Process Biochemistry 40 (2005) 681690

Direct fermentation of starch to l(+) lactic acid in SSF by Lactobacillus amylophilus GV6 using wheat bran as support and substrate: medium optimization using RSM
B.J. Naveena a , Md. Altaf a , K. Bhadrayya b , S.S. Madhavendra c , Gopal Reddy a,
a

Department of Microbiology, Osmania University, Hyderabad 500007, AP, India b Swaroop Tech. Consultancy, Secunderabad 500003, AP, India c Indian Institute of Chemical Technology (IICT), Hyderabad 500007, AP, India

Received 6 November 2003; received in revised form 2 January 2004; accepted 30 January 2004

Abstract Wheat bran is an underexploited cheap byproduct obtained during the milling process of wheat. The ability of Lactobacillus amylophilus GV6 to hydrolyze raw starch in wheat bran to produce l(+) lactic acid was studied in solid state fermentation (SSF), opening a novel method for the utilization of these agricultural byproducts. l(+) Lactic acid production by L. amylophilus has been reported by various groups from processed and unprocessed starch but there are no reports available from natural starches in SSF. All amylolytic wild strains reported so far have high yield efciencies at low substrate concentrations whereas at high substrate concentrations lactic acid production was low. L. amylophilus GV6 showed high yield efciency at high substrate concentration in SSF. To improve and optimize l(+) lactic acid production by L. amylophilus GV6, response surface methodology (RSM) using central composite rotatable design was adopted in SSF. MINITAB-13 was used for planning the experiments, data analysis, contour diagrams and response optimizations. The optimum media composition was obtained as peptone, 0.9%; yeast extract, 0.88%; tri-ammonium citrate, 0.379%; NaH2 PO4 2H2 O, 0.769% and Tween-80, 0.30 ml. Under these conditions a maximum of 36 g of lactic acid was produced per 100 g of wheat bran having 54 g of starch. The organism showed 90% yield efciency based on substrate consumed. Successful optimization of the selected ingredients, led to 100% increase in lactic acid production, i.e. from 18 to 36 g. Due to its high potentiality in conversion of starch to l(+) lactic acid, L. amylophilus GV6 can be exploited industrially for developing a novel technology using inexpensive renewable resources. 2004 Elsevier Ltd. All rights reserved.
Keywords: Lactobacillus amylophilus; Wheat bran; Lactic acid; RSM; SSF

1. Introduction Lactic acid fermentation has received extensive attention for a long time [1,2]. Lactic acid has wide applications in food, pharmaceutical, leather, textile industries and as a chemical feed stock. It has two enantiomers l(+) and d() of which l(+) isomer is used by human metabolism due to the presence of l lactate dehydrogenase and is preferred for food. In view of recent developments l(+) lactic acid is in great demand due to its use as starting material to produce biodegradable polymers used in medical, industrial and consumer products [36]. The lactic acid market is expected to
Corresponding author. Tel.: +91-4027682246; fax: +91-4027090020. E-mail addresses: navvi99@yahoo.co.in (B.J. Naveena), gopalred@hotmail.com (G. Reddy). 0032-9592/$ see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.01.045

grow 8.6% annually and in US alone its demand is expected to be 49,600 MT in future [7,8]. Lactic acid is produced by chemical synthesis and microbial fermentation. Chemical synthesis results in racemic mixture of lactic acid whereas specic stereo isomeric form can be obtained by microbial fermentation [9,10]. Rened sugars, although expensive are the most commonly used substrates for producing lactic acid by fermentation [2]. Lactic acid is also produced from cheaper substrate like starch in two-step process of saccharication followed by Lactobacillus fermentation [10]. Use of a microorganism which directly ferments starch to l(+) lactic acid would eliminate saccharication cost and Lactobacillus amylophilus GV6 is one such organism having high production efciency in submerged fermentation as reported earlier [1113].

