Bioresource Technology 101 (2010) 78957901

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Bioresource Technology
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Efcient production of L-lactic acid from cassava powder by Lactobacillus rhamnosus

Limin Wang a,1, Bo Zhao a,1, Bo Liu a, Chunyu Yang b, Bo Yu a, Qinggang Li b, Cuiqing Ma b, Ping Xu a,c,*,
Yanhe Ma a
a

Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, Peoples Republic of China
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, Peoples Republic of China
c
MOE Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, Peoples Republic of China
b

a r t i c l e

i n f o

Article history:
Received 26 February 2010
Received in revised form 5 May 2010
Accepted 6 May 2010
Available online 2 June 2010
Keywords:
Cassava powder
Lactobacillus rhamnosus
L-Lactic acid
Fermentation

a b s t r a c t
Cassava is one of the most efcient and rich crops in terms of carbohydrate production, which is a tropical
perennial plant that grows on poor or depleted soils. Microbial conversion of such a renewable raw material to useful products is an important objective in industrial biotechnology. L-Lactic acid was efciently
produced from cassava powder by a Lactobacillus rhamnosus strain CASL. The fermentation properties of
cassava powder were compared with those of glucose and corn powder. The efciencies of various
fermentation strategies for L-lactic acid production from cassava powder, including simultaneous saccharication and fermentation (SSF), two-step fermentation (TSF) and simultaneous liquefaction,
saccharication and fermentation (SLSF), were investigated. The high L-lactic acid concentration
(175.4 g/l) was obtained using 275 g/l of cassava powder concentration (total sugar of 222.5 g/l) in SSF
batch fermentation. This is the highest L-lactic acid concentration reported, from cassava source, and it provides an efcient L-lactic acid production process with cheap raw bioresources, such as cassava powder.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
Lactic acid has extensive applications in the food, cosmetic,
pharmaceutical, and textile industries (Dumbrepatil et al., 2008).
Recently, the renewable and biodegradable plastic, poly(lactic
acid) (PLA), is attracting more and more attentions. Since L-lactic
acid is a precursor of PLA, the demand for L-lactic acid is also continuously increasing. Lactic acid can be produced by chemical synthesis or microbial fermentation. The latter is better because it
leads to the production of optically pure lactic acid, while chemical
synthesis results in a racemic mixture of lactic acid (Oh et al., 2005;
Dumbrepatil et al., 2008). Currently, microbial fermentation accounts for approximately 90% of the total lactic acid production
worldwide.
Most studies on L-lactic acid production have focused on the use
of pure or easily fermentable substrates such as glucose, sucrose,
maltose, or xylose (Patel et al., 2006; Ilmen et al., 2007). Due to
the high costs of these pure materials, the process is less economic
for industrial applications. The production cost of L-lactic acid
might be signicantly reduced if cheap raw materials could be
used, such as starchy, cellulosic materials, and molasses (Ohkouchi

* Corresponding author at: MOE Key Laboratory of Microbial Metabolism and

School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai
200240, Peoples Republic of China. Tel.: +86 21 34206647; fax: +86 21 34206723.
E-mail address: pingxu@sjtu.edu.cn (P. Xu).
1
These authors contributed equally to this study.
0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.05.018

and Inoue, 2006; Dumbrepatil et al., 2008; Romani et al., 2008). Of

these, starchy and cellulosic materials are currently receiving a
great attention because they are cheap, abundant, and renewable.
The utilization of cellulosic resources such as cellulose, corncob,
wood, and waste paper for L-lactic acid production is considered
to be a promising approach. However, to date, many technical
problems, such as inhibition of the enzymes involved in cellulose
hydrolysis by intermediate products, have hindered the commercial application of these processes. Using simultaneous saccharication and fermentation (SSF) operation, the enzyme inhibition
could be removed (John et al., 2006a; Romani et al., 2008). Utilization of cheap starches could be another effective approach for
reducing the cost of L-lactic acid production. The major starchy resources are cereals (corn, wheat, and rice), cassava, sweet potatoes,
and potatoes. In 2002, the world production of starch amounted to
approximately 58 million tons (roughly 69% from corn, 10% from
cassava, 9% from sweet potatoes, 6% from wheat, 6% from potatoes,
and less than 1% from other sources) (Peters, 2007). Many starchy
materials such as barley, corn, wheat, and potato have been used as
carbon sources for lactic acid production (Hofvendahl and HahnHagerdal, 1997; Oh et al., 2005; Wee et al., 2008).
Cassava is one of the most efcient crops in terms of carbohydrate production. It is a tropical perennial plant that grows on poor
or depleted soils in which the yields of other crops are very low
(Peters, 2007). In the subtropical region of southern China, cassava
is the fth largest crop in term of production, after rice, sweet potato, sugar cane, and maize. Cassava roots are very rich in starch,

