Lactic acid production by Bacillus coagulansKinetic studies and optimization of culture medium for batch and continuous fermentations

T. Payot, Z. Chemaly, and M. Fick

Laboratoire des Sciences du Ge nie Chimique, CNRS, E.N.S.A.I.A. Vandoeuvre cedex, France
Bacillus coagulans is an atypical strain for lactic acid production; the thermophile character of this strain (growth at 52C) proves that it is particularly adapted for industrial production of lactate without sterile conditions. In the first step, continuous culture was performed to define the experimental domain of aeration and nitrogen supplementation. The aerobic condition showed a positive influence on growth and a negative effect on lactate production. At steady state for pH 6.4, the concentrations of biomass and lactic acid were 3.9 and 19.5 g l1, respectively, without aeration and 4.6 and 11 g l1 with aeration. The nitrogen source is essential for the fermentation process. Pulses of different types of yeast extract (liquid and powder) were added into the fermentor at steady state. After pulses, biomass concentration increased two and three times with powdered yeast extract and liquid yeast extract, respectively. Liquid yeast extract was more efficient for growth than powdered yeast extract probably due to degradation of vitamins in a spray dryer. Secondly, a factorial fractional experimental design was performed to optimize batch fermentation. Temperature and pH control, the initial concentration of sugar, and the nitrogen source were optimized. For the initial sucrose concentration of 60 g l1, productions of biomass and lactic acid were 3.1 and 55 g l1, respectively. The maximal specific production rate of lactic acid is high (6.1 0.3 g/l h/g/l cell) in comparison with mesophilic lactic acid bacteria. 1998 Elsevier Science Inc.
Keywords: Lactic acid; Bacillus coagulans; continuous fermentation; kinetic studies

Introduction
Lactic acid is one of the most widely used organic acids in the food industry and is a very common substrate for chemical synthesis.1 Recently, there has been an increased interest in lactate because it can be used as raw material for the production of biodegradable polymers with applications in medical (high resorption thread), pharmaceutical (low diffusion drug), and food industries (packaging).2 Now, production of lactic acid is achieved with a fermentation process using whey permeate3,4 or molasses5,6 as carbon source. Various mesophilic lactic bacterial strains4 are used to produce lactic acid such as Lactobacillus helveticus,4

Address reprint requests to Dr. M. Fick, CRNS E.N.S.A.I.A., Lab des Sciences du Genie Chimique, BP 172, 2 avenue la Foret de Haye, 54505 Vandoeuvre cedex, France. Received 1 May 1997; revised 29 June 1998; accepted 16 July 1998

Lactobacillus delbrueckii,7 and Lactobacillus plantarum;8,9 however, these mesophilic strains are not adapted for industrial production of lactate because high contamination risks are subversive. Thermophilic strains are ideal for this production because sterile conditions are not necessary anymore. For this reason, a number of studies used Bacillus coagulans as a lactic acid producer10 12 at high temperature10 (55C). Sucrose and yeast extract were supplemented with sulfate, phosphate, magnesium, manganese, and iron salts.11 In this paper, an atypical strain of B. coagulans was studied in batch and continuous mode to prove the real potentiality and the performance of this bacteria. Optimization of temperature, pH regulation, nitrogen source concentration, and salt supplementation was performed since these factors are essential for lactic acid producers.3,8,1315 First, continuous processes were performed to define the experimental domain conditions such as influence of aeration, nitrogen, and dilution rate. After that, an experimental design was developed to find the influence of nutrients such

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Table 1 Activation medium and culture medium for B. coagulans Activation medium Sucrose Tryptone Yeast extract NaCl (NH4)2SO4 (NH4)2HPO4 pH Temperature 20 g l1 16 g l1 10 g l1 5 g l1 6.4 50C Culture medium 100 g l1 2 g l1 1.7 g l1 1.0 g l1 6.4 50C

Sugar and organic acids analysis

Sugar and lactic concentrations were determined by offline high performance liquid chromatography (Waters Associate, Milford, MA). Sucrose, fructose, d-glucose and l-lactic acid concentrations were determined enzymatically with a sucrose/d-glucose diagnostic kit (Boehringer Mannheim 1113950, Mannheim, Germany); d-glucose/d-fructose diagnostic kit (Boehringer Mannheim 139106); and a l-lactic acid diagnostic kit (Boehringer Mannheim 139084). The l-lactic acid was measured by a second enzymatic method with a glucose-lactate analyzer YSI 2000 (Bioblock, Illkirch, France). HPLC was used as a qualitative screening of media composition and for estimation of by-products using a UV-RI detector. Enzymatic techniques were used to determine the concentration of metabolites.

as yeast extract, sugar, salts, and to optimize physical parameters (pH and temperature). Finally, advantages and the inconvenience of B. coagulans TB/04 were discussed and compared with another B. coagulans11 and with Lactobacillus rhamnosus studied in our laboratory.