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Solid-state fermentation (SSF) technology has gained renewed attention from industry, due to its advantages and reported to be the most appropriate process for developing countries [14,15]. Use of SSF in direct conversion of starch to l(+) lactic acid is not yet exploited. It is known that minimal preprocessing and supplementing of inorganic nutrients to inexpensive substrates, originated from waste materials (starch or cellulosic substances), would offer great advantage to increase lactic acid production [1618]. A number of cheaply available raw substrates (brans) have been screened for single step production of lactic acid by L. amylophilus GV6 in SSF. Wheat bran was selected as the best substrate and support in SSF in laboratory level fermentations [19]. In the present study, to achieve success in faster yield improvements, the media components peptone, yeast extract, tri-ammonium citrate, NaH2 PO4 2H2 O and Tween-80 were evaluated for lactic acid production in SSF and statistically optimized to enhance productivity. Each variable was tested based on our prior experience over a range and xed in central composite rotatable design of RSM. The role of each variable, their interactions and statistical analysis to obtain predicted yields of lactic acid was explained by applying second-order polynomial model. The data was analyzed statistically and response surface contour plots were constructed which indicated the possibility of enhancement in the production of lactic acid. The analysis was done using MINITAB-13.

and 0.1 M), dehydrated in alcohol and these dehydrated samples were placed on aluminium stubs gold coated in HUS-GB vacuum evaporator, observed in Hitachi S-520 scanning electron microscope (SEM). 2.4. Extraction and estimation of lactic acid Lactic acid formed after fermentation was extracted from fermented bran with 50 ml distilled water by vigorous shaking and ltering through cheesecloth after allowing it to stand for 3 h. The extract was cold centrifuged (8000 rpm for 20 min) and supernatant was taken for estimating the lactic acid. Total lactic acid was estimated according to the method of Kimberley and Tailor [20]. The concentration of lactic acid is expressed as gram per 10 g of the fermented bran. Total starch content in bran was determined by standard acid hydrolysis method [21]. 2.5. Response surface methodology (RSM) The central composite rotatable design [22] was used to develop mathematical models that relate the variables in the experiments and optimize them for lactic acid production. According to this design, 32 experiments were performed. Details of response surface methodology can be found in Neter et al. [23]. The matrix for this design along with observed results are shown in Table 1. 2.6. Statistical analysis RSM is used to study the variables independently for their interactions and quadratic effects. MINITAB-13 was used for analysis of observed data, contour diagrams, regression coefcients, t-statistics and response optimizations. Five variables were considered for application of RSM. The behaviour of the system was explained by the following second-order polynomial equation. Y = B0 + Bi X i +
2 Bii Xi +

2. Materials and methods 2.1. Microorganism Lactobacillus amylophilus GV6, a facultatively anaerobic, amylolytic lactic acid producing strain, isolated, characterized and maintained in this laboratory was used in the present SSF studies [1113]. 2.2. Fermentation media SSF was carried out anaerobically at 37 C, pH 6.75 in 120 ml serum vials with prereduced and sterilized medium composed of 10 g of wheat bran and appropriate volume of moistening liquid (83%). The moistening liquid contains sodium acetate 0.2%, magnesium sulphate 0.05%, calcium carbonate 8%, and the ve nutrients (peptone, yeast extract, tri-ammonium citrate, NaH2 PO4 2H2 O and Tween-80) selected for optimizations according to the requirement of experimental design. N2 is used as head space gas. Lactic acid extraction is done after 5 days of incubation. 2.3. Substratemicrobe interaction by scanning electron microscope Fermented and unfermented wheat bran samples were xed in 4% gluteraldehyde in phosphate buffer (pH 6.9

Bij Xi Xj

(1)

in which Y is predicted response which is a dependent variable, i.e. lactic acid production; B0 is an off set term (constant); Bi is linear effect; Bij is quadratic effect when i = j and interaction effect when i < j; Bii is a squared term; Xi is ith variable, which are called as independent variables. The second-order polynomial equations were used to estimate the response of the dependent variable, i.e. lactic acid. Later an experiment was run using the optimum values for variables given by response optimization for conrmation of the predicted value and the lactic acid production was conrmed.

