Journal of Bioscience and Bioengineering

VOL. 119 No. 1, 10e18, 2015

www.elsevier.com/locate/jbiosc

REVIEW

Fermentative production of lactic acid from renewable materials: Recent

achievements, prospects, and limits
Ying Wang,1 Yukihiro Tashiro,2, 3 and Kenji Sonomoto1, 4, *
Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture,
Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan,1 Institute of Advanced Study, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka
812-8581, Japan,2 Laboratory of Soil Microbiology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of
Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan,3 and Laboratory of Functional Food Design, Department of Functional
Metabolic Design, Bio-Architecture Centre, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan4
Received 16 April 2014; accepted 3 June 2014
Available online 27 July 2014

The development and implementation of renewable materials for the production of versatile chemical resources have
gained considerable attention recently, as this offers an alternative to the environmental problems caused by the petroleum industry and the limited supply of fossil resources. Therefore, the concept of utilizing biomass or wastes from
agricultural and industrial residues to produce useful chemical products has been widely accepted. Lactic acid plays an
important role due to its versatile application in the food, medical, and cosmetics industries and as a potential raw
material for the manufacture of biodegradable plastics. Currently, the fermentative production of optically pure lactic
acid has increased because of the prospects of environmental friendliness and cost-effectiveness. In order to produce
lactic acid with high yield and optical purity, many studies focus on wild microorganisms and metabolically engineered
strains. This article reviews the most recent advances in the biotechnological production of lactic acid mainly by lactic
acid bacteria, and discusses the feasibility and potential of various processes.
2014, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Lactic acid; Renewable materials; Microbial fermentation; Lactic acid bacteria; Metabolic pathways]

Lactic acid (2-hydroxypropanoic acid) is a naturally occurring

hydroxycarboxylic acid, rst rened from sour milk by the Swedish
chemist Scheele in 1780 (1). Subsequently, due to its versatile applications as an acidulant, avour enhancer, and preservative, lactic
acid has occupied a prime position in the food, pharmaceutical,
cosmetic and other chemical industries (2,3). Recently, new uses for
this compound are emerging. Lactic acid production has received a
signicant amount of interest because it can be used as a feedstock
for the production of poly-lactic acid (PLA), a polymer present in
medical applications and environmentally friendly biodegradable
plastics, which can be substituted for synthetic plastics derived
from petroleum resources (2e4). In nature, lactic acid occurs in two
optical isomers, D (
)- and L ()-lactic acids. L ()-Lactic acid is the
preferred isomer in the food and drug industries, because only this
form is adapted to be assimilated by the human body. Therefore, the
forms of lactic acid in pure isomers are more valuable for different
specic applications (4,5). Copolymerization of the D (
)- and L ()isomers results in amorphous materials, whereas homopolymers
form regular structures and are in a crystalline phase (6).
Lactic acid can be manufactured commercially by either chemical synthesis or biotechnological production by lactic acid

* Corresponding author at: Laboratory of Microbial Technology, Division of Applied

Molecular Microbiology and Biomass Chemistry, Department of Bioscience and
Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1
Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel./fax: 81 (0)92 642 3019.
E-mail address: sonomoto@agr.kyushu-u.ac.jp (K. Sonomoto).

fermentation. The most common method to synthesize lactic acid is

based on the hydrolysis of lactonitrile. However, chemical synthesis
always yields a racemic mixture of DL-lactic acid from petroleum
resources. On the other hand, an optically pure L ()- or D (
)-lactic
acid can be obtained by the microbial fermentative method.
Currently, approximate 90% of lactic acid is produced by the microbial fermentation. With the development of industrial bioconversion, microbial fermentation by the appropriate microorganism
has become the dominant method of lactic acid production due to
environmental concerns, low production temperature, low energy
requirements, and high purity (7).
In recent times, the consumption of lactic acid as a feedstock for
the production of PLA has increased considerably. However, the
amount of PLA production (450 million kilograms per year) is still
much lower than the total amount of plastics production (200
billion kilograms per year) (8). PLA production is restricted by high
production costs, although the annual industrial investment is
several million dollars (9). It has been reported that the cost of raw
materials for the fermentative production of lactic acid usually
accounts for greater than 34% of the total manufacturing cost (10).
Thus, the efciency and economics of lactic acid fermentation is still
a problem from many points of view, and the substrate plays a vital
role in the improvement of such a process. There have been various
attempts to produce lactic acid efciently from economic resources,
such as rice bran (11), paper sludge (12), and green microalga (13).
Nowadays, renewable materials such as lignocellulose and starch
from agricultural residues and forestry resources are generally

1389-1723/$ e see front matter 2014, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2014.06.003

