This article appeared in a journal published by Elsevier.

The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elseviers archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright

Author's personal copy

Bioresource Technology 101 (2010) 78957901

Contents lists available at ScienceDirect

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

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

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.

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

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,

Author's personal copy

7896

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. 2. Methods 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 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). 2.3. Effect of various carbon sources on L-lactic acid fermentation 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/

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. 2.4. Inuence of glucoamylase concentration and processing time on glucose release 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. 2.5. Optimization of concentrations of cassava powder and YE 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 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

Author's personal copy

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

7897

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. 2.7. Batch fermentation in a 5-l bioreactor 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

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. 2.8. Statistical optimization of fermentation medium 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

Cassava powder suspended in deionized water

SLSF

TSF

SSF

CSF

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

Liquefaction with -amylase

Liquefaction with -amylase

Dextrin Saccharification with glucoamylase

Dextrin

Dextrin

Glucose

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

L-lactic

acid

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

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

L-lactic

acid

L-lactic

acid

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.

Author's personal copy

7898

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

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. 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

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)

60 50 40 30 20 26 52 78 104 Glucoamylase (U/g cassava powder) 130

Table 1 Use of different carbon sources by strain CASL for L-lactic acid production. Carbon source Glucose Substrate concentration (g/l) 100 150 200 100 150 200 100 150 200
L-Lactic

90 80 70

acid

85.5 1.3 129.5 3.0 133.8 3.8 86.3 1.1 120.7 3.5 158.2 2.8 100.4 2.7 138.9 1.9 177.7 2.6

0.86 0.86a 0.67a 0.85b 0.81b 0.80b 0.96b 0.97b 0.94b

Glucose (g/l)

(g/l)

Yield (g/g)

Productivityc (g/l h) 1.8 1.8 1.9 0.9 1.7 1.7 1.4 1.9 2.5

60 50 40 30 20 10 0 0 2 4 6 Time (h) 8 10

Cassava powder Corn powder

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).

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.

Author's personal copy

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

7899

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

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 2 Kinetic parameters of L-lactic acid fermentation from cassava powder in SSF. Cassava powder (g/l) 75 100 125 150 175 200 225 250 275 300 325 350
L-Lactic

acid (g/l)

Yielda (g/g) 0.86 0.86 0.86 0.81 0.83 0.80 0.76 0.76 0.75 0.77 0.70 0.65

Productivityb (g/l h) 0.9 0.9 1.5 1.7 2.0 1.7 2.4 2.6 2.1 1.6 1.6 2.4

Table 3 Kinetic parameters of L-lactic acid fermentation in different fermentation strategies. Fermentation process SLSF TSF SSFc SSFd
L-Lactic acid (g/l)

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

Residual glucose (g/l) 0 37.3 2.5 0 0

Yielda (g/g) 0.68 0.53 0.57 0.66

Productivityb (g/l h) 1.6 1.0 1.1 1.3

187.2 6.6 145.1 9.6 155.8 8.5 180.9 3.7

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).

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.

Author's personal copy

7900

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

200 175 Glucose (g/l), L-Lactic acid (g/l) 150

30 25 20 15 10 5 OD (620 nm)

125 100 75 50 25 0 0 24 48 72 Time (h) 96 120

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.

0 144

3.6. Batch fermentation in a 5-l fermentor

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

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

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 Enterococcus faecalis RKY1 Carbon source (g/ l) + nitrogen source (g/l) 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) Fermentation process TSF Lactic acid (g/l) 129.9 126.7 123.3 123.2 SLSF SLSF TSF SLSF SSF 83.8 162 102.7 123 175.4 Yield (g/g) 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 Productivity (g/l h) 1.5 1.5 1.7 1.4 1.4 3.8 2.3 1.8 John et al. (2006a) Linko and Javanainen (1996) Hofvendahl and HahnHagerdal (1997) John et al. (2006b) This study Reference 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

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

Author's personal copy

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

7901

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. References
Akerberg, C., Zacchi, G., 2000. An economic evaluation of the fermentative production of lactic acid from wheat our. Bioresour. Technol. 75, 119126. Altaf, M., Naveena, B.J., Reddy, G., 2007. Use of inexpensive nitrogen sources and starch for L-lactic acid production in anaerobic submerged fermentation. Bioresour. Technol. 98, 498503. Dumbrepatil, A., Adsul, M., Chaudhari, S., Khire, J., Gokhale, D., 2008. Utilization of molasses sugar for lactic acid production by Lactobacillus delbrueckii subsp. delbrueckii mutant Uc-3 in batch fermentation. Appl. Environ. Microbiol. 74, 333335.