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Table 1 Central composite rotatable design for optimization of ve nutrients (each at ve levels) and mathematically predicted values and experimental values for the production of l(+) lactic acid by Lactobacillus amylophilus GV6 in SSF Run Peptone (x1 ) Yeast extract (x2 ) Triammonium citrate (x3 ) (1) 0.4 (1) 0.4 (1) 0.4 (1) 0.4 (1) 0.8 (1) 0.8 (1) 0.8 (1) 0.8 (1) 0.4 (1) 0.4 (1) 0.4 (1) 0.4 (1) 0.8 (1) 0.8 (1) 0.8 (1) 0.8 (0) 0.6 (0) 0.6 (0) 0.6 (0) 0.6 (2) 0.2 (2) 1.0 (0) 0.6 (0) 0.6 (0) 0.6 (0) 0.6 (0) 0.6 (0) 0.6 (0) 0.6 (0) 0.6 (0) 0.6 (0) 0.6 NaH2 PO4 2H2 O (x4 ) Tween-80 (x5 ) Lactic acid production gram per 10 g fba Experimental values (1) 0.55 (1) 0.55 (1) 0.55 (1) 0.55 (1) 0.55 (1) 0.55 (1) 0.55 (1) 0.55 (1) 1.45 (1) 1.45 (1) 1.45 (1) 1.45 (1) 1.45 (1) 1.45 (1) 1.45 (1) 1.45 (0) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (0) 0.10 (2) 1.90 (0) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (0) 1.00 (1) 0.25 (1) 0.15 (1) 0.15 (2) 0.25 (1) 0.15 (2) 0.25 (2) 0.25 (1) 0.15 (1) 0.15 (1) 0.25 (1) 0.25 (1) 0.15 (2) 0.25 (1) 0.15 (1) 0.15 (2) 0.25 (0) 0.20 (0) 0.20 (0) 0.20 (0) 0.20 (0) 0.20 (0) 0.20 (0) 0.20 (0) 0.20 (2) 0.10 (2) 0.30 (0) 0.20 (0) 0.20 (0) 0.20 (0) 0.20 (0) 0.20 (0) 0.20 2.90290 1.90190 2.23210 1.42890 1.78200 1.42230 1.80990 1.47280 1.02540 1.68280 1.55160 2.72520 2.00480 1.71130 1.57110 1.62050 0.88990 1.32000 1.63540 2.45590 1.79710 2.07260 1.54550 1.80880 2.10300 2.58505 1.93460 1.90360 1.80970 2.05280 1.85600 1.91300 Predicted values 2.7805 1.7369 2.0949 1.4821 1.6389 1.4693 1.8849 1.5773 0.8389 1.6865 1.5829 2.7861 2.0301 1.7661 1.6537 1.8933 1.1117 1.1029 1.9131 2.1831 2.0627 1.8119 1.7237 1.6349 2.2656 2.4271 1.9109 1.9109 1.9109 1.9109 1.9109 1.9109

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

(1) 0.875 (1) 1.625 (1) 0.875 (1) 1.625 (1) 0.875 (1) 1.625 (1) 0.875 (1) 1.625 (1) 0.875 (1) 1.625 (1) 0.875 (1) 1.625 (1) 0.875 (1) 1.625 (1) 0.875 (1) 1.625 (2) 0.500 (2) 2.000 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250

(1) 0.875 (1) 0.875 (1) 1.625 (1) 1.625 (1) 0.875 (1) 0.875 (1) 1.625 (1) 1.625 (1) 0.875 (1) 0.875 (1) 1.625 (1) 1.625 (1) 0.875 (1) 0.875 (1) 1.625 (1) 1.625 (0) 1.250 (0) 1.250 (2) 0.500 (2) 2.000 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250 (0) 1.250

Coded values are given in parenthesis. Real values are in percent w/w. a fb, fermented bran.