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L. Wang et al. / Bioresource Technology 101 (2010) 78957901

and it is the basis of many products, including food, animal feed

and starch-based products (Kostinek et al., 2007). Despite these
advantages, cassava has four major drawbacks, including low energy density, low protein content, rapid postharvest deterioration
and potential cyanide toxicity, and consumption of insufciently
processed cassava may cause konzo (also called mantakassa), a
paralytic neurological disease (Gegios et al., 2010). According to
the study of Oboh, about 42% of harvested cassava roots in West
and East Africa are processed into dried chips and our, and most
cassavas are unutilized (Oboh and Oladunmoye, 2007). Cassava
starch, cassava bagasse, and fresh cassava roots have been reported
for L-lactic acid production (Ghofar et al., 2005; John et al., 2006a;
Wee et al., 2008). Cassava powder, a low-cost raw material, is produced by grinding cassava to powder. It contains abundant starch
(more than 70%), and could be used for lactic acid production.
However, to the best of our knowledge, there were few studies
on L-lactic acid production from the raw material, cassava powder
(Yu and Hang, 1989). In this study, cassava powder was chosen as
the major nutrient source for L-lactic acid production. The efciencies of various fermentation strategies, including SSF, two-step fermentation (TSF) and simultaneous liquefaction, saccharication
and fermentation (SLSF), were investigated. The aim of this study
was to develop an efcient and economic L-lactic acid fermentation
process using a Lactobacillus rhamnosus strain.

l, 150 g/l and 200 g/l, respectively. Calcium carbonate was added as
60% (w/w) of the carbon resources to the broth.
Cassava powder and corn powder were enzymatically hydrolyzed to degrade the starch into soluble sugar. Liquefaction was
carried out in 1-l beakers containing 500 ml of the mixed suspension. The procedure used was performed as follows: cassava powder and corn powder were sieved through a mesh with the
diameter of 250 lm and then suspended in deionized water. The
pH of the suspension was adjusted to 6.0 using 2 mol/l of HCl. Excess a-amylase was added to the suspension, and the resulting
mixture was heated to 95 C in a water bath. The residual starch
was measured by the color reaction of iodine. Liquefaction was
considered to be completed when the blue coloration of the iodine
test faded. The suspension was diluted to the required substrate
concentration and transferred to Erlenmeyer asks together with
YE and CaCO3 for L-lactic acid fermentation. The media were autoclaved at 115 C for 20 min. Excess glucoamylase was added to ensure complete release of glucose from the liqueed cassava
powder. Fermentations were carried out in 500-ml Erlenmeyer
asks each containing 200 ml medium. All the fermentations were
carried out at 42 C under static conditions. The culture pH was
maintained at 5.66.0 using CaCO3. The well mixed samples were
taken every 24 h, and the concentration of L-lactic acid was
determined.

2. Methods

2.4. Inuence of glucoamylase concentration and processing time on

glucose release

2.1. Chemicals
Cassava powder with a starch content of 89.6% (w/w) was
kindly provided by Ally Chem Co., Ltd. (Lianyungang, China). Corn
powder with a starch content of 85.7% (w/w) was purchased from
Hong Da Food Stuff Machining Factory (Sanli, China). Commercially
available thermo-stable a-amylase and glucoamylase with activities of 4  104 U/ml and 2.6  104 U/ml, respectively, were purchased from An Ke Bioengineering Co., Ltd. (Shandong, China). All
other chemicals were of analytical grade and commercially
available.
2.2. Microorganism, inoculum preparation and culture conditions

Cassava powder was prepared and liqueed as described above.

