 1,6-bisphosphate
(FBP) by phosphofructokinase with ATP consumption.
FBP is then split into glyceraldehyde 3-phosphate
(GAP) and dihydroxyacetone phosphate (DHAP),
which is catalyzed by fructose bisphosphate aldolase.
GAP is converted to pyruvate via two substrate-level
phosphorylations. Ultimately, pyruvate is reduced
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together with NADH oxidation to produce L-LA or
D-LA by L-LDH or D-LDH, respectively. In some LAB,
lactate racemase, which catalyzes the interconversion of
L-LA/D-LA, has been reported to result in the formation
of a racemic mixture of LA in EMP.55) During EMP,
the theoretical yield of LA from glucose is 1 g/g
(2 mol/mol) in homolactic fermentation.
In PPP, pentose sugars, such as D-xylose, are con-
verted to D-xylulose by xylose isomerase, which is fur-
ther phosphorylated to xylulose 5-phosphate (X5P) by
xylulose kinase. X5P is subsequently metabolized by
transaldolase and transketolase to form GAP. GAP is
then converted to pyruvate and then to LA via EMP. In
the PPP, 3 mol of D-xylose is converted to 5 mol of
LA, with a theoretical yield of 1 g/g (1.67 mol/mol),60)
and the highest yield (0.98 g/g) of LA was reported for
B. coagulans JI12.41) In contrast, a few LAB can
metabolize pentose sugars (D-xylose).7) Most pentose-
utilizing LAB mainly utilize the phosphoketolase (PK)
pathway to convert pentose sugar to LA and byprod-
ucts (acetate or ethanol) with a theoretical yield of only
0.6 g/g (1 mol/mol), which leads to heterofermenta-
tion.61) As exceptions, the LAB Enterococcus mundtii
QU 25,26) E. faecium QU 50,41) and some metaboli-
cally engineered LAB such as L. plantarum62) have
been reported to produce L-LA from D-xylose in a
homofermentative manner via PPP/EMP with yields of
0.904, 0.89, and 0.89 g/g, respectively. In Rhizopus sp.,
pentose (xylose) sugar is metabolized by PPP to
produce LA with a yield of 0.54–0.65 g/g and while
hexose (D-glucose) is metabolized via EMP and tricar-
boxylic acid pathway to produce LA and other byprod-
ucts (ethanol, CO2, fumaric acid, citric acid, malic
acid) with LA of yield 0.71–0.79 g/g.22,63,64) Therefore,
EMP and PPP are much better suited for increasing LA
yield, which are common pathways in LA-producing
Bacillus strains.
Tongpim et al.56) recently proposed that L-LA-pro-
ducing thermotolerant B. coagulans strains formed a
distinct cluster in a phylogenetic tree based on their
16S rRNA gene sequence and showed heterogeneity in
sugar metabolism. Although it has been reported that a
few LAB utilize xylose as a carbon source, 8 of the 11
tested B. coagulans strains were able to metabolize
xylose (one of the major sugar components in lignocel-
luloses).56) In addition, De Clerck et al.65) also reported
that half of the B. coagulans strains (15 strains)
5
metabolize D-xylose. These phenomena suggest the
feasibility of efficient L-LA production from various
lignocelluloses using B. coagulans strains.
Additionally, much progress has been made in our
understanding of LA metabolism in Bacillus strains.
The draft genome sequences of potent L-LA-producing
B. coagulans strains (2–6, 36D1, DSM1, H-1, XZL4,
and XZL9) showed the presence of xylose-utilizing
genes encoding the enzymes xylose isomerase, ribulok-
inase, and ribulose 5-phosphate 4-epimerase involved
in PPP.66) In contrast, the genes involved in the PK
pathway were absent from all tested B. coagulans
strains, which may result in high LA yield from xylose.
Actually, Ye et al.42) reported that B. coagulans C106
produced 83.5 g/L of L-LA from 85 g/L of D-xylose,
with an L-LA yield of 0.98 g/g, productivity of
7.5 g/L h, optical purity of 99.6% via PPP.
II. Available biomass for LA production
by Bacillus species
LA production by Bacillus strains has mainly been
investigated using expensive refined sugars (e.g. D-glu-
cose) as a sole carbon source. However, substitutes for
these expensive refined sugars, including cheap and
widely abundant biomass resources, such as lignocellu-
lose (e.g. wood and crop residues), sucrose (e.g. sugar-
cane and beet molasses), and starches (e.g. tapioca
starch and corn starch) are rapidly gaining importance
for the production of LA.15)
II.i. Lignocellulosic biomass
Lignocelluloses consists of cellulose (insoluble fibers
of β-1,4-glucan), hemicellulose (non-cellulosic polysac-
charides of xylan, arabinose, glucan, and mannan), and
lignin (a complex aromatic polymer).26) The composi-
tions of cellulose (35–50%), hemicellulose (20–40%),
and lignin (10–30%) vary depending on plant type.67–70)
In enzyme hydrolysis of the lignocelluloses, cellulose
mainly liberates D-glucose and cellobiose, whereas
several sugars are obtained from hydrolysis of hemicel-
lulose, including hexoses (D-mannose, D-galactose, and
D-glucose) and pentoses (D-xylose and L-arabinose).
D-xylose is the dominant sugar in hemicellulose. Thus
far, wheat straw,71) paper sludge,72) corn fiber hydroly-
sate,73) corncob molasses,74) empty fruit bunches (EFB)
of palm oil,75,76) Jerusalem artichoke powder,77) and
Siberian larch52) have already been efficiently used as
raw materials for LA production by Bacillus strains. For
example, B. coagulans JI12 utilized EFB hydrolysate
(containing 4.7 g/L D-glucose, 48.8 g/L of D-xylose, and
9.6 g/L of L-arabinose) and produced 59.2 g/L of L-LA,
with an L-LA yield of 0.97 g/g and a productivity of
6.2 g/L h and optical purity of 99.6%.75)
However, one of the major challenges in fermenta-
tive production using lignocelluloses as substrates exist
in the pretreatment process required for generating
fermentable sugars and efficient LA production.
The pretreatment of lignocelluloses also liberates phe-
nolic compounds (furfural and 5-hydroxy methyl fur-
fural [HMF]), furan derivatives (ferulic acid and
vanillin), and weak organic acid (acetic acid).78) These
 6 P. Poudel et al.