Conductivity measurements
Samples and online measurements were performed with a conductivity probe WTW16,17 (Wissenschaftlich-Technische Werksta tten GmbH D-8120, Weilheim, Germany) LF 96 with a Tetracon 96 conductivity cell. This technique was used for the online estimation of lactic acid concentration.

Materials and methods Strains and medium

B. coagulans TB/04 is a characterized strain isolated by natural selection using continuous culture. In our experimental conditions, this Bacillus was shown to be a homofermentative lactic acid bacteria. The bioconversion capability was always superior to 90%. The bacterial was grown on sucrose.

Preparation of bacterial extract

Fermentation broth was recovered after batch fermentation and centrifugation (4,500 rpm for 20 min). Cells were washed three times with saline water (0.9% NaCl) and disintegrated in a bead-mill (5 min with 0.1 mm glass beads). Cell debris was washed two times with water the homogenate was hydrolyzed with 6 n H2SO4 at 90C for 2 h. The preparation was neutralized with 6 n NH3 and centrifuged at 4,000 rpm for 30 min. The supernatant was concentrated by atomization (100C and 150 ml h1). Concentrate was used as bacterial extract.

Selection of bacterial strain

Four strains characterized as B. coagulans were compared (industrial collection, Brussels Biotech, Brussels, Belgium). The most productive strain was used for this work and named B. coagulans TB/04.

Fermentation protocol
Sterile culture medium (200 ml) was inoculated with 15 ml activation medium (cell concentration about 3 g l1) and incubated for 12 h at 50C in a 250-ml mini-reactor (Wheaton Instrument, Millville, NJ). The agitation speed was set up at 200 rpm, and then

Biomass analysis
Cell concentration was estimated by measurement of optical density at 570 nm correlated to dry cell weights.

Figure 1 Continuous bioreactor

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(g l1), and lactic acid yield (%, lactate produced/initial sucrose concentration) were determined. Fermentation conditions. Lactic acid fermentation was performed at 50 or 52C. The broth was stirred at 300 rpm and the pH was maintained at 6.2 or 6.4 by automatic addition of 2 8 n NH3.

Results Continuous fermentation

Influence of dilution rate. A continuous fermentation was performed to find the influence of dilution rate on growth and lactic acid production at pH 6.4 and 50C. Medium was prepared with 100 g l sucrose, liquid yeast extract (4 g l), and ammonium salts (NH4)2HPO4 (1.0 g l) and NH4)2SO4 (1.7 g l). This study was performed to estimate maximal specific growth rate (max) at steady state. The continuous state was started after a batch phase of 18 h. Fresh medium was added into the reactor with a peristaltic pump at a very low dilution rate (0.03 h1). At steady state for each dilution rate, concentrations of biomass, sugars, lactic acid, and other organic acids were determined. The results showed a low consumption of sucrose at all dilution rates (25%), only a quarter of sucrose was used by the cells, the global yield did not exceed 20% (Table 3), and the lactate/sucrose yield decreased as the dilution rate increased. The highest biomass was obtained at a 0.15 h1 dilution rate while the maximum lactate was achieved at 0.03 h1. Fructose was not detected at low dilution rates (below 0.1 h1) but increased with the dilution rate. The total concentration of other products (acetate, pyruvate, ethanol, fumarate, formate, and citrate) did not exceed 2 g l1 at all dilution rates considered Decreased sucrose consumption could be due to a high concentration of sucrose or a deficiency of vitamins, peptides, or salts. Since sucrose is hydrolyzed to glucose and fructose, the fructose utilization pathway seems to be limited11 (fructose accumulation at high dilution rates). The homofermentative character of the strain for our conditions is confirmed by the amounts of acetate and ethanol produced ( 2 g l1). Finally, the maximal specific growth rate was estimated around 0.3 h1. Influence of pH regulation. The second continuous fermentation was performed at 50C to test pH influence on biomass and lactic acid production. In order to reduce inhibition pressure, a new continuous culture at high dilution rate (0.2 h1) was conducted between pH values of 6.0 7.0 with 2 g l1yeast extract and 60 g l1 sucrose (molasses) without ammonium salts. The results show that optimal pH for lactic acid production is defined around 6.5 (Table 4). Glucose and fructose were not detected for pH values between 6.2 6.8, but a very low concentration of fructose (2 g l1) was detected for pH values between 6.0 7.0 (data not shown). Other by-products such as acetate, pyruvate, ethanol, fumarate, formate, and citrate were detected at very low concentrations (total 2 g l1 0.5). Influence of aeration. This parameter was studied under the same continuous culture described for the previous study. For two different pH values, the culture medium was 193