3. Results and discussion 3.1. Substratemicrobe interaction An amylolytic strain, L. amylophilus GV6 is used for production of l(+) lactic acid in SSF using wheat bran as solid support and substrate having a particle size ranging 1.53 mm. Smaller particle size is generally used as it provides larger surface area, for diffusion of nutrients, both at the surface and in the pores of substrates having the same tortuosity [24] and wheat bran fullled the above requirements in the present study. As the substrate and microbial interactions are unique, chemical composition and physical properties of the substrate should be taken into consideration. Growth characteristics, physiology and production of metabolites by the organism are based on the extracellular enzymes in growth-associated metabolism. Interaction of the organism with wheat bran was observed using SEM. The surface structure of substrate before inoculation was

rough in texture with extensive compact and complex mesh (Fig. 1). After inoculation there was extensive growth of microbial cells seen adsorbed on to the wheat bran particles and dissolution of mesh bres were observed due to extra cellular amylopullulanase production leaving behind relatively smooth substrate particles (Fig. 2). This observation explains the conversion of raw starch to glucose, which in turn is converted to l(+) lactic acid by L. amylophilus GV6. There are certain reports explaining the effect of support for Lactobacillus sps. for production of lactic acid. Inert support of inorganic porous materials was used for lactic acid production by immobilized cells of Lactobacillus rhamnosus [25]. Lactobacillus delbruckii (mutant DP3), Lactobacillus casei (ATCC 11443) and L. amylophilus (NRRL B-4437) produced more lactic acid using glucose as carbon source in a trapped state on solid support (polypropylene chips) than in free form [26]. In the present study inexpensive wheat bran served both as solid support and substrate successfully.

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Fig. 1. Scanning electron microscope (SEM) photograph of unfermented wheat bran with compact starch bres.

3.2. Optimization for lactic acid production Signicantly contributing variables analyzed from previous experiments were used in RSM to check the best operating parameters and decide optimum operating conditions for the fermentation process. The Students t distribution and corresponding values, along with parameter estimate are given in Table 2. The probability (P) values were used as a tool to check the signicance of each of the coefcients. The results show that among the independent variables (x1 ), two variables (x2 ) yeast extract and (x3 ) tri-ammonium citrate have signicant effect. Among the interactions(x1 x4 ) peptone and NaH2 PO4 2H2 O (x1 x5 ) peptone and Tween-80 (x2 x4 ) yeast extract and NaH2 PO4 2H2 O (x2 x5 ) yeast

extract and Tween-80 (x3 x4 ) tri-ammonium citrate and NaH2 PO4 2H2 O are highly signicant (Table 2). From Fig. 3 it is evident that due to dominating interaction effects of peptone and NaH2 PO4 2H2 O higher levels of these nutrients give higher yields of lactic acid. It is also observed that decrease in peptone and increase in Tween-80 concentrations is giving higher yield of lactic acid (Fig. 4). Fig. 5 shows higher yield of lactic acid at both higher and lower levels of yeast extract and NaH2 PO4 2H2 O whereas yield is prominent at higher levels of these variables. Maximum lactic acid production is observed at lower yeast extract concentration with increase in Tween-80 concentration and vice versa due to interaction effect as shown in Fig. 6. At higher concentrations of both tri-ammonium citrate and NaH2 PO4 2H2 O

Fig. 2. Scanning electron microscope photograph of fermented wheat bran with bacterial cells showing the hydrolyzed starch bres.