(A) Starch Cellulose Hemicellulose (B) Starch Cellulose Hemicellulose

D-xylose L-arabinose D-xylose L-arabinose

D-glucose D-glucose
f f
ATP ATP
a D-xylulose a D-xylulose
ADP ADP ATP
ATP
D-glucose 6P g g
ADP D-glucose 6P k ADP
h D-xylulose 5P
b b D-xylulose 5P
6-phosphogluconate
D-ribose 5P
D-fructose 6P i D-fructose 6P l
D-ribulose-5P
ATP ATP
D-sedoheptulose 7P
c c
ADP ADP
GAP Acetyl-P
D-fructose 1,6BP D-fructose 1,6BP
j
D-erythrose 4P

DHAP GAP DHAP GAP

d i d
NAD+ 2ADP NAD+ 2ADP Ethanol
Downloaded by [NUS National University of Singapore] at 14:55 26 November 2015

NADH 2 ATP NADH 2 ATP

Acetic acid
H2 O H2 O
Pyruvate Pyruvate
NADH NADH
e e
NAD+ NAD+
L-lactic acid L-lactic acid/D-lactic acid
PPP /EMP (Homofermentative pathways) PK pathway (Heterofermentative pathways)

Fig. 1. Metabolic pathway for LA production from several sugars (xylose, glucose, arabinose), cellulose, and starch derived from various
biomasses via homofermentative (EMP/PPP) (A) and heterofermentative (PK) (B) pathways.
Note: Enzymes: (a) hexokinase; (b) glucose 6P isomerase; (c) 6-phosphofructo kinase; (d) triose phosphate isomerase; (e) lactate dehydrogenase;
(f) xylose isomerase; (g) xylulose kinase; (h) epimerase; (i) transaldolase; (j) transketolase; (k) glucose 6P dehydrogenase; (l) phosphoketolase.