Figure 2 Batch bioreactor

100 ml of this culture was aseptically transferred into 900 ml culture medium in a 2-l fermentor (Setric 2M SGI, Toulouse, France) with an Ingold pH probe.

Continuous fermentation
Medium feeding and the stage probe were activated with a peristaltic pump (Ismatec and Maton Lesquin, Santa Clara, CA, TH 50LF52) after a batch phase of 1214 h. The activation and culture media were defined for B. coagulans BB/ZVHB (Table 1). Fermentations (1 l) were performed to study the most important parameters for growth of and the lactic acid production by B. coagulans (Figure 1). At steady state, pulse and shift techniques were used to study the influence of different types of nitrogen sources. Pulses were performed with liquid yeast extract (Biokar ref. 112002, Beauvais, France) and powder yeast extract (Biospringer ref. 103022, Maisons-Alfort, France).

Batch fermentation and experimental design

The influence of different nutrients, pH, and temperature on the batch production (Figure 2) of lactic acid by B. coagulans were 5 studied (Table 2). A factorial fractional experimental design 29 IV was used and the maximal specific lactic acid production rate (g lactic acid g1 cells h1), maximal specific growth rate (h1), maximal specific sugar consumption rate (g g1 cells h1), maximal concentration of biomass (g l1), end lactic concentration

Table 2 Parameters studied using a factorial fractional experimental design Level (1) 120 g l1 0 g l1 0 g l1 0 g l1 0 g l1 0 g l1 6.1 50C 0 ml l1
a

N 1 2 3 4 5 6 7 8 9

Parameter Molasses Powder yeast extract Liquid yeast extract Bacterial extract (NH4)2 SO4 (NH4)2 HPO4 pH Temperature Tween 80

Level (1) 60 g l1 2 g l1 2 g l1 dw 2 g l1 dw 2 g l1 1 g l1 6.4 52C 1 ml l1

a

Nine parameters studied for two levels (1) (1). Dry weight, dw a Sucrose equivalent concentration

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Table 3 Influence of dilution rate on the production of biomass, lactic acid, and other by-products produced by B. coagulans and residual sugars left in the fermentor Sucrose, residual (g l1) 77 78 79 79 79 79.5 80.5 81 83 83 83 82

Dilution rate (h1) 0.03 0.05 0.1 0.125 0.15 0.175 0.185 0.2 0.225 0.25 0.275 0.3b
a

Biomass (g l1) 0.7 0.9 0.9 1.1 1.1 1.05 1.02 1 0.95 0.92 0.88 0.5

Lactic acid (g l1) 20.1 19.5 19 18.5 18 17.6 16.4 14.9 13.7 12.5 12.3 10

Glucose (g l1) 0 0 0 0 0 0 0 0 0 0 0 0.9

Fructose (g l1) 0 0 0 0.2 0.5 0.6 0.7 0.9 1.7 2.4 2.9 4.8

Yield (%) 93 92 93 90 89 90 88 87 88 86 84 83

By-productsa (g l1) 1 1.1 0.9 0.9 1 1.2 1.3 1.4 1.3 1.4 1.6 1.9

By-products such as acetate, pyruvate, ethanol, formate, citrate, and fumarate max evaluation (washout dilution rate)