According to the product specications, the optimum pH and temperature for glucoamylase activity are 4.2 and 60 C, respectively.
The liqueed solution was diluted to the concentration of 100 g/l,
and the pH was adjusted to 4.2. To study the amount of glucoamylase required for glucose complete release, 26, 52, 78, 104, and
130 U glucoamylase per gram of cassava powder were added,
and the mixture was maintained at 60 C in a water bath and agitated at 100 rpm for 24 h. To study the reaction time required for
glucose complete release, 104 U glucoamylase per gram of cassava
powder was used and samples were taken at 0 h, 1 h, 2 h, 4 h, 6 h,
8 h, 9 h, and 10 h. The reactions were carried out in 100-ml Erlenmeyer asks each containing 50 ml of the mixed suspension.

L. rhamnosus strain CASL, isolated from the soil samples in a

milk-producing factory in China, was used in this study (Xu
et al., 2007). The strain was deposited in China General Microbiological Culture Collection Center (CGMCC No. 2183). It was characterized at Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH (DSMZ), Germany. Strain CASL is rod shaped
Gram positive, oxidase and hemolysis negative. Its carbon utilization pattern and cellular fatty acid pattern are also typical for the
species L. rhamnosus. Strain CASL is a homofermentative L-lactic
acid producer. The optical purity (ee) of L-lactic acid produced by
strain CASL is 9899% (Xu et al., 2007). It was maintained on the
agar slant with the following medium (in g/l): glucose, 50; yeast
extract (YE), 15; and CaCO3, 30. It was regenerated by three consecutive propagations at 42 C for 24 h in the above-mentioned medium without agitation. The seed culture was prepared by
incubating the strain in 200 ml of the above medium in 500-ml
Erlenmeyer asks for 24 h under static conditions. The inoculum
volume was 10% (v/v).

2.5. Optimization of concentrations of cassava powder and YE

2.3. Effect of various carbon sources on L-lactic acid fermentation

SSF, TSF, and SLSF are common strategies used for the fermentative production of lactic acid from starchy materials (Linko and
Javanainen, 1996; Oh et al., 2005; John et al., 2006a; Wee et al.,
2008). In this study, the fermentation medium used for developing
the appropriate strategy contained 275 g/l cassava powder, 5 g/l

The fermentation medium for studying the effect of various carbon sources on L-lactic acid production contained 15 g/l of YE. The
amounts of glucose, cassava powder, and corn powder were 100 g/

The fermentation medium used for optimizing the cassava powder concentrations contained 75350 g/l cassava powder and 15 g/
l YE. Calcium carbonate was added as 60% (w/w) of cassava powder
to the medium. The fermentation medium for optimizing the YE
concentration contained 275 g/l cassava powder, 015 g/l YE, and
165 g/l CaCO3. Excess a-amylase and 104 U glucoamylase per gram
of cassava powder were utilized to release the fermentable sugar
from the cassava powder. The enzyme pretreatment and fermentation processes were the same as those described above. Fermentations were carried out in 500-ml Erlenmeyer asks each containing
200 ml medium at 42 C under static conditions. The culture pH
was maintained at 5.66.0 using CaCO3. The well mixed samples
were taken every 6 h, 12 h, and 24 h, and the concentration of Llactic acid was determined.
2.6. Fermentation strategies

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L. Wang et al. / Bioresource Technology 101 (2010) 78957901

YE, and 165 g/l CaCO3. The steps involved in the various fermentation strategies are summarized in Fig. 1.
Fermentations were carried out in 500-ml Erlenmeyer asks
each containing 200 ml medium at 42 C under static conditions.
The culture pH was maintained at 5.66.0 by CaCO3 present in
the medium. The well mixed samples were taken every 24 h, and
the residual glucose and L-lactic acid concentrations were
determined.

was used. The culture pH was maintained at 5.66.0 by CaCO3

present in the fermentation medium. Enzymatic pretreatment of
the pre-culture was carried out by the same procedure as that used
in the ask, and the inoculum volume was 10% (v/v). Samples were
taken every 3 h, and the optical density (OD) together with residual
glucose and L-lactic acid concentrations were determined.