compounds are known to inhibit microbial growth and
LA production at concentrations ranging from 0.08 g/L
to 5.3 g/L.78) Three main approaches have been investi-
gated to improve LA production from pretreated ligno-
celluloses containing inhibitors: usage of LA-producing
Bacillus strains that are tolerant to these inhibitors,
introduction of removal process before fermentation,
and conversion of these inhibitors into less-toxic sub-
stances by LA-producing Bacillus strains. Some B.
coagulans strains have been reported to be resistant to
certain levels of these inhibitors,50,73,76,79) for instance,
Bacillus sp. P38, which tolerates 6 g/L 2-furfural in
corn stover hydrolysate, produced 180 g/L of L-LA with
a productivity of 2.4 g/L h and a yield of 0.96 g/g
under a fed-batch fermentation process (first
approach).50) By using LAB, there are a few reports on
tolerance to fermentation inhibitors,80,81) for instance,
Lactobacillus brevis S3F4 with the tolerance of 10 mM
(ca. 1.94 g/L) ferulic acid and 15 mM (ca. 1.44 g/L)
furfural could produce 18.7 g/L LA from 25 g/L
xylose.80) Meantime, removal processes using chemi-
cals, such as overliming,73,76) prior to fermentation
have also been reported to remove inhibitors from pre-
treated lignocelluloses for LA fermentation using Bacil-
lus strains (second approach). Overliming decreased to
ca. 27% furfural in EFB hydrolysate (63.1 g/L total
sugar), which resulted in 59.2 g/L LA with 0.97 g/g
yield by B. coagulans JI12.76) To our knowledge, there
is no report on LA fermentation using LAB by similar
approach. As the third approach, successful simultane-
ous detoxification, saccharification, and fermentation
has only been reported using B. coagulans JI12 by
converting furfural (an inhibitor) to 2-furoin acid (a
lessor inhibitor), producing 73.9 g/L of L-LA, with an
L-LA yield of 1.09 g/g from EFB hydrolysate,
75)
while
we found no reports of LAB converting inhibitors to
non-inhibitors. This process has become attractive in
terms of not only overcoming the crucial issue of tox-
ins produced by pretreatment of lignocelluloses, but
also simplifying the overall processes in an environ-
ment-friendly method that requires less energy.67)
II.ii. Sucrose-based biomass
Molasses from sugarcane and beet are sucrose-rich
biomasses obtained from sucrose-based industries as
waste byproducts. Sugarcane molasses contains 40–
60% sucrose that can be utilized by micro-organisms to
produce LA.82) A few researchers have investigated the
feasibility of using sucrose as a raw material for LA
production using Bacillus strains.83,84) Payot et al.83)
reported that B. coagulans TB/04 produced 55 g/L of
LA from 60 g/L of sucrose derived from sugarcane
molasses. In contrast, beet molasses containing 47%
sucrose, 0.5% nitrogen, and 3% betaine has been
shown to be used not only as a fermentation substrate,
but also as an enhancer of LA fermentation by B. coag-
ulans H-1.84) The addition of beet molasses to a glu-
cose solution increased L-LA productivity by 22%
compared to that observed with fermentation using only
D-glucose as a sole carbon source. Unlike other carbo-
hydrates (such as cellulose and starch), these sucrose-
containing molasses have a significant advantage: direct
utilization for LA production without any requirements
 Bacillus strains for optically pure L-lactic acid production
for saccharification because many B. coagulans strains
can convert sucrose to LA.56)
II.iii. Starchy biomass
Starch is a polysaccharide consisting of D-glucose
with α-1,4 or α-1,6 bonds. It is usually found in cereal-
based products (rice, corn, and wheat) and tuber plants
(potato and cassava). Starchy biomasses are cheap
alternatives to refined sugars. Inexpensive starches,
starch-containing wastes, and starch derivatives
obtained from many starch industries are attractive car-
bon sources for LA production even though starch
requires saccharification by enzymes such as α-amylase
and glucoamylase before fermentation.85,86) Several
enzymatic hydrolysates of corn starch87) and tapioca
starch39) have been used as substrates for LA produc-
Downloaded by [NUS National University of Singapore] at 14:55 26 November 2015
tion by Bacillus strains. Similarly, a considerable num-
ber of starch-containing wastes such as food wastes
have also been utilized for LA production by B. coagu-
lans and Bacillus licheniformis strains.88)
Conversely, direct fermentation from starch using
amylolytic LA producers has the advantage of skipping
the saccharification process. Although some amylolytic
LAB and fungi (R. oryzae) have been reported to pro-
duce LA from starch without any enzymatic hydroly-
sis,15,22) there are still the several problems regarding
low optical purity of LA (for LAB; <98%), low yield
(for Rhizopus; 0.79 g/g), mesophilic conditions
(≤45 °C), and requirement for abundant nutrients (for
LAB).26) Recently, Poudel et al.33) have established a
direct starch fermentation process using Bacillus sp.