Franceschin, G., Zamboni, A., Bezzo, F., Bertucco, A., 2008. Ethanol from corn: a technical and economical assessment based on different scenarios. Chem. Eng. Res. Des. 86, 488498. Gegios, A., Amthor, R., Dixon, B.M., Egesi, C., Mallowa, S., Nungo, R., Gichuki, S., Mbanaso, A., Manary, M.J., 2010. Children consuming cassava as a staple food are at risk for inadequate zinc, iron, and vitamin A intake. Plant Foods Hum. Nutr.. doi:10.1007/s11130-010-0157-5. Ghofar, A., Ogawa, S., Kokugan, T., 2005. Production of L-lactic acid from fresh cassava roots slurried with tofu liquid waste by Streptococcus bovis. J. Biosci. Bioeng. 100, 606612. Hofvendahl, K., HahnHagerdal, B., 1997. L-Lactic acid production from whole wheat our hydrolysate using strains of Lactobacilli and Lactococci. Enzyme Microb. Technol. 20, 301307. Ilmen, M., Koivuranta, K., Ruohonen, L., Suominen, P., Penttila, M., 2007. Efcient production of L-lactic acid from xylose by Pichia stipitis. Appl. Environ. Microbiol. 73, 117123. John, R.P., Nampoothiri, K.M., Pandey, A., 2006a. Simultaneous saccharication and fermentation of cassava bagasse for L-(+)-lactic acid production using Lactobacilli. Appl. Biochem. Biotechnol. 134, 263272. John, R.P., Nampoothiri, K.M., Pandey, A., 2006b. Simultaneous saccharication and L-(+)-lactic acid fermentation of protease-treated wheat bran using mixed culture of Lactobacilli. Biotechnol. Lett. 28, 18231826. Kostinek, M., Specht, I., Edward, V.A., Pinto, C., Egounlety, M., Sossa, C., Mbugua, S., Dortu, C., Thonart, P., Taljaard, L., Mengu, M., Franz, C.M.A.P., Holzapfel, W.H., 2007. Characterisation and biochemical properties of predominant lactic acid bacteria from fermenting cassava for selection as starter cultures. Int. J. Food Microbiol. 114, 342351. Linko, Y.Y., Javanainen, P., 1996. Simultaneous liquefaction, saccharication, and lactic acid fermentation on barley starch. Enzyme Microb. Technol. 19, 118 123. Nancib, N., Nancib, A., Boudjelal, A., Benslimane, C., Blanchard, F., et al., 2001. The effect of supplementation by different nitrogen sources on the production of lactic acid from date juice by Lactobacillus casei subsp. rhamnosus. Bioresour. Technol. 78, 149153. Oboh, G., Oladunmoye, M.K., 2007. Biochemical changes in micro-fungi fermented cassava our produced from low- and medium-cyanide variety of cassava tubers. Nutr. Health 18, 355367. Oh, H., Wee, Y.J., Yun, J.S., Han, S.H., Jung, S.W., Ryu, H.W., 2005. Lactic acid production from agricultural resources as cheap raw materials. Bioresour. Technol. 96, 14921498. Ohkouchi, Y., Inoue, Y., 2006. Direct production of L-lactic acid from starch and food wastes using Lactobacillus manihotivorans LMG 18011. Bioresour. Technol. 97, 15541562. Patel, M.A., Ou, M.S., Harbrucker, R., Aldrich, H.C., Buszko, M.L., Ingram, L.O., Shanmugam, K.T., 2006. Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid. Appl. Environ. Microbiol. 72, 32283235. Peters, D., 2007. Raw materials. Adv. Biochem. Eng. Biotechnol. 105, 130. Romani, A., Yanez, R., Garrote, G., Alonso, J.L., 2008. SSF production of lactic acid from cellulosic biosludges. Bioresour. Technol. 99, 42474254. Wang, F.R., 2005. Bioengineering analysis and test. China Light Industry Press, Beijing, China. Wee, Y.J., Reddy, L.V.A., Ryu, H.W., 2008. Fermentative production of L-(+)-lactic acid from starch hydrolyzate and corn steep liquor as inexpensive nutrients by batch culture of Enterococcus faecalis RKY1. J. Chem. Technol. Biotechnol. 83, 1387 1393. Xu, P., Zhao, B., Qin, J.Y., Ma, C.Q., Zhou, S., 2007. Method and a Lactobacillus rhamnosus strain CASL CGMCC No. 2183 for the production of L-lactic acid. Chinese Patent, ZL 200710176057.7. Yu, R.C., Hang, Y.D., 1989. Kinetics of direct fermentation of agricultural commodities to L-(+) lactic acid by Rhizopus oryzae. Biotechnol. Lett. 11, 597 600.