2.7. Batch fermentation in a 5-l bioreactor

The response surface methodology was also used to verify the

used fermentation medium. SAS package (version 9.0, SAS Institute
Inc., USA) was used for all the statistical analysis and the response
surface plotting. Besides the studied components, the fermentation
media for all the statistical optimization experiments contained
excess a-amylase and 200 g/l of CaCO3. SSF was used here, and

Batch fermentations were carried out in a 5-l bioreactor (Biostat

B., B. Braun Biotech International GmbH, Melsungen, Germany)
containing 2 l of clarifying solution medium at 42 C under static
conditions. The claried solution fermentation in SSF strategy

2.8. Statistical optimization of fermentation medium

Cassava powder suspended in deionized water

SLSF

TSF

Liquefaction
with
-amylase
Autoclaved at
121C for 15
min; -amylase
and
glucoamylase
were added to
the medium
together with
the inoculum.

Dextrin
Saccharification
with
glucoamylase

Glucose

L-lactic

Autoclaved at
115C for 20
min and
fermented with
strain CASL

acid

L-lactic

acid

SSF

CSF

Liquefaction
with
-amylase

Dextrin

Autoclaved at
115C for 20
min;
glucoamylase
was added to the
medium with the
inoculum

L-lactic

acid

Liquefaction
with
-amylase

Dextrin

Suspended
particles were
removed by
centrifugation,
and the
clarifying
solution was
used as the
feedstock
Clarifying
solution

Autoclaved at
115C for 20
min;
glucoamylase
was added to
the medium
with the
inoculum
L-lactic

acid

Fig. 1. Different types of fermentation strategies. SSF: simultaneous saccharication and fermentation; TSF: two-step fermentation; SLSF: simultaneous liquefaction,
saccharication and fermentation; CSF: clarifying solution fermentation in SSF.

L. Wang et al. / Bioresource Technology 101 (2010) 78957901

2.9. Analyses
The starch content of the cassava powder and corn powder was
determined by the anthronesulphuric acid colorimetry assay, and
the color of the samples was measured at 640 nm using a 7200 visible spectrophotometer (UNICO Instruments Co., Ltd., Shanghai,
China) (Wang, 2005). The fermentation broths were centrifuged
at 6000 rpm for 5 min and the supernatants were diluted to the desired extent with deionized water. The glucose and L-lactic acid
concentrations were measured by SBA-80C biosensor analyzer
(Institute of Biology, Shandong Academy of Sciences, China), which
could provide quick measurements of L-lactic acid and glucose
based on technology of the immobilized oxidases. The OD was
measured using a 7200 visible spectrophotometer at 620 nm.
Hydrochloric acid (2 mol/l) was added to neutralize the CaCO3
present in the fermentation medium. All the experiments were carried out in triplicates.
3. Results and discussion
3.1. L-Lactic acid production from various carbon sources in SSF
Glucose, cassava powder, and corn powder were used for L-lactic acid production. All the above carbon sources could be used by
strain CASL to produce L-lactic acid. L-Lactic acid production, yield
and average productivity are summarized in Table 1.
At glucose concentrations of 100 g/l and 150 g/l, the yields of Llactic acid (0.86 g/g) were the same. A higher glucose concentration (200 g/l) resulted in a lower L-lactic acid yield (0.67 g/g). With
the cassava powder as carbon source, the yield (0.85 g/g) was obtained at the cassava powder concentration of 100 g/L. Higher cassava powder concentration resulted in low L-lactic acid yield. The
average yield of L-lactic acid from corn powder was at around
0.96 g/g, which was higher than those of glucose and cassava powder. The maximum concentration of L-lactic acid in the fermentation with cassava powder and corn powder was 158.2 g/l and
177.7 g/l, respectively.
L-Lactic acid production from corn powder and cassava powder
was as efcient as that from glucose. Higher concentration of L-lactic acid was obtained from corn powder than that from cassava
powder. The reason may be due to more abundant nutrients in

Table 1
Use of different carbon sources by strain CASL for L-lactic acid production.
Carbon
source
Glucose

Substrate
concentration (g/l)
100
150
200

L-Lactic

acid

(g/l)

Yield
(g/g)
a

Productivityc
(g/l h)