aerated (0.06 m3 h1). The biomass concentration increased under aerobic conditions (Table 5) with a decrease in lactic acid production. Temperature effect. This continuous culture was identical to that of previous studies (same medium composition and dilution rate) with a pH regulation at 6.5. The temperature was tested between 50 58C. The optimal temperature for growth and lactic acid production was found between 50 52C. B. coagulans grew at 58C. This shows its thermophile character (Table 6). Influence of yeast extract. A continuous culture was performed with a low concentration of nitrogen source: sucrose, 60 g l1; yeast extract, 2 g l1; without ammonium
Table 4 Influence of pH on the growth of and lactic acid production by B. coagulans TB/04 in continuous fermentation at 0.2 h1 dilution rate and 50C pH regulation (3 N ammonia) 6.0 6.2 6.4 6.5 6.6 6.8 7.0 Biomass (g l1) 3.8 3.9 3.9 4 3.95 4 3.8 Lactic acid (g l1) 13 17.5 19.5 22.5 18.8 18.5 15.4 Sucrose residual (g l1) 41.5 37.6 35.5 32.3 35.7 36.5 40.2

salts, pH 6.5, and a temperature of 50C. Two pulses of yeast extract (powder and liquid yeast extract, respectively) were successively added into the fermentor. A first pulse of powder yeast extract was added at steady state. After waiting for the steady state to be reached again, a second pulse of liquid yeast extract was then added. Figure 3 shows the influence of the two types of yeast extract on growth. The single yeast extract concentration curve corresponds to both types since they have the same dry weight. Yeast extracts considerably increase biomass (two times and three times, respectively, with powdered yeast extract and liquid yeast extract). Liquid yeast extract is more efficient than the powder at the same dry weight. Supplementation of the medium with liquid yeast extract and powdered yeast extract increased the lactic acid production from about 9 to 19 g l1 and 13.5 g l1, respectively (data not shown).

Batch fermentation
Eighteen batch fermentations were performed to study the influence of nine parameters at two different levels of experimental design (Tables 2 and 7). Experiment n15 was repeated (15A, B, and C) for statistical evaluation of this study. These experiments were performed to evaluate the influence of a high concentration of molasses (diluted 5 times 12Bricks 120 g l1 equivalent sucrose), different nitrogen sources (yeast extract of liquid and powdered types and bacterial extract), ammonium salts and

Table 6 Temperature influence on the growth of and lactic acid production by B. coagulans TB/04 Table 5 Influence of aeration on the growth of and lactic acid produced by B. coagulans TB/04 pH 6.0 6.0 6.4 6.4 O2 Biomass(g l1) 3.8 4.5 3.9 4.6 Lactic acid(g l1) 13 9 19.5 11 Temperature (C) 50 52 54 56 58 Biomass (g l1) 4.1 4.2 3.6 3.4 3 Lactic acid (g l1) 22 21.9 19 16.3 13 Sucrose (g l1) 32.3 32.2 34.6 38.1 41.4

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Figure 3 Pulses of yeast extract. Effect on growth: yeast extract theoretical concentration given by Eq. (1) (s); concentration of biomass after the pulse of powder yeast extract (E); and concentration of biomass after the pulse of liquid yeast extract () Y Y 0 ( tD ) Eq. (1) where Y is the concentration of yeast extract; Y 0 , the initial concentration of yeast extract; t , the time after the pulse, and D the dilution rate

Tween 80 (as surfactant to increase transfer of sugar and lactic acid through membrane cells). The effect of the bacterial extract was tested to evaluate the feasibility of substituting yeast extract. High sugar concentration was added to demonstrate sucrose inhibition. Further small variations in pH and temperature were tested to determine the process stability with a view to industrial scaleup. The overall results obtained under the 15 different experimental conditions were represented in Table 8. Under condition n15A, biomass (3.1 g l1) and lactic acid (55 g l1) were obtained without accumulation of fructose (Figures 4 and 5). Analysis errors were estimated for lactic acid and sucrose concentration at 5% (HPLC, enzymatic technique, and conductivity) and 10% (HPLC), respectively. The total concentration of the by-products did not exceed 1 g l1 with a very low accumulation of acetate and ethanol (Figure 6). These results agree with the results obtained in chemostatic
Table 7 Factorial fractional experimental design Powder Y.E. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Liquid Y.E. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Bacterial extract 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

studies: the same maximum specific growth rate (0.29 h1) was obtained in both studies (Batch and Chemostat). (Figure 7). The mean kinetics of the three optimum experiments (n15A, B, and C) were calculated and summarized in Table 9. The results obtained with these three experiments show that error does not exceed 10% for all the parameters considered; moreover, the results of this culture could be interpreted after global realization of experimental design. Errors were determined with statistic studies given by experimental design.18 The effect of each parameter tested is summarized in Table 10.