B.J. Naveena et al. / Process Biochemistry 40 (2005) 681690 Table 2 Coefcients and t-values for l(+) lactic acid production in SSF using central composite rotatable design Variable Constant Peptone Yeast extract (NH4 )3 citrate NaH2 PO4 2H2 O Tween-80 Peptone peptone Yeast extract yeast extract (NH4 )3 citrate (NH4 )3 citrate NaH2 PO4 2H2 O NaH2 PO4 2H2 O Tween-80 Tween-80 Peptone yeast extract Peptone (NH4 )3 citrate Peptone NaH2 PO4 .2H2 O Peptone Tween-80 Yeast extract (NH4 )3 citrate Yeast extract NaH2 PO4 2H2 O Yeast extract Tween-80 (NH4 )3 citrate NaH2 PO4 2H2 O (NH4 )3 citrate Tween-80 NaH2 PO4 2H2 O Tween-80 S, 0.2670; R2 , 6.7%. Designate I x1 x2 x3 x4 x5 2 x1 2 x2 2 x3 2 x4 2 x5 x1 x 2 x1 x3 x 1 x4 x1 x 5 x2 x 3 x 2 x4 x2 x 5 x 3 x4 x 3 x5 x4 x5 Coefcients 1.9109 0.0022 0.0675 0.0627 0.0222 0.0402 0.2009 0.0343 0.0066 0.0579 0.1088 0.0675 0.0605 0.2555 0.2072 0.0544 0.1318 0.1989 0.1188 0.0399 0.0218 Standard error of coefcients 0.10649 0.05450 0.0545 0.05450 0.05450 0.05450 0.04930 0.04930 0.04930 0.04930 0.04930 0.06675 0.06675 0.06675 0.06675 0.06675 0.06675 0.06675 0.06675 0.06675 0.06675 t-value 17.944 0.041 1.238 1.151 0.738 0.788 4.076 0.695 0.133 1.174 2.208 1.011 0.906 3.828 3.104 0.815 1.975 2.980 1.780 0.598 0.326

685

Probability 0.000 0.968 0.241 0.274 0.476 0.476 0.002 0.502 0.897 0.265 0.049 0.334 0.384 0.003 0.010 0.432 0.074 0.013 0.103 0.562 0.750

lactic acid production was observed to be high (Fig. 7). Fig. 8 is showing the effect of Tween-80 and tri-ammonium citrate on lactic acid production. Either lower or higher concentrations of tri-ammonium citrate and Tween-80 is favouring higher yield of lactic acid. Overall it is observed that Tween-80 is inuencing the production of lactic acid to maximum extent. At higher concentrations of Tween-80 the organism might be utilizing certain nutrients present in solid substrate whereas, when concentration of Tween-80 is reduced it is dependent on supplemented nutrients. It is reported that use of Tween-80, a surfactant, increased the pro-

duction of enzymes [27,28] considerably in turn increasing the biomass and this may lead to increase in lactic acid production. NaH2 PO4 2H2 O is also observed to have inuence on lactic acid production as seen in Figs. 3, 5, and 7 where the organism requires inorganic phosphate for higher yields of lactic acid. It is reported that inorganic phosphate has effect on lactic acid production by Lactobacillus helvictus [29]. By applying the multiple regression analysis on experimental data, a second-order polynomial model (1) explains the role of each variable and their second-order interactions

Fig. 3. Response surface showing the effect of peptone and NaH2 PO4 2H2 O concentrations on lactic acid production.

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Fig. 4. Response surface showing the effect of peptone and Tween-80 concentrations on lactic acid production.

Fig. 5. Response surface showing the effect of yeast extract and NaH2 PO4 2H2 O concentrations on lactic acid production.

Fig. 6. Response surface showing the effect of yeast extract and Tween-80 concentrations on lactic acid production.

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Fig. 7. Response surface showing the effect of (NH4 )3 citrate and NaH2 PO4 2H2 O concentrations on lactic acid production.

in producing lactic acid. Y = 1.91090.0022x1 +0.0675x2 0.0627x3 0.0222x4 + 0.0403x5 +0.0675x1 x2 0.0605x1 x3 + 0.2555x1 x4 0.2072x1 x5 0.0544x2 x3 +0.1318x2 x4 0.1989x2 x5 + 0.1188x3 x4 + 0.0399x3 x5
2 2 2 0.0218x4 x5 0.2009x1 + 0.0343x2 + 0.0066x3 2 2 0.0579x4 + 0.1089x5

The model equation was modied to reduced tted model (3).

2 2 Y = 1.9109 0.2009x1 + 0.1088x5 + 0.2555x1 x4

0.2072x1 x5 0.1989x2 x5

(3)

(2)

The quadratic model in Eq. (2) with 20 terms contains 5 linear terms, 5 quadratic terms and 10, two-factor interactions. Out of these, insignicant terms (on the basis of P-values which are more than 0.05) are neglected (Table 2).