VOL. 119, 2015

considered to represent an attractive substrate as feedstock for the
production of lactic acid due to their abundance (5). However, one
bottleneck for lactic acid production utilizing renewable materials
is the cost of pretreatment. Most renewable materials are not
directly available for lactic acid fermentation without pretreatment
due to their intimate association with lignin and the lack of hydrolytic enzyme production by lactic acid-producing strains (5).
Another limiting factor is the recovery and purication of lactic acid
from the fermentation broth, because complex media consisting of
various nutrients hampers not only the separation but also the
purication of lactic acid. Therefore, there are many challenges in
the industrial bioconversion of renewable materials to lactic acid.
This article presents a review of recent advances in the
biotechnological production of lactic acid using renewable materials from the aspects of metabolic and enzymatic mechanisms, and
metabolic engineering associated with lactic acid production. The
major production processes, renewable materials, bioreactor systems, and fermentation modes are reviewed. We also describe
recent achievements and limitations in simultaneous saccharication and fermentation (SSF) and molecular genetic approaches in
the production of lactic acid from renewable materials.
MICROORGANISMS INVOLVED IN BIOTECHNOLOGICAL
PRODUCTION OF LACTIC ACID
Wild type of LAB
Lactic acid bacteria (LAB) are dened as
facultative anaerobic or micro-aerophilic organisms and characterized by the following aspects: (i) can grow at temperatures as
low as 5 C or as high as 45 C, (ii) can grow at pH 4.0e4.5, some also
proliferate at pH 3.2 or 9.6, and (iii) generally require complex nitrogens, vitamins, and minerals for growth and lactic acid production (14). Studies on LAB constitute approximately 90% of the
literature on lactic acid production because they can produce
lactic acid with high yield and high productivity. The most
common LAB species belong to the genera Lactobacillus,
Lactococcus, Pediococcus, Aerococcus, Carnobacterium, Oenococcus,
Tetragenococcus, Vagococcus, Weisella, Leuconostoc, Streptococcus,
and Enterococcus. Among them, optically pure L ()-lactic acid is
produced by several species such as Enterococcus mundtii (15e17)
and Lactococcus lactis (18), while D (
)-lactic acid can be
produced by Lactobacillus delbrueckii (19).
Wild-type LAB, isolated and screened from various sources, are
always the most powerful source for obtaining fermentable and
genetically stable strains, which are widely utilized in lactic acid
production. Two Lactobacillus strains of OND 32T and YAM 1 were
isolated from sour cassava starch fermentation, and can directly
ferment starchy biomass to lactic acid (20). Abdel-Rahman et al.
(16,17) presented a novel wild-type strain of E. mundtii QU 25,
which is a very attractive candidate for efciently metabolizing
lignocellulose-derived sugars into optically pure homo L ()-lactic
acid. The strain can produce lactic acid from a high concentration of
xylose via the pentose phosphate pathway (16) as described in
detail in next section, through which 3 mol of xylose yield 5 mol
of lactic acid. This means that the theoretical yield is close to
100%, and few by-products are formed through fermentation by
this strain (16).
Wild type of Bacillus genus and fungi Although LAB are
widely used in lactic acid production, some other strains such as
those of the genus Bacillus, as well as fungi, also produce lactic acid.
Patel et al. (21) isolated Bacillus sp. strains 17C5 and 36D1 from soil
and geysers, and proved their ability to produce L ()-lactic acid
from sugarcane bagasse with a maximum productivity of
6.7 mmol L
1 h
1 in SSF. Another group focused on the
bioconversion of paper sludge to lactic acid by the thermophilic
Bacillus coagulans strain P4-102B (12).

LACTIC ACID PRODUCTION FROM RENEWABLE MATERIALS

11

The best-known fungal source as a lactic acid producer is

Rhizopus oryzae. In general, R. oryzae has relatively lower nutritional demands but the mycelial morphology and oxygen supply
are considered to inuence lactic acid productivity. The rst report
on efcient D (
)-lactic acid fermentation by R. oryzae was in 1936
(22). R. oryzae NRRL 395 has been recognized as one of the most
suitable fungi for lactic acid fermentation (23). Guo et al. (24)
described a fermentation process involving the simultaneous utilization of hemicellulose and cellulose in corncobs by a newly
isolated R. oryzae.
Engineered microorganisms by mutagenesis and metabolic
engineering Studies also focus on engineered microorganisms
in order to meet the commercial requirements including improved
optical purity of the product, reduction of nutritional supply,
improved yield and productivity, a broader substrate specicity,
and the elimination of plasmids and antibiotic markers. The initial
efforts of genetic modications were mainly on improving LAB by
traditional approaches, which involve exposing the bacterium to
mutagens such as ethylmethyl sulphonate, N-methyl-N0 -nitro-Nnitrosoguanidine, and ultraviolet radiations (25,26).
On the other hand, efcient genetic tools targeting several microorganisms have been developed over the past few decades.
Amongst these, metabolic engineering is an important tool for industrial biotechnology (27). According to the manipulation of
enzyme functions, transcription, and the regulatory system in the
microorganisms, metabolic engineering redirects metabolic ux,
changes cellular protein levels, and regulates gene expression in
several hosts such as Saccharomyces cerevisiae (28), Escherichia coli
(29), Corynebacterium glutamicum (30), R. oryzae (31), and Lactococcus lactis (32). Table 1 shows a summary of recent studies on
engineering approaches for lactic acid production.
STUDIES ON METABOLIC PATHWAYS WITH LAB
LAB ferment sugars such as hexose and pentose via different
metabolic pathways that lead to homo- or hetero-fermentation
(Fig. 1). Homo-fermentation produces virtually only lactic acid as
the end product via the EmbdeneMeyerhofeParnas (EMP)
pathway and the pentose phosphate (PP)/glycolic pathway from
hexose and pentose, respectively. The theoretical yield of lactic acid
from glucose is 1.0 g g
1 (2.0 mol mol
1) via the EMP pathway
while pentose exhibits a theoretical yield of 1.0 g g
1
(1.67 mol mol
1) of lactic acid via the PP/glycolic pathway (43,44).
In the EMP pathway, the rst steps of glycolysis are the phosphorylation of glucose to fructose 1,6-diphosphate (FDP) and its
subsequent cleavage into dihydroxyacetone phosphate (DHAP) and
glyceraldehyde 3-phosphate (GAP). The GAP is then converted to
pyruvate via a route that includes 2 substrate-level phosphorylation steps. Finally, pyruvate is reduced to lactic acid by L-LDH or DLDH with the oxidation of NADH to NAD for the redox balancing.
In the PP/glycolic pathway, 3 mol of xylulose 5-phosphate (xylulose
5-P; 5 carbons), generated by the phosphorylation of pentose
sugars such as xylose and arabinose, is converted to 5 mol of GAP
(3 carbons) via 2 key enzymes: transketolase and transaldolase. The
resulting GAP is converted to pyruvate and then to lactic acid
(3 carbons) as the nal product, thereby providing a theoretical
yield of lactic acid from pentose of 1.0 g g
1 (1.67 mol mol
1).
On the other hand, in hetero-fermentation, equimolar amounts
of lactic acid, carbon dioxide, and ethanol (or acetate) are formed
via the phosphoketolase (PK) pathway, in which glucose 6-phosphate (6 carbons) is initially converted to ribulose 5-phosphate
(5 carbons) and carbon dioxide (1 carbon) in a reaction catalyzed by
several enzymes (45). Then, the resulting xylulose 5-P from ribulose 5-phosphate is cleaved to an equimolar amount of GAP and
acetyl phosphate (acetyl-P). The acetyl-P is reduced to ethanol