85.5 1.3
129.5 3.0
133.8 3.8

0.86
0.86a
0.67a

1.8
1.8
1.9

Cassava
powder

100
150
200

86.3 1.1
120.7 3.5
158.2 2.8

0.85
0.81b
0.80b

0.9
1.7
1.7

Corn
powder

100
150
200

100.4 2.7
138.9 1.9
177.7 2.6

0.96b
0.97b
0.94b

1.4
1.9
2.5

Data presented are the means of three replicates; denotes the standard deviation
of the replicates.
a
g L-lactic acid/g initial glucose.
b
g L-lactic acid/(g initial starch  1.11).
c
Concentration of L-lactic acid (in g/l)/fermentation time (in h).

corn powder such as various amino acids and mineral substances

than those in cassava powder, and these nutrients could serve as
nitrogen sources during lactic acid production (Oh et al., 2005).
Furthermore, the potential cyanide toxicity of cassava powder
may inuence the propagation and metabolism of bacteria (Kostinek et al., 2007). For a long time, corn has been widely used for
productions of lactic acid and other chemicals by fermentation
(Oh et al., 2005; Franceschin et al., 2008). In order to rstly meet
humans demand for food, other cheap and applicable bioresource
should be alternative for lactic acid production. Raw cassava powder is such a low-cost and non-grain feedstock material, and it was
therefore used for L-lactic acid production in our study.
3.2. Inuence of glucoamylase concentration and processing time on
glucose release
Prior to L-lactic acid production, excess a-amylase was added to
liquefy the cassava powder solution. To ensure complete conversion of the starch present in cassava powder to fermentable sugar,
the effects of glucoamylase concentration and pretreatment time
on the release of fermentable sugar were studied (Linko and Javanainen, 1996). In the previous study, we found that the sugar released in the cassava powder solution was glucose (data not
shown). As shown in Fig. 2, the glucose concentration gradually increased with the increase in the enzyme concentration and pretreatment time. The maximal concentration of glucose (69.3 g/l)
was obtained when 104 U of glucoamylase per gram of cassava
powder was added. Further addition of glucoamylase had no effect

80
70

Glucose (g/l)

all of the experiments in this part were conducted in 300-ml

Erlenmeyer asks each containing 100 ml of medium at 42 C
under static conditions. Samples were taken at 96 h and the concentration of L-lactic acid was determined.

60
50
40
30
20
26

52
78
104
Glucoamylase (U/g cassava powder)

130

90
80
70

Glucose (g/l)

7898

60
50
40
30
20
10
0
0

6
Time (h)

10

Fig. 2. Effect of glucoamylase on glucose production from cassava powder. (a)

Effect of the glucoamylase concentration on glucose production and (b) effect of the
reaction time on glucose production.

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L. Wang et al. / Bioresource Technology 101 (2010) 78957901

on glucose release (Fig. 2a). The amount of released

reached 77.9 g/l at 9 h, and there was no further increase
increase in saccharication time (Fig. 2b). Therefore, the
of glucoamylase was 104 U per gram of cassava powder,
saccharication time was 9 h.

glucose
with an
amount
and the

3.3. Effect of the cassava powder concentration on L-lactic acid

production in SSF
Different concentrations of cassava powder were utilized by
strain CASL in SSF progress to produce L-lactic acid. The kinetic
parameters of L-lactic acid fermentation from cassava powder are
shown in Table 2.
L-Lactic acid production increased with an increase in the substrate concentration. When the initial concentration of cassava
powder was 300 g/l, the L-lactic acid concentration reached
232.4 g/l. Further addition of cassava powder did not improve Llactic acid production. The maximum yield of L-lactic acid was
0.86 g/g (from initial cassava powder concentrations of 100 g/l
and 125 g/l) and it decreased with an increase in the cassava powder concentration. The average productivity of L-lactic acid uctuated, and the highest value (2.6 g/l h) was obtained with 250 g/l
cassava powder. To the best of our knowledge, such a high L-lactic
acid concentration has rarely been reported in batch fermentation.
On one hand, strain CASL is a homofermentative L-lactic acid producer, which means that the carbon resource in the medium is
metabolized into only L-lactic acid by the homofermentative pathway through EMP (Xu et al., 2007). On the other hand, the highconcentration starch segments of cassava powder were gradually
degraded to fermentable sugar in SSF, and the inhibition by high
substrate concentrations was overcome (John et al., 2006a). Furthermore, when the cassava powder concentration was high (more
than 275 g/l), the medium solidied readily. This led to difculties
in the subsequent L-lactic acid fermentation process.
3.4. Effect of the YE concentrations on L-lactic acid production in SSF
YE is the most commonly used nutrient source in fermentations, but high price hinders its use in large quantities. In an economic analysis, the largest contributor for lactic acid production
was found to be YE, which accounted for about 38% of total production medium cost (Altaf et al., 2007). To study the effect of the YE
concentration on L-lactic acid production, different concentrations
of YE (0, 2.5, 5, 7.5, and 10 g/l) were added to the medium containing 275 g/l cassava powder. L-Lactic acid concentration increased
with the addition of YE, and 173.4 g/l of L-lactic acid was obtained