This reduced tted model is considerably simpler and ts the data almost as well the model (2) with all terms. Hence it can be used for further exploration and validation. Analysis of variance (ANOVA) was done by MINITAB-13. ANOVA (Table 3) indicates that the effect of interactions of variables and their quadratic effects are highly signicant and the regression between independent variables and out put is also quite signicant. The observed and predicted experimental results are given in Table 1. The quadratic

Fig. 8. Response surface showing the effect of (NH4 )3 citrate and Tween-80 concentrations on lactic acid production.

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Table 3 Analysis of variance for the production of l(+) lactic acid by Lactobacillus amylophilus GV6 in SSF Serial number 1 2 3 4 5 6 7 8 Source Regression Linear Square Interaction Residual error Model Pure error Total DF 20 5 5 10 11 6 5 31 SS 5.1282 0.2547 1.7933 3.0803 0.7842 0.7501 0.0340 5.9123 MS 0.2564 0.0529 0.3586 0.3080 0.0712 0.1250 0.0068 F-value 3.60 0.71 5.03 4.32 18.38 P-value 0.017 0.626 0.012 0.012 0.003

model in Eq. (2) was used to predict the output of lactic acid with planned parameters and compared with observed values. The coefcient of determination (R2 ) for production of lactic acid is 0.8670 (Table 2). This observed variation in production of lactic acid (86.7%) can be explained by the tted model Eq. (2). The multiple regression (R) for the production of lactic acid is 0.9311. This value shows a good agreement between experimental observations and predicted values. It can be seen that while the highest out put was observed at run 1 (2.9029g/10g of bacterial bran) (Table 1). The overlaid contour plot, response surface plot of yield for certain pairs of variables shown at Figs. 6, 8 and 9 indicated that there are regions where the out put could be more than 3.000 g. By running the optimization program with MINITAB-13 within the experimental range investigated, the following optimum values were obtained: peptone, 0.9%; yeast extract, 0.88%; tri-ammonium citrate, 0.379%;

NaH2 PO4 2H2 O, 0.769% and Tween-80, 0.30 ml. The predicted lactic acid production was 32 g/100 g of wheat bran having 54 g of starch. Hence the parameters given by response optimization were used for conrmation of the predicted value of 32 g lactic acid/100 g of wheat bran. The organism produced 36 g of l(+) lactic acid from 54 g of starch present in 100 g of wheat bran. Lactobacillus amylophilus JCM 1125 produced 53.4 g/l of lactic acid using 100 g/l, liqueed starch as reported by Yumoto and Ikeda [10]. Lactobacillusplantarum NCIM 2084 produced 72.9 g/l of lactic acid when provided with 100 g/l of liqueed starch [30]. L. amylophilus (NRRL B4437) produced 29 g/l lactic acid from 45 g/l of corn starch and L. amylovorous was used in conversion of 120 g/l liqueed starch to 92.5 g/l lactic acid in submerged fermentation [31,32]. In the above reports, enzymatic hydrolysis of starch was done prior to Lactobacillus fermentation

Contour Plot of Lactic acid(

2.0 Lower Bound Upper Bound White area: feas ible region Lac tic ac id(
2 4

1.5

YE
1.0 0.5 0.5 1.0 1.5 2.0

Peptone Hold values: Tri-Amm: 0.6 Sod Di H: 1.0 Tween-80: 0.2

Fig. 9. Response surface showing the effect of peptone and yeast extract (YE) concentrations on lactic acid production.