Bacteria
Bacteria
Bacteria

Fungi

E. coli W3110 SZ40

Lactococcus lactis IL 1403 ptk::tkt
Lactobacillus plantarum NCIMB 8826 DldhL1xpk1::tkt
Rhizopus oryzae NRRL 395 pLdhA71X

e, not determined. ldh, lactate dehydrogenase gene; pdc, pyruvate decarboxylase gene; cupdc1, pyruvate decarboxylase gene; p, pyruvate formate lyase gene; msg, methylglyoxal synthase gene; cydAB, cyoABCD, cbdAB, cytochrome
oxidases genes; glpK-glpD, glycerol kinase and glycerol-3-phosphate dehydrogenase gene; dld, D-lactate dehydrogenase gene; xylA, xylRAB, xylose isomerase genes; xylB, xylulokinase gene; bgl, b-glucosidase gene; als, acetolactate
synthase gene; xpk, phosphoketolase gene; pta, phosphate acetyltransferase gene; adhE, alcohol/acetaldehyde dehydrogenase gene; frdA, fumarate reductase gene; tkt, transketolase gene; LDH, lactate dehydrogenase.

31
e
77.5
Glucose
Increase LDH activity

Bacteria
Bacteria
E. coli ATCC 11303 TG114
E. coli ATCC 700926 LA02Ddld

expression
L-ldhA

29
32
42
51
50.1
38.6
Glucose
Xylose
Arabinose
Improved yield of D-lactic acid
L-Lactic acid fermentation from xylose
D-Lactic acid fermentation from arabinose

0.97e0.99
0.79
0.82

40
41

Yeast
Yeast
Yeast
Yeast
Yeast
Bacteria
Bacteria
Bacteria
Bacteria
Candida utilis NBRC0988 cupdc1 D4-ldh2
Kluyveromyces lactis CBS2359 pEPL2
Pichia stipitis CBS6054 VTT-C-04590
Saccharomyces cerevisiae CEN.PK182 RWB850-2
S. cerevisiae OC2 YILM-2B
Bacillus coagulans P4e102B QZ19
Corynebacterium glutamicum X5C1
C. glutamicum R DldhA/pCRB204
Escherichia coli ATCC 700926 ECOM3

118
32
Glucose
Glycerol

msgA deletion
pta, adhE, frdA, dld deletion and glpK-glpD
expression
pB mutation
xylRAB expression
D-ldhL1and xpk1 deletion, tkt expression

0.98
0.85

0.95
0.60
0.58/0.44
0.83  0.02
0.61
e
e
0.87
0.8
103.3
109
58/41
122
61.5
90
z40.5
120
e
Glucose
Glucose
Xylose/Glucose
Glucose
Glucose
Glucose
Cellobiose/Glucose/Xylose
Glucose
Glucose

L-Lactic acid production

Improved yield of lactic acid
Reduced by-product
Homo fermentative lactate production
D-Lactic acid production
D-Lactate production
Broaden sugar utilization range
Improved productivity of D-lactic acid
Lactate production under oxic and anoxic
growth conditions
Improved lactate productivity and cell yield
D-Lactic acid production

Genus

L-ldhL

cupdc1 deletion, L-ldh2 expression

expression
L-ldhL expression
pdc deletion
pdc1 deletion
D-ldh and als deletion
xylA, xylB, bglF317Aand bglA expression
D-ldhA expression
cydAB, cyoABCD, and cbdAB deletion

Yield
Y (g g
1)
Lactic acid
g (g L
1)
Substrate
Outcome
Approach

J. BIOSCI. BIOENG.,

Metabolically engineered microorganism

TABLE 1. Metabolic engineering approaches for lactic acid production by several microorganisms.