MC-07 to obtain optically pure L-LA (purity 100%,
yield 0.977 g/g). Furthermore, fermentation using strain
MC-07 could be performed in a simple mineral salt
medium containing only 0.001% yeast extract as an
organic nitrogen source, at a high temperature (50 °C).
This finding shows that some low-cost starchy bio-
masses can be directly fermented to L-LA with high
yield and optical purity by omitting the addition of
enzymes and nitrogen sources, a relatively simple
methodology for LA fermentation.
III. Fermentative production of LA by
Bacillus strains
III.i. Bacillus strains
As shown in Table 2, B. coagulans, B. licheniformis,
B. subtilis, B. thermoamylovorans and some yet to be
identified Bacillus species have been reported to be
potent L-LA producers. All these Bacillus strains accu-
mulated optically pure L-LA (97–100%). Among these
reported Bacillus species, B. coagulans accumulated a
higher amount of L-LA and showed thermotolerant
behavior (Table 2).
III.ii. Fermentation modes
During LA fermentation, high LA yield, productivity,
and the concentration of the final accumulated product
are important factors for evaluating fermentation effi-
ciency. To achieve such economically important param-
eters, numerous investigations on LA fermentation
have been conducted under batch, fed-batch, and con-
tinuous fermentation processes (Tables 2 and 3).
7
Batch fermentation is the most frequently used for
LA production.25) The advantage of using batch fer-
mentation is the low risk of contamination compared
with other fermentation approaches because it is a
closed system in which no nutrients are added during
the fermentation period.93,94) High LA production
(210 g/L from D-glucose87) and 140.9 g/L from D-xy-
lose)42) has been obtained using Bacillus strains in
batch fermentation under non-sterile conditions
(Table 2). These LA concentrations are the highest in
the published literature using Bacillus strains under
batch fermentation. In repeated batch mode, microbial
cells can be repeatedly used for subsequent batch fer-
mentations.79) This method led to improved LA pro-
ductivity and reduced fermentation time and does not
require inoculum preparation. B. coagulans strain 2–6
accumulated the highest L-LA concentration (107 g/L,
with an optical purity of 99.8%) under open repeated
batch fermentation from D-glucose, in which the yield
and productivity were improved to 0.95 g/g and 2.9 g/
L h, respectively, compared to batch fermentation
(63 g/L, yield up to 0.94 g/g, productivity of 1.4 g/
L h).51) However, various problems, such as substrate
inhibition due to a high initial concentration of sugar,
carbon catabolite repression (CCR) in mixed sugar fer-
mentation caused by the consumption of more prefer-
able sugars and lagging of the less preferable sugars,
and product inhibition caused by LA accumulation still
exist in batch fermentation.
In fed-batch fermentation, fresh medium or nutrients
is continuously or sequentially fed into the fermentation
broth without removal of culture broth mainly to over-
come the problems of substrate inhibition and nutrient
deficiencies that occur in batch fermentation.26)
Fermentative LA production by Bacillus strains under
fed-batch fermentation is illustrated in Table 3. Several
feeding strategies, including pulse feeding, constant
feeding, and exponential feeding, have been developed
to increase the LA concentration in fed-batch fermenta-
tion. Among these, pulse feeding is considered easier
and more suitable for industrial scale.93) When a pulse
feeding strategy was used, the highest reported L-LA
concentration (225 g/L) was obtained using Bacillus sp.
WL-S20 under fed-batch fermentation.37)
Compared to batch and fed-batch fermentation, con-
tinuous fermentation in Bacillus strains has been less
studied. Continuous fermentation is adopted to over-
come the issue of product inhibition that can occur in
batch and fed-batch fermentation. In continuous fer-
mentation, products, such as inhibitors, are constantly
diluted by adding fresh medium at the same rate as that
of the outflow of the broth, to maintain a steady vol-
ume in the fermentor. In addition, cells can be main-
tained in a stable physiological state and at a constant
growth rate; thereby, attaining steady maximum pro-
ductivity. Continuous fermentations using B. laevolacti-
cus NCIB 10269,17) B. coagulans TB/04,83) and B.
subtilis MUR135) have been investigated. Gao and
Ho35) reported an average LA productivity of 16.8 g/
L h at a dilution rate of 0.4 h−1, using glucose under
continuous fermentation, which resulted in 4.8-fold
increase in productivity compared to that obtained in
fed-batch fermentation.35,36) De Boer et al.17) also
showed that Bacillus laevolaticus NCIB 10269 have
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Table 2. Lactic acid production efficiencies among the Bacillus strains under batch and repeated batch fermentation modes.