Table 2
Kinetic parameters of L-lactic acid fermentation from cassava powder in SSF.
Cassava powder (g/l)

L-Lactic

75
100
125
150
175
200
225
250
275
300
325
350

64 3.0
86.3 1.1
107.6 1.8
120.7 3.5
143.9 0.8
158.2 2.8
169.8 4.1
188.9 2.8
204.9 4.7
232.4 0
225.1 4.7
226.0 2.3

acid (g/l)

Yielda (g/g)

Productivityb (g/l h)

0.86
0.86
0.86
0.81
0.83
0.80
0.76
0.76
0.75
0.77
0.70
0.65

0.9
0.9
1.5
1.7
2.0
1.7
2.4
2.6
2.1
1.6
1.6
2.4

Data presented are the means of three replicates; denotes the standard deviation
of the replicates.
a
g L-lactic acid/(g initial starch  1.11).
b
Concentration of L-lactic acid (in g/l)/fermentation time (in h).

with 5 g/l of YE. Further addition did not signicantly improve the
L-lactic acid production (data not shown).
In general, lactic acid bacteria are fastidious microorganisms
that require substrates with high nitrogen content, and have a particular demand for B vitamins (Hofvendahl and HahnHagerdal,
1997). YE is rich in B vitamins and could enhance L-lactic acid production rates by lactic acid bacteria. Various less expensive nitrogen sources, including peptone, corn-steep liquor, and urea, were
compared with YE in terms of their efciency for lactic acid production. None of these nitrogen sources was reported to give lactic
acid concentration as high as that obtained with YE (Nancib et al.,
2001). In our study, low amounts of YE (5 g/l) could meet the demand for high L-lactic acid production.
3.5. L-Lactic acid production in different fermentation strategies
To develop an efcient process for L-lactic acid production, the
SSF, TSF, and SLSF strategies were compared with respect to L-lactic
acid production. The fermentation parameters are summarized in
Table 3.
In SLSF, 187.2 g/l of L-lactic acid, with a yield of 0.68 g/g and an
average productivity of 1.6 g/l h was obtained. Although high L-lactic acid concentration and productivity was obtained in SLSF, the
viscosity of the medium dramatically increased after autoclaving,
which negatively inuenced the down-stream processing. Our results were similar to those of John et al., who recently reported that
the optimal initial concentration of cassava bagasse was 15.5% (w/
v) in SLSF and that further increase in the concentration of the cassava bagasse made the medium slurry, which negatively inuenced the mixing of enzymes and inoculum (John et al., 2006a).
While in TSF and SSF, liquefaction before autoclaving overcame
the viscosity problem and led to the rapid transformation of starch
to dextrin.
As shown in Table 3, L-lactic acid concentration in SSF reached
155.8 g/l and no glucose remained, while 145.1 g/l of L-lactic acid
was obtained in TSF and 37.3 g/l of glucose remained in the medium. Enzymatic pretreatment for liquefaction and saccharication
in TSF resulted in complete glucose release in the nal hydrolysate.
Thus, the high initial concentration of reduced sugar inhibited Llactic acid production. During saccharication in SSF, hydrolyzing
enzymes were added together with the inoculum. The glucose produced by the stepwise action of glucoamylase at the non-reducing
ends of dextrins was fermented by the microorganisms to L-lactic
acid (Hofvendahl and HahnHagerdal, 1997).
TSF, as a kind of conventional L-lactic acid production strategy
from starchy materials, requires gelatinization, liquefaction, and
saccharication prior to fermentation. This strategy is not economical because of the high-energy required for the pretreatment of

Table 3
Kinetic parameters of L-lactic acid fermentation in different fermentation strategies.
Fermentation
process