33
34
35
28
36
37
38
29
39

WANG ET AL.
Reference

12

(2 carbons) via acetyl-CoA and acetaldehyde intermediates, or

converted to acetate via acetate kinase, while GAP further enters to
EMP pathway to form lactic acid (3 carbons) (5). As the result, the
theoretical yield of lactic acid from glucose reaches only 0.5 g g
1
(1.0 mol mol
1) with hetero-fermentative LAB. The ratio of ethanol
to acetate is dependent on the redox potential in the cells. In terms
of the metabolism of pentose sugars such as xylose and arabinose,
xylulose 5-P (5 carbons) is the common intermediate, which is then
cleaved to GAP and acetyl-P via the PK pathway. The resulting
acetyl-P is metabolized to synthesize acetic acid or ethanol (both 2
carbons), whereas the GAP is converted to pyruvic acid and then to
lactic acid (3 carbons) as the nal product, with a theoretical yield
of lactic acid from pentose sugars of 0.6 g g
1 or 1.0 mol mol
1 via
the PK pathway (16,21). Therefore, the EMP and PP pathways are
more effective than the PK pathway in improving lactic acid yield
and sugar utilization (44). A few wild-type and metabolically
engineered strains showed the ability to metabolize pentose by
homo-lactic acid fermentation, such as E. mundtii QU 25 (16,17) and
Lactobacillus plantarum NCIMB 8826 DldhL1-xpk1::tkt (42,46). In
addition, the concentration of pentose sugars also affected the
metabolic ux with LAB. It was reported that when xylose was used
as the sole carbon source, a higher yield of lactic acid was obtained
with a high concentration of initial xylose than with a low concentration (16,43).
EFFECTS OF SEVERAL FACTORS ON LACTIC ACID FERMENTATION
BY LAB
Effects of mixed sugars as carbon source
Co-fermentation
by mixed sugars has been recognized as effective for lactic acid
fermentation. In particular, simultaneous and efcient fermentation using mixed sugars of hydrolyzates derived from renewable
materials including pentoses and hexoses is a signicant hurdle
(47). Various rened sugars such as glucose, xylose, sucrose,
cellobiose, and lactose have been used in the process of lactic
acid production (48e51). In general, glucose is the most favoured
sugar for most lactic acid-producing strains, which results in
many studies reporting lactic acid fermentation from glucose as
the sole carbon source (30,39,52). However, glucose has been
shown to limit the ability to effectively utilize other sugars to
produce lactic acid, a phenomenon known as carbon catabolite
repression (49). To date, it has been reported that several studies
have aimed at investigating efcient lactic acid fermentation
using mixed sugars in the presence of glucose by wild-type or
metabolically engineered strains.
Abdel-Rahman et al. (5) isolated a wild-type LAB, E. mundtii QU
25, which was able to produce optically pure L-lactic acid from
glucose, cellobiose, and xylose. E. mundtii QU 25 has been shown to
metabolize glucose and cellobiose simultaneously with a high yield
of lactic acid (>0.9 g g
1) without by-products.
Yoshida et al. (51) reported co-fermentation with glucose,
xylose, and arabinose for homo-D-lactic acid production. By introducing xylA (xylose isomerase) and xylB (xylulokinase) into the
genome of Lactobacillus plantarum NCIMB 8826, replacing the xpk1
gene (PK) with the tkt gene (TK), and by deleting the ldhL1 (L-LDH)
and xpk2 (PK) genes, Lactobacillus plantarum DldhL1::PxylABxpk1::tkt-Dxpk2::PxylAB was rendered capable of utilizing xylose
and arabinose in the presence of glucose. This is the rst report that
accomplished homo-D-lactic acid fermentation from mixed sugars
including hexose and pentose, without carbon catabolite repression
by metabolically engineered LAB.
Yun and Ryu (49) studied lactic acid production using Enterococcus faecalis RKY1 in co-fermentations with different sugar
mixtures of glucose/fructose, glucose/maltose, and fructose/
maltose as the carbon sources. The strain grown on a mixture
of glucose/fructose simultaneously consumed these sugars to

VOL. 119, 2015

LACTIC ACID PRODUCTION FROM RENEWABLE MATERIALS

Exo--(14)-glucanase

Cellobiose

Cellooligosaccharides

Arabinose

Galactose
(1)

13

Xylose
(3)

(2)

-glucosidase

Glucose1-P

Ribulose

ATP

ATP

(20)

ADP

ADP

NAD(P)H

ADP

NAD(P)

6-Phosphogluconate

ADP

(8)

NAD(P)+

(5)

NAD(P)H

ATP

(21)

Ribulose 5-P

(19)
(17)
ATP

Xylulose

UDP-glucose

(18)

ADP

Glucose

Galactose1-P

UDP-galactose

ATP

(7)

CO2

Xylulose 5-P

Glucose 6-P
(6)

Mannose
Ribose 5-P

(22)

(9)

(16)

(4)

Xylulose 5-P

Xylulose 5-P

PEP

Pyr

GAP
Mannose 6-P

(15)

Sedoheptulose 7-P
(10)

Fructose 6-P
ATP

(9)

(11)

Erythrose 4-P

ADP

Acetyl-P

GAP
NAD+
NADH

ATP

ADP

CoA

(24)

ATP

Pyruvate

(12)

Dihydroxyacetone
phophate

(13)
NAD

ADP

(14)
NAD

Acetic acid

Acetyl-CoA

NADH

GAP

(23)

Pi

H20

Fructose1,6-P

ADP

NADH

(25)

NAD

ATP

NADH

H20

Pyruvate

Lactic acid

Acetaldehyde

NADH

NADH

NAD+

NAD

(14)

(26)

Lactic acid

PP/Glycolytic Pathway
(Homo-lactic acid metabolism)

Ethanol

PK Pathway
(Hetero-lactic acid metabolism)

FIG. 1. Pathways for lactic acid production from renewable materials-derived sugars (glucose, xylose, cellobiose, arabinose, mannose, and galactose) by lactic acid bacteria (4,7,16,19).
GAP, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate. Enzymes: 1, galactokinase; 2, arabinose isomerase; 3, xylose isomerase; 4, mannose phosphotransferase system; 5, hexokinase; 6, glucose-6-phosphate dehydrogenase; 7, 6-phosphogluconate dehydrogenase; 8, ribulose-5-phosphate 3-epimerase; 9, transketolase;
10, transaldolase; 11, 6-phosphofructokinase; 12, fructose-bisphosphate aldolase; 13, triosephosphate isomerase; 14, lactate dehydrogenase; 15, phosphomannose isomerase;
16, phosphoglucose isomerase; 17, phosphoglucomutase; 18, galactose-1-phosphate uridyl transferase; 19, glucosyltransferase; 20, ribulokinase; 21, xylulokinase; 22, phosphoketolase; 23, acetate kinase; 24, phosphotransacetylase; 25, aldehyde dehydrogenase; 26, alcohol dehydrogenase.