Discussion
B. coagulans TB/04 is an atypical strain for lactic acid production. The optimum temperature for growth and lactic acid production was around 52C as with other thermophilic

Sucrose 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15A 15B 15C 16 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

(NH4)2SO4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

(NH4)2HPO4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

pH 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Temperature 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Tween 80 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Part of planning chosen for experiments, 116 experimental design; AC, replicate experiments

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Table 8 Comparative table of parameters obtained for each experiment planned Experiment n 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15A 15B 15C 16 Average Repetitive Error % max (h1) 0.15 0.16 0.27 0.25 0.26 0.3 0.26 0.32 0.31 0.19 0.32 0.32 0.29 0.26 0.28 0.28 0.29 0.29 0.265 5

R p / X (g/l h/g/l)
2.5 2.5 3 2.6 2.6 2.3 2.3 2.4 1.9 1.8 2.9 2.6 3.4 3.8 6.1 6.3 5.8 5.6 3 5

R s / X (g/l h/g/l)
3.9 3.5 3.1 6.6 5.8 5.2 4.7 2.9 4.9 3.8 5.9 5.1 5.4 5.2 9.5 9.2 9.2 7.6 5.2 5

X max (g/l1)
1.3 1.4 1.65 2.35 2.55 1.35 2.75 2.2 1.5 0.8 2.0 1.2 2.8 2.6 3.1 3 3.2 2.5 2 510

P max (g l1)
18 24 41 33 32 24 50 31 20 17 25 20 48 38 55 56 53 55 33.2 510

Y p/s (%)
109 94 99 98 80 80 102 97 87 80 96 91 104 95 92 94 91 92 93.5 10

Sixteen experiments (116) and three experiments of replicates (AC). Evolution of maximal specific growth rate, maximal specific lactic acid production rate, maximal specific sugar consumption rate, maximal biomass and lactic acid concentration and yield

strains.11 The thermophilic nature of the microorganism is useful in the industrial production of lactic acid because it will reduce the cost by avoiding sterile conditions. Continuous fermentation studies were less affected by pH and temperature variations; thus, handling in industrial processes becomes easy. Ammonium salt addition into culture medium was recommended by many authors,3,11 but their effects were not proven in our experiments. In the same way, Tween 80 as surfactant had no significant effect which was contrary to the Lactobacillus sp. The reason is probably due to the different membrane structure of the Bacillus strain.19 Biomass increased under aerobic conditions, but lactic acid concentration dramatically decreased. This distinctive feature was described for B.sp.SHO-1 by Ohara and Yahata10 in 1996 and was also confirmed by this experiment. Experimental design indicates that high concentrations of molasses led to decreased lactic acid production, indicating the inhibitory effect of high sugar concentrations. A

fermentation with high concentrations of pure sucrose gave the same results (data not shown); moreover, under stressed conditions (high pH, high dilution rate, high temperature), cells accumulate low amounts of fructose. The fructose assimilation pathway is probably a limiting stage for B. coagulans TB/04. This result confirms studies of Heriban et al.11 which have shown that the fructose hydrolysis pathway is indicative in lactic bacteria. The most important parameter for lactic acid production by B. coagulans TB/04 is the nitrogen source. Yeast extract is essential for a good fermentation performance. It is assimilated as the nitrogen source and contains vitamins and cofactors for growth.15 The maximum biomass, specific growth rate, final concentration of lactic acid, specific lactic acid production rate, and specific sugar consumption rate were increased with yeast extract supplementation. Liquid

Figure 4 The best fermentation obtained during the experimental design near optimal conditions

Figure 5 The best fermentation obtained during the experimental design near optimal conditions

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Figure 6 The best fermentation obtained during the experimental design near optimal conditions