Lactic acid
Fermentation
Fermentation substrate Strains C (g/L) Y (g/g) P (g/L h) OP (%)† Fermentation parameters modes References
89)
Glucose B. thermoamylovorans CNCM I- 11.8 nd ca. 0.49 100 pH control at 7.0; Ferm.temp., 50 °C; Yeast extract, 20 g/L Batch
1378T
83)
Molasses B. coagulans TB/04 55.0 0.93 nd 99 pH control at 6.4; Ferm.temp., 52 °C; Yeast extract, 2 g/L Batch
69)
Soft wood hydrolysate B. coagulans BS121 13.7 nd ca. 0.19 99 pH control at 6.0; Ferm.temp., 60 °C; Yeast extract, 4 g/L Batch
60)
Acid hydrolysis sugarcane B. coagulans 17C5 55.8 ca. 0.93 ca. 0.80 99.5 pH control at 5.0; Ferm.temp., 50 °C; Corn steep liquor, 5 g/L Batch
baggase
59)
Acid hydrolysis of sugarcane B. coagulans 36D1 36.0 nd nd 99.0 pH control at 5.0; Ferm.temp., 50 °C Batch
18)
Saccharified food waste B. coagulans NBRC 12583T 86.0 0.97 0.72 97.0 pH control at 6.5; Ferm.temp., 55 °C Batch
90)
Saccharified food waste B. licheniformis TY7 40.0 1.35 2.50 97.0 pH control at 6.5; Ferm.temp., 55 °C Batch
72)
Paper sludge hydrolysates B. coagulans 36D1 92.8 0.77 0.96 99.5 pH control at 5.0; Ferm.temp., 50; Corn steep liquor, 5 g/L Batch
72)
Paper sludge hydrolysate B. coagulans P4–102B 91.78 0.78 0.82 99.5 pH control at 5.0; Ferm.temp., 50 °C; Corn steep liquor, 5 g/L Batch
73)
Corn fiber hydrolysate B. coagulans MXL-9 40.2 0.58 0.54 99.5 pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L Batch
52)
Hot water-extracted Siberian B. coagulans MXL-9 33.0 0.94 0.55 99 pH control at 7.0; Ferm.temp., 50 °C; Yeast extract, 5 g/L; Tryptone, Batch
larch 10 g/L
51)
Glucose B. coagulans 2–6 63 0.94 1.4 99.4 pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L Batch
51)
Glucose B. coagulans 2–6 107.0 0.95 2.90 99.8 pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L Repeated batch
34)
Cellobiose B. licheniformis BL1 4.4 0.86 0.30 >99.0 pH control at 7.0; Ferm.temp., 50 °C; Yeast extract, 5 g/L Batch
91)
P. Poudel et al.