produce lactic acid without carbon catabolite repression, whereas

utilization of maltose was repressed in the presence of glucose or
fructose. Therefore, carbon catabolite repression of maltose metabolism was suggested to be caused by preferentially metabolized
sugars.
Taniguchi et al. (50) described the production of optically pure Llactic acid with a yield of 0.63 g g
1 from a model lignocellulose
hydrolyzate including xylose and glucose by a mixed culture of
Enterococcus casseliavus IFO 12256 and Lactobacillus casei 2218.
Although the mixed culture exhibited carbon catabolite repression,
the complete consumption of 50 g L
1 xylose and 100 g L
1 glucose
was achieved.
Effects of nutrients on lactic acid fermentation by LAB LAB
generally have complex nutritional requirements due to their
limited ability to synthesize elements for their own growth. While

carbon sources are used to generate energy for proliferation,

LAB require other nutrients such as nitrogen sources, vitamins,
and minerals for maintenance, cellular growth, and lactic acid
secretion.
The ratio of carbon source to nitrogen source (C/N ratio) is a
major factor that affects the lactic acid conversion process.
Generally, a proper C/N ratio, achieved by the addition of complex
nitrogen sources such as yeast extract, peptone, and meat extract,
has a positive effect on lactic acid production (53). In particular,
yeast extract is an efcient nutrient for high lactic acid production
by LAB, although the utilization of yeast extract results in a high
cost of lactic acid production. Therefore, the replacement of yeast
extract with an economic alternative nitrogen source would
reduce the cost of nutrients. To date, various cheap nitrogen
sources have been investigated for use as an alternative to yeast
extract, such as hydrolyzate of sh waste (54), corn steep liquor

14

WANG ET AL.

(55), wheat bran extract (56), and silkworm larvae (57). By using
corn steep liquor as the cheap nitrogen sources, the lactic acid
productivity increased with decreasing the C/N ratio (mol/mol)
from 37:1 to 19:1, while the further decrease to 12:1 had no positive impact on lactic acid production by Lactobacillus sp. MKTLC878 (58).
In addition to carbon sources and nitrogen sources, vitamins and
minerals have signicant effects on lactic acid fermentation by LAB.
For example, pyridoxine stimulated the growth of some LAB, while
neither 2,4,5-trimethyl-3-hydroxypyridine nor 2,4-dimethyl-3hydroxy-5-hydroxymethyl pyridine showed the same effect (59).
Chauhan et al. (60) screened media components for high lactic acid
production with Lactobacillus sp. KCP01. They found that
MgSO4$7H2O, KH2PO4, sodium citrate, NaCl, and sodium succinate
had less of an effect on lactic acid production, and that sodium
sulphate, sodium acetate, K2HPO4, MnSO4$4H2O and FeSO4$7H2O
were found to be signicant.
Effects of fermentation condition on lactic acid
fermentation by LAB In the eld of fermentation technology,
fermentation conditions such as pH, temperature, and inoculum
size were considered important factors for cell growth, lactic acid
concentration, lactic acid productivity, and lactic acid yield.
The initial or controlled optimal pH value for lactic acid production varies between 5.0 and 7.0, depending on the used LAB.
Without pH control, the pH of the fermentation broth decreases
with increasing lactic acid production, resulting in the inhibition of
cell growth and its production. Idris and Suzana (61) reported the
effect of initial pH from 4.5 to 8.5 on lactic acid production during
batch fermentation by Lactobacillus delbrueckii ATCC 9646. An
initial pH of 6.5 caused the early induction of sugar consumption,
maximal rate of sugar consumption, and maximal lactic acid concentration. Guyot et al. (62) studied lactic acid production in batch
fermentation with pH control by Lactobacillus manihotivorans LMG
18010T at different pH values (4.5). Compared to non-pHcontrolled batch fermentation, pH-controlled batch fermentation
at 6.0 reduced the fermentation period by half, and increased the
lactic acid concentration to 12.6 g L
1 and the OD600 value to 5.1
from 7.6 g L
1 and 1.65, respectively.
Since LAB are mesophilic bacteria, LAB can grow and produce
lactic acid at a maximum temperature of approximately 50 C. In
addition, the growth rates of the LAB used differed based on the
fermentation temperature, and the optimum temperature for lactic
acid concentration did not correspond to lactic acid yield or lactic
acid productivity. For Lactobacillus amylophilus ATCC 49845,
Yumoto and Ikeda (63) reported optimal temperatures of 25 C and
35 C for maximum lactic acid productivity and yield, respectively.
Abdel-Rahman et al. (17) examined the effect of temperature on the
production of lactic acid from 20 g L
1 cellobiose by E. mundtii QU
25. Both the maximum lactic acid concentration and the highest
yield were obtained at 30e43 C.
An inoculum size of 5e10% (v v
1) is desirable to arrest heterolactic fermentation and to reduce the duration of the lag phase (64).
Because an inoculum size of more than 5% is costly and constrains
the operation, the inoculum size should be decreased. To investigate the inuence of inoculum size on lactic acid production,
different amounts (1e5% v v
1) were inoculated into the fermentation medium. The maximum lactic acid concentration of
33.72 g L
1 by Lactobacillus casei NBIMCC 1013 was observed with
an inoculum size of 2e4% (v v
1), whereas a low lactic acid production was attributed to a low density of starter culture (1% v v
1)
(65). Qi and Yao (66) studied the effect of inoculum size from 3% to
8% on lactic acid fermentation from rice straw with Lactobacillus
casei GIM 1.159. An inoculum size of 6% was found to be optimal for
the production of lactic acid. Lower inoculum sizes resulted in
insufcient biomass for lactic acid conversion, whereas higher