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which is expensive. Wheat straw hemicellulose pretreatment through enzymes/acid released 1112 g/l fermentable sugars and were used for production of lactic acid in co-culture fermentation using Lactobacillus brevis and Lactobacillus pentosus [33]. L. casei subsp. casei CFTRI 2022, L. helviticus CFTRI 2026 and Streptococcus thermophilus CFTRI 2034 were used in SSF with sugarcane pressmud as a substrate containing 14% total sugars on dry weight basis and reported 6.14% lactic acid production [34]. At high starch concentrations, the lactic acid production was low [5,10,31,32]. In the present study L. amylophilus GV6 was found to produce 36 g of lactic acid from a high concentration of raw starch (54 g) present in 100 g of wheat bran. Wheat bran was utilized as both support and substrate by L. amylophilus GV6 in single step conversion of raw starch to l(+) lactic acid. Compared with other reported strains, L. amylophilus GV6 showed higher amylolytic and lactic acid production efciencies which ensured elimination of saccharication steps of raw starch. A detailed study was made to nd out the lactic acid production yield (i.e. gram of lactic acid produced per gram of starch utilized). The organism could produce 90% lactic acid yield, which is comparable with that of submerged fermentation reported earlier for this organism [13]. Expensive pure starch could be replaced with cheaper underutilized abundantly available byproduct like wheat bran. The replacement of pure starch with wheat bran will bring down the cost of substrate 100 times and will make the whole process more economical in terms of substrate costs. When compared to sucrose and hydrolyzed starch, the substrate costs will be still lowered to many folds. Successful application of RSM to optimize the selected ingredients led to two-fold (100%) increase in lactic acid production. L. amylophilus GV6 is found to be the most efcient in production of l(+) lactic acid both in submerged and solid state fermentations at high substrate concentrations as per the available reports. Also there are no reports on use of amylolytic Lactobacillus in SSF. Thus, L. amylophilus GV6 with its 90% lactic acid yield efciency with cheap renewable natural starchy substrates could turn out to be a novel strain to develop a potential technology.

[3] [4] [5] [6] [7] [8] [9]

[10] [11] [12]

[13]

[14]

[15] [16] [17]

[18]

[19]

[20] [21] [22]

Acknowledgements
[23]

BJN thanks the Council of Scientic and Industrial Research (CSIR) Government of India, New Delhi, for providing Senior Research Fellowship (SRF) to carryout this work.

[24] [25]

References
[1] Beninga H. A history of lactic acid making. Boston: Kluwer Academic Publishers; 1990. [2] Vickroy TB. Lactic acid. In: Blanch HW, Drew S, editors. Comprehensive biotechnologythe principles, applications and regulation of

[26]

[27]