L-Lactic acid
(g/l)

Residual
glucose (g/l)

Yielda
(g/g)

Productivityb
(g/l h)

SLSF
TSF
SSFc
SSFd

187.2 6.6
145.1 9.6
155.8 8.5
180.9 3.7

0
37.3 2.5
0
0

0.68
0.53
0.57
0.66

1.6
1.0
1.1
1.3

The fermentation was carried out in a medium containing cassava powder (275 g/l).
Data presented are the means of three replicates; denotes the standard deviation
of the replicates.
SLSF: simultaneous liquefaction, saccharication and fermentation. TSF: two-step
fermentation. SSF: simultaneous saccharication and fermentation.
a
g L-lactic acid/(g initial starch  1.11).
b
Concentration of L-lactic acid (in g/l)/fermentation time (in h).
c
SSF: turbid hydrolysate solution fermentation in SSF.
d
SSF: claried solution fermentation in SSF.

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L. Wang et al. / Bioresource Technology 101 (2010) 78957901

30

200

25

150
20

125

15

100
75

10

OD (620 nm)

Glucose (g/l), L-Lactic acid (g/l)

175

50
5

solution fermentation in SSF than those obtained in the turbid

hydrolysate fermentation in SSF (155.8 g/l and 0.57 g/g). The average productivities in the claried solution fermentation in SSF and
the turbid hydrolysate fermentation in SSF were 1.3 g/l h and 1.1 g/
l h, respectively. The possible reason is that the suspended particles inuence the glucoamylase activity and the glucose release
in the hydrolysate. Moreover, lactic acid production from the turbid solution required more steps and led to difculties in product
purication. Therefore, in our study, the claried solution from
the liqueed cassava powder hydrolysate, which contained the
same amount of soluble dextrin as turbid hydrolysate, was used
as the feedstock.

25

3.6. Batch fermentation in a 5-l fermentor

0
0

24

48

72
Time (h)

96

120

144

Fig. 3. Time course of L-lactic acid production by strain CASL in batch fermentation.
Symbols: L-lactic acid production (j), glucose consumption (N), and OD620 (d). The
fermentation was carried out in a medium containing cassava powder (275 g/l).

starchy materials. Moreover, autoclaving at high temperature and

high pressure results in the Maillard reaction, leading to caramelization and the consumption of some nutrient sources. Compared
with TSF, SSF has many benets. Because saccharication occurs
during fermentation, it eliminates the need for complete hydrolysis of starch to glucose prior to fermentation. The cost savings
are substantial because the requirements in terms of energy input,
reactor tanks, and time are reduced (Romani et al., 2008). Based on
the above results and discussion, SSF appeared to be more suitable
than TSF and SLSF for producing L-lactic acid at an industrial scale.
During L-lactic acid production, there was a phenomenon that
large amounts of particles were suspended in the cassava powder
hydrolysate. Our preliminary study indicated that the volume of
suspended particles was 35% (v/v) that of the liqueed cassava
powder (data were not shown). The suspended particles in SSF
were removed by centrifugation and the claried solution was
used as the feedstock. The comparison between the claried solution fermentation in SSF and turbid hydrolysate fermentation in
SSF was conducted under the same experimental conditions, i.e.,
with the same amounts of cassava powder and YE, same pH and
a constant C/N ratio. As shown in Table 3, higher concentration
(180.9 g/l) and yield (0.66 g/g) were obtained in the claried

L-Lactic acid production with cassava powder was carried out in

a 5-l fermentor by the claried solution fermentation in SSF strategy. According to the results in Sections 2.4 and 2.5, the medium,
containing 275 g/l cassava powder, 5 g/l YE, and 104 U glucoamylase per gram of cassava powder, was chosen for the batch fermentation. The formation of L-lactic acid nished at 96 h when the
residual glucose was completely consumed, and 175.4 g/l of L-lactic acid was obtained, with a productivity of 1.8 g/l h and a yield of
0.71 g/g based on initial starch (Fig. 3). Compared with the previous published results, our study showed that high concentration
and productivity of L-lactic acid could be obtained from cassava
powder by strain CASL (Table 4). The OD value at 620 nm increased
with the fermentation time and maintained at the value around 20
after 72-h incubation, which showed that the bacterial cell density
maintained at relatively high levels during the later period of fermentation progress.
The response surface methodology conrmed the verication of
the fermentation medium used. Cassava powder, glucoamylase,
and YE were used to compose the Box-Behnken design (Tables S1
and S2 in Supplementary data). The results showed YE (X3) had signicant positive inuence on L-lactic acid production. The response
surfaces and corresponding contour plots described by the multiple regression model shows that the maximum value of L-lactic
acid (166.7 g/l) was predicted using a optimized medium at cassava powder, glucoamylase, and YE concentrations of 284.3 g/l,
95.2 U/g, and 6.1 g/l, respectively. Using the optimized medium,
the production of L-lactic acid was 167.0 g/l by the batch fermentation, with a productivity of 1.7 g/l h and a yield of 0.71 g/g based on