J. BIOSCI. BIOENG.,
inoculum sizes caused excessive depletion of the nutrients necessary for cell growth.
DIFFICULTIES AND PROBLEMS IN LACTIC ACID FERMENTATION
BY LAB
Although approximately 90% of worldwide lactic acid is produced by microbial fermentation, there are still many challenges for
more effective and economical production. One of the obstacles in
the process of co-culture using mixed sugars is carbon catabolite
repression when using renewable materials containing various
components (16,67). A few wild-type and metabolically engineered
strains have been shown to simultaneously consume different
sugars such as glucose/cellobiose (17), xylose/arabinose/glucose
(68), and cellobiose/glucose/xylose (38). Moreover, the strategy of
mixed cultures with different microorganisms has been studied for
improving product yield and sugar consumption (50,69).
Another obstacle is end-product inhibition in the lactic acid
production process by LAB. As fermentation progresses along with
substrate utilization, the concentration of lactic acid gradually increases, causing acidication of fermentation broth and leading to
slowing of the fermentation process, including cell growth, substrate utilization, and lactic acid production. Thus, to alleviate the
end-product inhibition effect caused by lactic acid during fermentation, lactic acid should be removed selectively in situ from the
fermentation broth by a separation method such as electrodialysis
(70), or lactic acid-resistant LAB should be selected (7).
By-products such as ethanol and acetic acid are usually formed in
hetero-fermentation using pentoses, which results in a theoretical
yield of only 0.60 g g
1 of lactic acid from pentose (16,42,43). Cost of
separation and purication also increases with the lowered purity of
the lactic acid product. Some potential pentose-utilizing strains are
reported to overcome the problem, e.g., the wild-type of E. mundtii QU
25 can consume xylose homofermenatively with little or no acetate
production and with high yield (16). A metabolically engineered
Lactobacillus plantarum DldhL1-xpk1::tkt was also reportedly able to
convert xylose (46) and arabinose (42) to lactic acid in homofermentation.
The most serious obstacle for lactic acid production commercially is the availability and cost of feedstock for lactic acid
fermentation (4,7). Particularly, carbon sources are signicant,
should be consumed by the LAB, and are divided into 2 groups:
puried sugars such as glucose, xylose, sucrose, and sugar-containing materials. However, the use of puried sugars as feedstock
for lactic acid production is very expensive. From the perspective of
the economics of the feedstock in terms of its geographic availability, domestic renewable materials such as wastes and unutilized
resources from the agro-industry and forestry are preferred due to
their availability and low cost.
LACTIC ACID PRODUCTION FROM RENEWABLE MATERIALS
In most cases, glucose is the preferred carbon source for lactic
acid fermentation by LAB. However, as cheap and widely existing
renewable materials, starch (e.g., wheat starch, corn starch, sago
starch, potato starch) and lignocellulose (e.g., woody materials, crop
residues) are recognized to meet the requirements for the biotechnological production of lactic acid economically and efciently.
Starch from various plant products is a potentially renewable
material in terms of cost and availability. As a type of polysaccharide
of glucose, starch mainly exists in tubers such as potatoes and
cassava, and seeds of grains including wheat, corn, and rice. On the
other hand, lignocellulose, another carbohydrate source, is available
in large quantities, with widespread distribution and comparably
lower cost (6,71). The major constituents of lignocellulose are

VOL. 119, 2015

LACTIC ACID PRODUCTION FROM RENEWABLE MATERIALS

15

TABLE 2. Recent investigations on lactic acid production from renewable materials by different fermentation modes and methods.
Microorganism

Substrate

0.50
0.93
e
0.76
0.93
0.83

0.38
1.105  0.06
1.56
3.04
1.7
1.2

68
15

39.1
83.8
141.5
162.5
180
35.4
90.0
48.7
44.2
166
28.0
37.11
24.0
46.4
32.5
57.61  1.37
118

0.70
0.96
0.936
0.897
0.903
0.35
0.90
0.95
0.92
0.87
0.28
0.46
0.76
0.46
0.88
0.46
0.94

0.81
1.40
4.7
1.69
2.14
0.75
2.3
1.01
5.7
4.2
0.78
1.03
0.51
0.64
5.4
1.60  0.12
1.71

88
89
13
90
72
91
53
92

97
129
27.0
10.9

0.89
0.95
0.90
0.36

1.74
3.1
6.7
0.17

93
94
18

Fed-batch
Batch
Continuous
Continuous (with high cell density)
Batch
Batch

74.7
16.6  0.8
10.4
11.7
93.0
62.8

Corncob molasses
Sago starch

E. faecalis RKY1
Lactobacillus bifermentans DSM
20003T
L. brevis S3F4
L. casei NCIMB 3254
L. casei G-02
L. casei LA-04-1

Wood hydrolyzate
Wheat bran hydrolyzate

L. defbrneckii NRRL B-445

L. delbrueckii mutant Uc-3
L. delbrueckii ZU-S2

Alfalfa bers
Cellobiose and cellotriose
Corncob residue

L. delbrueckii mutant Uc-3

L. delbrueckii IFO 3202
L. paracasei LA104
L. pentosus CECT-4023T
L. plantarum 14431
L. rhamnosus CECT-288
L. rhamnosus HG 09
Lactobacillus sp. MKT-878 NCAIM
B02375

Molasses
Rice bran
Green microalga
Trimming vine shoots
Alfalfa bers
Apple pomace
Hydrolyzed acorn starch
Wheat starch

Batch
SSF
Fed-batch, SSF
Fed-batch (fed with CSL)
Fed-batch (fed with YE)
SSF
Batch
Batch
Continuous
Batch
SSF
SSF
Batch
SSF
Batch
Batch
SHF

Lactobacillus sp. RKY2

Rice and wheat bran

Lignocellulosic hydrolyzates
Sugarcane bagasse

SSF
Batch
Cell-recycle
Batch

Lactococcus lactis IO-1

Reference

Lactic acid
g (g L
1)

Bacillus sp. strain XZL9

Enterococcus faecium No. 78

Corncob
Cassava bagasse
Jerusalem artichoke tubers
Soybean meal hydrolyzate

Productivity
P (g L
1 h
1)