Corn stover hydrolyzate B. coagulans XZL4 81.0 0.98 1.86 99.5 pH control at 6.0; Ferm.temp., 50 °C Batch
36)
Glucose B. subtilis MUR1 143.2 0.90 2.75 99.5 pH control at 7.5; Ferm. temp., 37 °C; Air flow rate, 50 vvm; Yeast Batch
extract, 75 g/L
92)
Corn stover hydrolysate/Xylose B. coagulans NL01 75.0 0.75 1.04 99.8 pH control at 6.5; Ferm.temp., 50 °C; Yeast extract, 2.5 g/L Batch
75)
Oil palm empty fruit bunch B. coagulans JI12 59.2 0.97 6.2 99.6 pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L Batch
hydrolysate
42)
Xylose B. coagulans C106 83.6 0.98 7.5 99.6 pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 20 g/L Batch
140.9 0.98 4.8 Batch
87)
Glucose B. coagulans WCP10–4 210.0 0.99 3.50 100 pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 20 g/L Batch
76)
Oil palm empty fruit bunch B. coagulans JI12 80.6 ca. 1.19 3.40 99.6 pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L Batch
hydrolysate
50)
Glucosea/Food waste B. coagulans M21 12.5/35.1 0.62/ 0.31/0.58 100/100 pH control at 7.0; Ferm.temp., 50 °C; Yeast extract, 5 g/L Batch
0.86
79)
Wheat straw hydrolysate B. coagulans IPE22 56.4 0.96 2.35 100 pH control at 6.0; Ferm.temp., 52 °C; Yeast extract, 4 g/L Repeated batch
33)
Soluble starch Bacillus sp. MC-07 16.6 0.977 0.70 100 pH control at 7.0; Ferm. temp., 50 °C; Yeast extract, 0.01 g/L Batch
Notes: C, concentration; Y, yield; P, productivity; nd, not described. The optical purity (%) of L-LA was defined as ([L]−[D]) × 100/([L]+[D]), where [L] and [D] denote the concentrations of L-LA and D-LA, respectively.
†
OP, optical purity (only L-isomer)
a
Under aerobic condition (KLa = 1.43 min−1 see Ref.48)
 Downloaded by [NUS National University of Singapore] at 14:55 26 November 2015