Table 4
Comparison of L-lactic acid production from some starchy materials based on the results reported in the literature and those of this study.
Organism

Carbon source (g/

l) + nitrogen source (g/l)

Fermentation
process

Lactic
acid (g/l)

Yield (g/g)

Productivity
(g/l h)

Reference

Enterococcus faecalis RKY1

Corn starch (125) + CSL

(30) + YE (1)
Tapioca starch (125) + CSL
(30) + YE (1)
Potato starch (125) + CSL
(30) + YE (1)
Wheat starch (125) + CSL
(30) + YE (1)
Cassava bagasse
(155)+NH4Cl (5) + YE (5)
Barley starch (170) + YE
(10) + peptone (10)
Whole wheat our
(200) + CSL (15)
Wheat with protease
pretreatment (200)+ YE (1)
Cassava powder (275) + YE
(5)

TSF

129.9

1.04 based on initial

substrate
1.01 based on initial
substrate
0.99 based on initial
substrate
0.99 based on initial
substrate
0.96 based on initial
starch
0.87 based on initial
available glucose
0.920.94 based on
consumed glucose
0.95 based on initial
starch
0.71 based on initial
starch

1.5

Wee et al. (2008)

Lactobacillus casei NCIMB 3254

Lactobacillus casei NRRL B-441
Enterococcus faecalis RKY1
Lactobacillus delbrueckii NCIM 2025 and
Lactobacillus casei NCIMB 3254
Lactobacillus rhamnosus strain CASL

126.7
123.3
123.2
SLSF

83.8

SLSF

162

TSF

102.7

SLSF

123

SSF

175.4

The values in parentheses represent the concentrations of the carbon and nitrogen sources; CSL: corn-steep liquor.

1.5
1.7
1.4
1.4

John et al. (2006a)

2.3

Linko and Javanainen

(1996)
Hofvendahl and
HahnHagerdal (1997)
John et al. (2006b)

1.8

This study

3.8

L. Wang et al. / Bioresource Technology 101 (2010) 78957901

initial starch. Thus, the used media components could meet the
requirement on the fermentation for L-lactic acid production.
Since lactic acid production involved different processes, such
as the upstream processing, and the down-stream processing, the
lactic acid production cost ranged from 0.1 to 2.0 US$/kg lactic
acid. Among this, the cost of raw materials accounted for more
than 34% of total production-cost (Akerberg and Zacchi, 2000).
The unit price of raw cassava powder was more inexpensive than
that of the fresh corn and processed cassava products (Ghofar
et al., 2005; http://www.fao.org/). Results in this study implied
that the L-lactic acid production method presented should be feasible for the industrial-scale lactate production.
4. Conclusions
L. rhamnosus strain CASL was shown to be capable of producing
higher concentration and yield of L-lactic acid from cassava powder
than conventionally used rened sugars such as glucose in batch
fermentation. Considering both economy and convenience, SSF is
more suitable than TSF and SLSF. The claried solution fermentation in SSF enhanced the performance of L-lactic acid production
by reducing the cost of the medium and simplifying the fermentation process. The present study proposes a novel and economic
method for L-lactic acid production.
Acknowledgements
This study was supported by the National Basic Research Program of China (2007CB707803), Chinese National Programs for
High Technology Research and Development (2006AA020102 and
2007AA10Z360), Knowledge Innovation Program of the Chinese
Academy of Sciences (KSCX2-YW-G-005), and National Natural Science Foundation of China (30900022).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biortech.2010.05.018.
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