Fermentation mode

Yield
Y (g g
1)

81
82
83
79
84
85
72
86
87

SSF, simultaneous saccharication and fermentation; SHF, separate hydrolysis and fermentation; CSL, corn steep liquor; YE, yeast extract; e, not determined.

cellulose (linear b-D-glucan), hemicellulose (hetero-polysaccharides including xylose, glucose, mannose, galactose, and
arabinose), and lignin (a polymer of three closely-related phenylpropane moieties). Basically, cellulose forms a skeleton, which is
surrounded by hemicellulose and lignin (6).
Pretreatment of raw renewable materials
Enzymatic hydrolysis of raw renewable materials easily exposes the fermentative
sugars consumed by the used strain, and the hydrolyzates can then be
used in SSF, or direct fermentation (72). However, enzymatic
hydrolysis of lignocellulosic biomass without pretreatment is usually
not very effective because of the high stability caused by the
association of cellulose and hemicellulose with lignin (73). Therefore,
pretreatment of renewable materials is performed to remove lignin,
separate cellulose and hemicellulose, increase the accessible surface
area, partially depolymerise cellulose, and increase the porosity of
the material to aid in the subsequent access of the hydrolytic
enzymes. Pretreatment methods include physical (mechanical),
physico-chemical (steam pretreatment, hydrothermolysis, wet
oxidation, ammonia ber expansion), chemical (dilute acid, alkaline,
ionic liquids), and biological methods (microorganisms, enzymes)
(5,73). As the result of pretreatment, the size of the material is
reduced and its physical structure is opened. On the other hand,
when starchy materials are used as the substrate for lactic acid
production, the bioconversion requires a pretreatment process
including the gelatinization and liquefaction of starch, which is
carried out at a temperature between 90 C and 130 C for 15 min (74).
Acid pretreatment is extensively applied in the hydrolysis of
starchy and lignocellulose materials (6). Pretreatment with dilute
acid at high temperature, or strong acid (H2SO4, SO2, H3PO4) at low
pH, usually results in the hydrolysis of hemicellulose to monomeric
sugars (e.g., xylose, arabinose) and minimizes the need for hemicellulases (75). However, the utilization of various chemicals in the
pretreatment procedures is a major drawback and affects the total
cost of the fermentation. Physical or physico-chemical methods of
pretreatment such as milling, steam explosion, and irradiation
reduce the particle size, thereby increasing the available surface area

for enzymatic attack. Sasaki et al. (76) pretreated sugarcane bagasse

using steam explosion, prior to the processes of enzymatic hydrolysis. After steam explosion at 20 atm, enzymatic saccharication of
pretreated raw bagasse and the water-insoluble residue provided the
highest recovery rates of glucose (73.3% and 94.9%, respectively).
Interestingly, washing of the residue with water after steam explosion removed inhibitors in the hydrolyzate of steam-exploded
bagasse. Biological delignication is another pretreatment method
that can enhance enzymatic hydrolysis. Kurakake et al. (77) evaluated the biological pretreatment of ofce paper for enzymatic hydrolysis by Sphingomonas paucimobilis and Bacillus circulans.
Pretreatment with the combination of these 2 strains improved the
efciency of enzymatic hydrolysis of ofce paper, and exhibited 94%
sugar recovery under optimal conditions.
Enzymatic hydrolysis The bioconversion of renewable materials to lactic acid requires fermentable components as the carbon
sources by the LAB strains used, and enzymatic hydrolysis can
improve the efciency of the subsequent fermentation process
drastically (78). The purpose of enzymatic hydrolysis is to break
down polysaccharides into easily fermentable sugars, even in the
water-insoluble solid fraction that remains after pretreatment (5).
a-Amylase (EC 3.2.1.1), b-amylase (EC 3.2.1.2), and glucoamylase
(EC 3.2.1.3) are well-known amylolytic enzymes that catalyze the
hydrolysis of a-glucosidic bonds in starch and related saccharides.
In the process of enzymatic hydrolysis of cassava bagasse for L-lactic
acid production, a-amylase and glucoamylase were used in SSF
(79). A lactic acid yield of 96% was obtained from starch using
15.5% w v
1 of cassava bagasse as the substrate.
Enzymatic hydrolysis of cellulose is generally carried out by a
mixture of several cellulases. At least 3 major groups of cellulases
are involved in this process: (i) endo-b-1,4-glucanases (EG, EC
3.2.1.3), which attack regions of low crystallinity in the cellulose
ber, creating a new reducing and non-reducing chain end pair; (ii)
exo-b-1,4-glucanases (or cellobiohydrolases) (CBH, EC 3.2.1.91),
which degrade the molecule further by cleaving cellobiose units
from the free chain-ends; and (iii) b-glucosidases (b-G, EC 3.2.1.21),

16

WANG ET AL.

J. BIOSCI. BIOENG.,

which hydrolyze cellobiose into 2 glucose molecules (5). Unlike

cellulose, hemicelluloses are not chemically homogeneous. Xylans
are the most abundant among hemicelluloses. Besides xylose, xylans contain arabinose, glucuronic acid or its 4-o-methyl ether,
acetic acid, ferulic acid, and p-coumaric acid. There are a number of
enzymes that attack hemicellulose, such as glucuronidase, xylanase, b-xylosidase, galactomannanase, and glucomannanase. Berlin
et al. (80) optimized enzyme complexes for the efcient enzymatic
hydrolysis of cellulose and xylan components of lignocellulose by
supplying cellulase with crude enzyme preparations enriched in
xylanase, pectinase, and b-glucosidase.

cells, removes excess calcium carbonate, and helps to decompose any

residual sugar in the medium (4). Lactic acid is further recovered by
hydrolysis, esterication, and distillation. The solvent is removed by
evaporation, and then the salt is decomposed to yield free lactic acid.
Meanwhile, other alternative methods for lactic acid purication
without using calcium carbonate such as electrodialysis (95), the
use of a membrane bioreactor (96), liquid surfactant membrane
extraction (97), and adsorption (98) exhibited good potential and
had the advantage of simultaneous separation and concentration
of lactic acid. The choice of separation process is based on the
efcient and economical usage of these extractants.