Table 3. Lactic acid production in fed-batch and continuous fermentation processes.

Lactic acid

Fermentation substrate Strains C (g/L) Y (g/g) P (g/L h) OP (%) Fermentation mode Fermentation parameters References
−1 17)
Glucose Bacillus laevolacticus 45.0 1.0 ca 13.1 D-isomer, >99 Continuous pH control at 6.0; Dilution rate, 0.07 h ; Ferm. temp., 30 °C; Yeast extract,
NCIB 10269 2 g/L; Anaerobic condition by sparging N2 gas
83)
Sucrose B. coagulans TB/04 20.1 0.93 6.1 >99 Continuous pH control at 6.4; Dilution rate, 0.03 h−1; Ferm.temp., 50 °C, Yeast extract,
4 g/L
35)
Glucose B. subtilis MUR1 55.9 ca. 16.8 99.5 Continuous pH control at 7.5; Dilution rate, 0.3 h−1; Ferm.temp., 37 °C; Agitation speed,
0.95 50 rpm; Air flow rate, 3 vvm
36)
Glucose B. subtilis MUR1 183.5 0.98 3.52 99.5 Fed-batch with pH control at 7.5; Ferm.temp., 37 °C; Air flow rate, 50 vvm; Yeast extract,
exponential feeding 75 g/L
72)
Lime treated wheat straw B. coagulans 40.7 0.43 0.74 97.2 Fed-batch with pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L
DSM2314 constant feeding
74)
Corncob molasses B. coagulans XZL9 74.7 0.50 0.37 99.5 Fed-batch with pulse pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 10 g/L
feeding
43)
Cellulose B. coagulans 36D1 80.0 0.88 ca. 0.84 99.0 Fed-batch with pH control at 5.0; Ferm.temp., 50 °C; Corn steep liquor, 7.5 g/L
constant feeding
37)
Glucose Bacillus sp. WL-S20 225.0 0.99 1.04 100 Fed-batch with pulse pH controlled at 9.0; Ferm.temp., 45 °C; Peanut meal, 20 g/L
feeding
42)
Xylose B. coagulans C106 215.7 0.95 4.0 99.6 Fed-batch with pulse pH control at 6.0; Ferm.temp., 50 °C; Yeast extract, 20 g/L
feeding
92)
Corn stover hydrolysate B. coagulans NL01 75.0 0.75 1.04 >99 Fed-batch with pulse pH controlled at 6.5; Ferm.temp., 50 °C; Yeast extract, 2.5 g/L
feeding
50)
Acid treated corn stover B. coagulans P38 180.0 0.98 2.40 100 Fed-batch with pulse pH controlled at 4.8–5.0; Ferm.temp., 42 °C
Bacillus strains for optically pure L-lactic acid production