Figure 7 The best fermentation obtained during the experimental design near optimal conditions

and powdered yeast extract have the same effects on growth and the lactate production rate. Bacterial extract was prepared from the Bacillus strain. This source is very promising as a partial substitute for yeast extract in order to reduce the costs of medium. The experimental design shows the potentiality of bacterial extract to increase maximal specific growth rate and the lactic acid production rate. The maximum biomass and product formed did not show significant difference. This could be due to a defect in the preparation of the bacterial extract. The results obtained with a combination of the three types of nitrogen sources (batch 15 A, B, and C) are given in Table 11. Even though the sugar concentration used under our experimental conditions was lower than that used for Lactobacillus rhammosus cultures, the mean specific lactic acid production rate () was higher for B. coagulans TB/04. This parameter corresponds to the capacity of the cells to produce lactic acid. All the three lactic bacterial strains compared in Table 11 are homofermentative. Sugar is only for growth and lactate production. Bacillus strains are thermophilic (contrary to Lactobacillus) and are adapted for industrial production; moreover, high disparities exist in the same species of B. coagulans and the fermentation conditions. Heriban et al.11 demonstrated the key role of fructophosphokinase and pyruvate kinase in the glycolic flux of cells. A high concentration of yeast extract (15 g l1) and salt supplementation considerably increases biomass concentration,15 but the specific production rate was fourfold less efficient than B. coagulans TB/04.

Conclusions
A lactic acid producer was isolated and characterized as B. coagulans TB/04 (industrial classification). The termophilic character is ideal for industrial production of lactic acid. The specific lactic acid production rate is high in comparison with other lactic acid bacteria due to limited growth; however, the final concentration of lactic acid is low when compared with L. rhamnosus cultures because the high initial concentration of sugar cannot be used with B. coagulans TB/04. This limiting state, moreover, could be considerably reduced using an adapted process: fedbatch or high cell-density reactor coupling fermentation and microfiltration module20,21 (Figure 8). Productivity and performances could be increased if the biomass of B. coagulans is increased. Ammonium supplementation is not a key parameter in contrast with the yeast extract. Medium costs could be reduced by substituting yeast extract with bacterial extract or corn steep liquor. This work allows for the consideration of B. coagulans for the industrial production of lactic acid.

Acknowledgments
We thank the European Union for financial support to the AAIR PL94 2285 project. We also thank all the members involved in this project and especially Brussels Biotech S.A, the project coordinator, for supplying the bacterial strain.

Table 9Summary of performances of lactate production for optimal conditions such as maximal specific lactic acid production rate, maximal specific growth rate, maximal specific sugar consumption rate, concentrations of biomass, and lactic acid and yield Maximal specific production rate of lactic acid Maximal specific growth rate Maximal specific consumption rate of sucrose Maximal concentration of biomass End concentration of lactic acid Yield (lactate produced/sucrose consumed)

R p /X max R s /X X max P max Y p/s

6.1 0.3 h1 0.28 0.02 h1 9.5 0.5 h1 3.1 0.2 gl1 55 2.5 gl1 92 9%

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Table 10 Comparative effect of each parameter on maximal specific growth rate, maximal specific lactic acid production rate, maximal specific sugar consumption rate, maximal biomass, and lactic acid concentration and yield Pure error Y.E. powder NS Y.E. liquid NS Bacterial extract NS NS NS

Sucrose NS NS NS NS

(NH4)2SO4 NS NS NS NS NS

(NH4)2HPO4 NS NS NS NS NS

pH NS NS NS NS

Temperature NS NS NS NS

Tween 80 NS NS NS NS NS NS

X max P max max Rp /X R s /X Y p/s

10% 10% 5% 10% 10% 10%

Very positive (, effect was superior than twice as global error), positive (, effect superior than global error), negative (, effect superior than global error) and not significant (NS: effect inferior or of the order of global error). The pure error (total error) was calculated from analyses error (standard error of mean) for each analysis, repetitive error (standard error of mean), experimental error, and smoothing using statistical tools. The value of pure error was overestimated to be sure that the parameter effect was significant. The acceptation degree is 99.5%. For this plan, analysis standard error is of the order of repetitive standard error (%)

Table 11 Comparison of kinetic parameters between three lactic acid producers

Lactobacillus rhamnosusa
Growth temperature Nitrogen source (g l1) Sugar (g l1) max (h1) X max (g/l1) P max (g/l1) (g/l h/g/l cell) 38C Y.E.bb Whey permeate 100c 0.45 8 80 0.286

Bacillus coagulans Heriban et al.11

55C Y.E.15 Sucrose 80 ND 14.8 65 0.098

Bacillus coagulans TB/04

52C Y.E.4 B.E.d2 Molasses 60e 0.28 3 55 0.382

Not determined, ND Mean specific lactic acid production rate, a Equivalent concentration of lactose b Equivalent concentration of sucrose c Bacterial extract d Yeast extract e Unpublished results

Figure 8 Scheme of lactic acid production process using high-cell density reactor

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