biotechnology in industry, agriculture and medicine, vol. 3. Toronto: Pergamon Press; 1985. Gross RA, Kalra B. Biodegradable polymers for the environment. Science 2002;2(5582):80307. Malhotra VP, Raina RK, Sanjay R. Application of polymers in innovative medical devices. Techno-Econ News Mag 2000:3. Litcheld JH. Microbial production of lactic acid. Adv Appl Microbiol 1996;42:4895. Greg, Bohlmann. and Yuka, Yoshida. Biodegradable polymers. Chemical economic handbook (CEH) report, 2000. Lisa J. Lactic acid outlook up as poly lactide nears market. Chem Market Rep 2001;26:14. Felzia M. Lactic acid prices falter as competition toughens. Chem Market Rep 1999. Datta RS, Sai PT, Patric B, Moon SH, Frank JR. Technological and economic potential of polylactic acid and lactic acid derivatives. In: Proceedings of the International Congress on Chemicals from Biotechnology, Hannover, Germany, 1993. p. 1-18. Yumoto J, Ikeda K. Direct fermentation of starch to l(+) lactic acid using Lactobacillus amylophilus. Biotechnol Lett 1995;17:5436. Vishnu C, Sudha Rani K, Gopal Reddy , Seenayya G. Amylolytic bacteria producing lactic acid. J Sci Ind Res 1998;57:6003. Vishnu C, Seenayya G, Gopal R. Direct fermentation of starch to l(+) lactic acid by amylase producing Lactobacillus amylophilus GV6. Bioprocess Eng 2000;23:1558. Vishnu C, Seenayya G, Gopal R. Direct fermentation of various pure and crude starchy substrates to l(+) lactic acid using Lactobacillus amylophilus GV6. World J Microbiol Biotechnol 2000;18:42933. Pandey A, Carlos RS, Jose A, Rodriguez L, Nigam P. General considerations about solid state fermentation processes. In. Solid state fermentation in biotechnology: fundamentals and Applications. New Delhi: Asiatech publishers; 2001. p. 118. Carrizales V, Jaffe W. Solid state fermentation an appropriate biotechnology for developing countries. Interscience 1986;11:915. Zhang DX, Cheryan M. Starch to lactic acid in a continuous membrane bioreactor. Process Biochem. 29:14550. Teleyadi S, Cheryan M. Lactic acid from cheese whey permeate. Production and economics of continuous membrane bioreactor. Appl Microbiol Biotechnol 1995;43:2428. Melzoch K, Votruba J, Habora V, Rychtera M. Lactic acid production in a cell retention continuous culture using lignocellulosic hydrolysate as a substrate. J Biotechnol 1997;56:25031. Naveena BJ, Vishnu C, Altaf Md, Gopal R. Wheat bran an inexpensive substrate for production of lactic acid in solid state fermentation by Lactobacillus amylophilus GV6-Optimization of fermentation conditions. J Sci Ind Res 2003;62:4536. Kimberley AC, Taylor C. A simple calorimetric assay for muramic acid and lactic acid. Appl Biochem. Biotechnol 1996;56:4958. Methods of test for edible straches and strach products. Indian Stand. 1978;4706(2):106. Deming SN, Morgan SL. Experimental design: a chemomatric approach. Oxford: Elsevier; 1987. Neter J, Kutner MH, Nachtsheim CJ, Wasserman W. Applied linear statistical models. Chicago: McGraw-Hill; 1996. p. 1280310. ISBN 0256117365. Hesseltine CW. Solid state fermentation. Biotechnol Bioeng 1972;14:51732. Goncalves LMD, Barreto MTO, Xavier AMBR, Carrondo MJT, Klein J. Innert support for lactic acid productiona technology assessment. Appl Microbiol Biotechnol 1992;38:30611. Demirei A, Anthony LP III, Kenneth EJ. Evaluation of biolm reactor solid support for mixed culture lactic acid production. Appl Microbiol Biotechnol 1993;38:72833. Oten-Gyang K, Moulin G, Galzi P. Effect of medium composition on biosynthesis and excretion of the amylase of Schwamiomyces castellii. Eur J Appl Biotechnol 1980;9:12932.

690

B.J. Naveena et al. / Process Biochemistry 40 (2005) 681690 [31] Mercier P, Yerushalami L, Rouleau D, Dochania D. Kinetics of lactic acid fermentations on glucose and corn by Lactobacillus amylophilus. J Chem Technol Biotechnol 1992;55:11121. [32] Zhang DX, Cheryan M. Direct fermentation of starch to lactic acid by Lactobacillus amylovorus. Biotechnol Lett 1991;10:7338. [33] Grade A, Jonsson G, Schmidt AS, Ahring BK. Lactic acid production from wheat straw hemicellulose hydrolysate by Lactobacillus pentosus and Lactobacillus brevis. Biores Technol 2002;81(3):21723. [34] Xavier S, Lonsane BK. Sugarcane pressmud as a novel and inexpensive substrate for production of lactic acid in a solid state fermentation system. Appl Microbiol Biotechnol 1994;41:2915.

[28] Conto SR, Isabel R, Sanroman A. Design of different bioreactor congurations: application to ligninolytic enzyme production in semi-solid state cultivation. J Chem Technol Biotechnol 2001;76:78 82. [29] Abdeltif A. Effect of inorganic phosphate on lactate production by Lactobacillus helviticus grown on supplemented whey permeate. J Chem Technol Biotechnol 2000;75:2238. [30] Krishnan S, Bhattacharya S, Karanth NG. Media optimization for production of lactic acid by Lactobacillus plantarum NCIM 2084 using response surface methodology. Food Biotechnol 1998;12(1/2): 10521.