Fermentation modes and methods Several fermentation

modes were investigated for lactic acid production by various lactic
acid-producing strains using puried sugars and renewable materials (Table 2) as the substrates, including batch fermentation, fedbatch fermentation, semi-continuous/repeated batch fermentation,
continuous fermentation, SSF, and separate hydrolysis and
fermentation (SHF). In general, batch fermentation exhibited
higher lactic acid concentration and yield, but lower lactic acid
productivity than did continuous fermentation (15,81,88). In batch
fermentation, most of the substrate in the fermentor is consumed,
whereas the residual substrate would become the efuent in
continuous fermentation, resulting in a higher yield and
concentration of lactic acid in the former. On the other hand, endproduct inhibition of lactic acid is repressed by diluting the
fermentation broth in continuous fermentation; the reducing
inhibition effect leads to higher productivity (7). By integrating a
cell recycling system with continuous fermentation, higher cell
density than seen in conventional continuous fermentation can be
achieved, using modules such as microltration and ultraltration,
which would drastically increase lactic acid productivity. Tashiro
et al. (3) rst reported a continuous fermentation system for
D-lactic acid production using high cell density by cell recycling, in
which they obtained a high productivity of 18.0 g L
1 h
1 at a
dilution rate of 0.87 h
1.
SHF from renewable materials was also investigated, in which
the processes of enzymatic hydrolysis and fermentation were
conducted separately; each process could thereby be carried out
under optimal conditions (5). In the enzymatic hydrolysis process,
however, sugars yielded from renewable materials may strongly
inhibit hydrolytic enzyme activity (feedback inhibition), leading to
a requirement for more enzyme loads or a longer total period. On
the other hand, SSF is an effective method by which enzymatic
hydrolysis and fermentation are conducted in the same reactor
under the same conditions. The SSF process has several advantages
over SHF such as the usage of a single reactor for both steps, rapid
processing time, reduced feedback inhibition by the generated
sugars, and increased productivity (72). Nevertheless, the requirement for different optimal temperatures and pHs for saccharication and fermentation is the main limiting factor for SSF (5). The
optimal conditions for enzymatic hydrolysis are a temperature of
w50 C and a pH below 5.0, whereas the optimum conditions for
lactic acid fermentation are a temperature of 37e43 C and a pH
value of 5.0e7.0. Some compromises between the conditions for
enzymatic hydrolysis and fermentation are necessary in order to
obtain high overall lactic acid concentration, yield, and productivity
using SSF (5,19).

Conclusions and future scope

Fermentative production of
lactic acid has generated a signicant amount of interest because it
also offers solutions to the environmental pollution caused by the
petrochemical industry and the limited supply of petrochemical
resources. Two studies have been conducted to achieve efcient
fermentations for valuable substances from renewable materials.
First, most of the recent studies select the targeted renewable
materials initially, and then acquires an excellent strain with high
abilities of degrading and converting them to valuable substances
efciently by screening, mutagenesis, and molecular breeding
methods. Furthermore, an establishment of efcient production
process has been performed by the obtained strain.
On the other hand, we have recently proposed another study,
termed as designed biomass study (5). Designed biomass refers to
competent substances that can be designed for the corresponding
fermentation processes. In this type of study, all the technologies
previously developed in the recent studies (i.e., excellent strains or
efcient processes) can be applied; thereafter, the researchers
would identify or modify the renewable materials to be suitable for
the existing technologies. Of course, our concept involves not only
single sugar but also mixed sugars derived from various renewable
materials such as lignocellulosic biomasses after pretreatments and
enzymatic hydrolysis. One of the goals in designed biomass study is
to construct an adaptive production process by using the excellent
strain, efcient process, and designed biomass. Based on our
concept, we recently reported that the designed mixed sugars with
the ratio of glucose/cellobiose/xylose at 1:8:4 could achieve
simultaneous utilization of sugars by an excellent LAB of E. mundtii
QU 25 without carbon catabolite repression (99), which has been
serious drawback using mixed sugars in lactic acid fermentation as
described in the section effects of several factors on lactic acid
fermentation by LAB. In addition, we established the adaptive lactic
acid production process in the fed-batch fermentation using the
designed mixed sugars and E. mundtii QU 25 with much higher
lactic acid concentration (99) than those using the respective sole
sugar of glucose (48), cellobiose (17), and xylose (16).
In this review article, we have summarized many recent studies
on lactic acid fermentation from various renewable materials and
the production processes developed by the microbiologists in the
broad elds including fermentation technology, molecular microbiology, and metabolic engineering, and so on, and have corroborated the designed biomass study. Recently, genetically engineered
lignocellulosic biomasses such as rice straw have been reported by
the researchers in the eld of plant breeding, being improved in
enzymatic hydrolysis efciency (100) and being modied in compositions of cellulose and hemicellulose (101). Therefore, the active
collaboration with researchers between microbiology and plant
breeding should motivate the designed biomass study further and
would contribute to effective and economical lactic acid production
from renewable materials.

Separation and purication of lactic acid produced in lactic

acid fermentation Efcient separation and purication technologies are very important steps because they have a signicant
inuence on the nal quality and cost of lactic acid. For the purication of lactic acid, calcium carbonate is usually added to the
fermentation broth, and the pH is adjusted to approximately 10,
followed by heating and ltering. The procedure converts all of the
lactic acid to calcium lactate, coagulates protein in the medium, kills

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