hydrolysate feeding
76)
Jeruselem antichoke B. coagulans XZL4 134.0 0.96 2.50 >99.0 Fed-batch with pulse pH controlled at 5.2–5.5; Ferm.temp., 50 °C; Corn steep powder, 10 g/L
hydrolysate feeding
93)
Glucose B. coagulans 2–6 122.0 nd 2.50 99.2 Fed-batch with pH controlled at 5.6; Ferm.temp., 50 °C for 36 h and increased up to 55 °C
exponential feeding at 0.2 °C/h; Yeast extract, 5 g/L
94)
Mixture of untreated cane B. coagulans H-1 168.3 0.88 2.10 >99 Fed-batch with pH controlled at 6.2; Ferm.temp., 52 °C; Yeast extract, 10 g/L
molasses and glucose constant feeding
Note: C, concentration; Y, yield; P, productivity; OP, optical purity (only L-isomer unless indicated); nd, not described.
9
 10 P. Poudel et al.
the ability to produce D-LA with a productivity of
13.1 g/L h at dilution rate of 0.07 h−1. In continuous
mode, selection of the optimum dilution rate for attain-
ing maximum cell growth and LA productivity is
important in Bacillus strains. LA productivity was
highly improved for a long period in continuous
fermentation compared to that in batch and fed-batch
fermentation.
Bacillus strains have been also employed to produce
LA from mixed sugars derived from lignocellu-
loses.52,59,60,75–77,79) However, a major obstacle for
using mixed sugars is the difficulty in simultaneous uti-
lization of each sugar during fermentation. Several
researchers have reported on CCR in B. coagulans
strains, i.e. initial consumption of more preferable sug-
ars like glucose and lagging utilization of less prefer-
able sugars like D-xylose and L-arabinose.44,72,75) CCR
Downloaded by [NUS National University of Singapore] at 14:55 26 November 2015
with mixed sugars has mainly been reported for batch
fermentation using LA-producing Bacillus strains.75)
This indicated that repeated batch fermentation by B.
coagulans strains showed simultaneous utilization of
mixed sugars (D-glucose, D-xylose, and L-arabinose) to
some extent.59,77) Zhang et al.79) investigated repeated
batch fermentation using various substrates (D-glucose,
D-xylose, and L-arabinose) by B. coagulans IPE22 for
up to six consecutive batches. In the first batch, fer-
mentation exhibited a lag phase of 5 h, and then pen-
tose sugars (D-xylose and L-arabinose) were utilized to
produce 54.14 g/L of LA in 24 h, whereas in the sixth
batch, the lag phase was eliminated and all sugars were
simultaneously utilized to produce 56.27 g/L of LA in
17 h. However, complete avoidance of CCR has not
been solved using LA-producing Bacillus strains.
Therefore, more potent CCR-negative LA-producers
must be procured, either by isolation of wild strains or
through genetic manipulations, or by optimizing the
fermentation conditions.
IV. Metabolic engineering of Bacillus
species for LA production
Metabolic engineering is being applied to improve
the cellular properties of Bacillus strains through alter-
ation of specific biochemical reactions or introduction
of new genes by DNA recombination.95–99) However,
attempts to engineer the genus Bacillus metabolically
for LA production are far fewer than those with
LAB,62) Escherichia coli,97) and fungi.98) This section
is focused on recent studies on the establishment of
genetically modified Bacillus strains to improve LA
fermentation.
Among the LA-producing Bacillus strains, B. subtilis
is being widely studied for its good performance and
established genetic engineering methods.100) Zhang
et al.96) reported the direct fermentation of cellulose to
L-LA by recombinant cellulolytic B. subtilis XZ7. In
this study, overexpression of β-endoglucanase-coding
genes by two-round directed evolution was performed,
and the gene encoding α-acetolactate synthase (alsS) in
the 2, 3-butanediol pathway was knocked out to
increase the L-LA yield. As a result, B. subtilis XZ7
could digest up to 92% of the insoluble regenerated
amorphous cellulose and produced L-LA with a yield of
up to 0.63 g/g. This study demonstrated that B. subtilis
is useful for genetic manipulation to generate excellent
LA-producing Bacillus strains for the desired
processes.
Genetic tools for L-LA-producing B. coagulans have
been gradually developing; however, compared to B.
subtilis, highly efficient genetic tools are still lacking.
Basically, wild-type B. coagulans strains produce L-iso-
mer efficiently from several sugars, while D-LA-produc-
ers are also required for the synthesis of stereocomplex
PLA, consisting of PLLA and PDLA.101,102) For the
fermentative production of D-LA, some B. coagulans
strains have been genetically modified using specific
genetic tools.95,99) Kovács et al.95) successfully applied
the widely used Cre-lox system for genomic modifica-
tions and removal of specific genes. The native ldhL
gene of B. coagulans DSM1 (a potent L-LA producer)
was disrupted, and the D-lactate dehydrogenase (ldhD)
gene was overexpressed to generate D-LA. The engi-
neered strain could produce 16.9% D-LA and 83.1% L-
LA. However, optically pure D-LA could not be
obtained from the engineered strain; therefore, further
improvements are required. Conversely, Wang et al.99)
engineered a thermotolerant B. coagulans strain
P4-102B (a potent L-LA producer, optical purity >99%)
by targeting the genes encoding the enzyme D-LDH for
D-LA production with high yield by deleting the native
ldhL (L-lactate dehydrogenase) and alsS (acetolactate
synthase) genes. The engineered strain produced opti-
cally pure D-LA (90 g/L of D-LA, optical purity >99%)
from D-glucose at 50 °C, and use of this strain can be
expected to reduce the cost of LA production for
biopolymers.
Aside from genetic engineering, another approach
that has been investigated is to breed thermotolerant L-
LA-producing B. licheniformis, as the wild-type BL1
strain could not efficiently ferment xylose to LA. Wang
et al.34) transferred BL1 strain into medium containing
xylose as the sole carbon source, and the evolved BL2
strain, obtained after 13 transfers, showed improved L-
LA production (concentration, 24.5 g/L; yield, 79.5%;
maximum LA productivity, 7.0 g/L h) from 30 g/L
xylose compared with the parent strain (0.6 g/L, 60.0%,
and 0, respectively). Furthermore, transcriptional analy-
sis revealed 5.1- and 6.2-fold higher mRNA expression
levels of xylA (xylose isomerase) and xylB (xylulose
kinase), respectively, in the BL2 strain compared to the
levels in the BL1 strain using xylose. Although further
work is needed to understand the mechanism, including
other genes and enzymes in this strain, this method is
also a new and easy technique for modifying the meta-
bolism of LA-producing Bacillus strains.
In future, progress toward more efficient and easy
methods for genetic manipulation and evolution will
lead to more studies in the field of metabolic engineer-
ing on LA-producing Bacillus strains.
V. Future prospects for LA-producing
Bacillus species
Today’s society is more concerned with climate
change because the majority of the industrial economies
are largely dependent on fossil resources, which provide
 Bacillus strains for optically pure L-lactic acid production
the basis for almost all of our energy and chemical feed-
stocks. In order to develop a sustainable society, we
need to substantially reduce our dependence on fossil
resources by establishing bio-based systems.103) Thus,
microbial technologies to produce value-added chemi-
cals, such as optically pure LA, would be the best alter-
native strategy to overcome our dependency on fossil
resources.104)
Recently, many Bacillus strains have been shown to
possess the ability to utilize a wide variety of biomass
resources to produce optically pure L-LA. In particular,
B. coagulans, B. subtilis, B. licheniformis, and B. ther-
moamylovorans strains are promising LA producers.
These strains also have the ability to tolerate high tem-
peratures, form spores, grow at high growth rates, and
to produce highly optically pure L-LA. The basic physi-
ological features of these strains indicate that Bacillus
Downloaded by [NUS National University of Singapore] at 14:55 26 November 2015
species can be an important L-LA producer. In addition,
many Bacillus strains have advantages for the homofer-
mentative conversion of the pentose sugars predomi-
nantly available in lignocelluloses. Most recently, B.
coagulans strains have also been determined to be for
use as safe probiotics.105) In addition, many Bacillus
strains have been adapted for the production of com-
mercially valuable products such as thermostable
enzymes and antimicrobial peptides.66) However, due
to limited studies on the metabolic pathways and meta-
bolic engineering in LA-producing Bacillus strains,
more investigations should be done to enhance the effi-
ciency of LA production. Besides, there are extremely
few investigations in terms of selection of nutrients
(i.e. carbon and nitrogen sources) and available bio-
masses followed by several pretreatment and saccharifi-
cation methods and improvement of production
processes dealing with LA-producing Bacillus strains,
compared with studies on LA production using LAB.
Further studies should focus on the development of
highly efficient strains and production processes that
are able to produce optically pure L-LA as well as D-
LA either by selective isolation and screening or
through genetic engineering approaches.
Authors contribution
Poudel P, Tashiro Y, Sakai K, designed this review
and Poudel P, drafted the review manuscript.
Disclosure statement
No potential conflict of interest was reported by the
authors.
Funding
This work was supported partly by the JSPS
KAKENHI [grant number 26740050].
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