Lactic acid production by Bacillus

coagulans—Kinetic studies and

optimization of culture medium for
batch and continuous fermentations
T. Payot, Z. Chemaly, and M. Fick

Laboratoire des Sciences du Génie 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 52°C) 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 l21,
respectively, without aeration and 4.6 and 11 g l21 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 l21, productions of
biomass and lactic acid were 3.1 and 55 g l21, respectively. The maximal specific production rate of lactic acid
is high (6.1 6 0.3 g/l z 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 Lactobacillus delbrueckii,7 and Lactobacillus plantarum;8,9

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
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 tempera-
ture10 (55°C). 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. Optimiza-
tion of temperature, pH regulation, nitrogen source concen-
tration, and salt supplementation was performed since these
factors are essential for lactic acid producers.3,8,13–15 First,
continuous processes were performed to define the experi-
mental 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

Enzyme and Microbial Technology 24:191–199, 1999

© 1998 Elsevier Science Inc. All rights reserved. 0141-0229/99/$–see front matter
655 Avenue of the Americas, New York, NY 10010 PII S0141-0229(98)00098-2
Papers
Table 1 Activation medium and culture medium for B. Sugar and organic acids analysis
coagulans
Sugar and lactic concentrations were determined by offline high
Activation medium Culture medium performance liquid chromatography (Waters Associate, Milford,
MA). Sucrose, fructose, d-glucose and l-lactic acid concentrations
were determined enzymatically with a sucrose/d-glucose diagnos-
Sucrose 20 g l21 100 g l21
Tryptone 16 g l21 —
tic kit (Boehringer Mannheim 1113950, Mannheim, Germany);
Yeast extract 10 g l21 2 g l21 d-glucose/d-fructose diagnostic kit (Boehringer Mannheim
NaCl 5 g l21 — 139106); and a l-lactic acid diagnostic kit (Boehringer Mannheim
(NH4)2SO4 — 1.7 g l21 139084). The l-lactic acid was measured by a second enzymatic
(NH4)2HPO4 — 1.0 g l21 method with a glucose-lactate analyzer YSI 2000 (Bioblock,
pH 6.4 6.4 Illkirch, France). HPLC was used as a qualitative screening of
Temperature 50°C 50°C 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 Conductivity measurements
the inconvenience of B. coagulans TB/04 were discussed
and compared with another B. coagulans11 and with Lac- Samples and online measurements were performed with a conduc-
tobacillus rhamnosus studied in our laboratory. tivity probe WTW16,17 (Wissenschaftlich-Technische Werkstätten
GmbH D-8120, Weilheim, Germany) LF 96 with a Tetracon 96
conductivity cell. This technique was used for the online estima-
Materials and methods tion of lactic acid concentration.

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.
Selection of bacterial strain
Four strains characterized as B. coagulans were compared (indus-
trial collection, Brussels Biotech, Brussels, Belgium). The most
productive strain was used for this work and named B. coagulans
TB/04.
Biomass analysis
Cell concentration was estimated by measurement of optical
density at 570 nm correlated to dry cell weights.
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 90°C 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 (100°C and 150 ml h21).
Concentrate was used as bacterial extract.
Fermentation protocol
Sterile culture medium (200 ml) was inoculated with 15 ml
activation medium (cell concentration about 3 g l21) and incubated
for 12 h at 50°C in a 250-ml mini-reactor (Wheaton Instrument,
Millville, NJ). The agitation speed was set up at 200 rpm, and then

Figure 1 Continuous bioreactor

192 Enzyme Microb. Technol., 1999, vol. 24, February 15/March

 Lactic acid production by B. coagulans: T. Payot et al.
(g l21), and lactic acid yield (%, lactate produced/initial sucrose
concentration) were determined.

Fermentation conditions. Lactic acid fermentation was per-

formed at 50 or 52°C. 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 50°C. Medium was
prepared with 100 g l2 sucrose, liquid yeast extract (4 g l2),
and ammonium salts (NH4)2HPO4 (1.0 g l2) and NH4)2SO4
Figure 2 Batch bioreactor (1.7 g l2). This study was performed to estimate maximal
specific growth rate (mmax) at steady state.
The continuous state was started after a batch phase of
100 ml of this culture was aseptically transferred into 900 ml 18 h. Fresh medium was added into the reactor with a
culture medium in a 2-l fermentor (Setric 2M SGI, Toulouse, peristaltic pump at a very low dilution rate (0.03 h21). At
France) with an Ingold pH probe. steady state for each dilution rate, concentrations of bio-
mass, sugars, lactic acid, and other organic acids were
Continuous fermentation determined. The results showed a low consumption of
Medium feeding and the stage probe were activated with a sucrose at all dilution rates (,25%), only a quarter of
peristaltic pump (Ismatec and Maton Lesquin, Santa Clara, CA, sucrose was used by the cells, the global yield did not
TH 50LF52) after a batch phase of 12–14 h. The activation and exceed 20% (Table 3), and the lactate/sucrose yield de-
culture media were defined for B. coagulans BB/ZVHB (Table 1). creased as the dilution rate increased. The highest biomass
Fermentations (1 l) were performed to study the most important was obtained at a 0.15 h21 dilution rate while the maximum
parameters for growth of and the lactic acid production by B. lactate was achieved at 0.03 h21. Fructose was not detected
coagulans (Figure 1). at low dilution rates (below 0.1 h21) but increased with the
At steady state, pulse and shift techniques were used to study
the influence of different types of nitrogen sources. Pulses were
dilution rate. The total concentration of other products
performed with liquid yeast extract (Biokar ref. 112002, Beauvais, (acetate, pyruvate, ethanol, fumarate, formate, and citrate)
France) and powder yeast extract (Biospringer ref. 103022, Mai- did not exceed 2 g l21 at all dilution rates considered
sons-Alfort, France). Decreased sucrose consumption could be due to a high
concentration of sucrose or a deficiency of vitamins, pep-
Batch fermentation and experimental design tides, or salts. Since sucrose is hydrolyzed to glucose and
fructose, the fructose utilization pathway seems to be
The influence of different nutrients, pH, and temperature on the
limited11 (fructose accumulation at high dilution rates). The
batch production (Figure 2) of lactic acid by B. coagulans were
studied (Table 2). A factorial fractional experimental design 29IV–5 homofermentative character of the strain for our conditions
was used and the maximal specific lactic acid production rate (g is confirmed by the amounts of acetate and ethanol pro-
lactic acid g21 cells h21), maximal specific growth rate (h21), duced (, 2 g l21). Finally, the maximal specific growth rate
maximal specific sugar consumption rate (g g21 cells h21), was estimated around 0.3 h21.
maximal concentration of biomass (g l21), end lactic concentration
Influence of pH regulation. The second continuous fer-
mentation was performed at 50°C to test pH influence on
Table 2 Parameters studied using a factorial fractional experi- biomass and lactic acid production. In order to reduce
mental design inhibition pressure, a new continuous culture at high dilu-
tion rate (0.2 h21) was conducted between pH values of
Level Level 6.0 –7.0 with 2 g l21yeast extract and 60 g l21 sucrose
N° Parameter (21) (1)
(molasses) without ammonium salts. The results show that
a a optimal pH for lactic acid production is defined around 6.5
1 Molasses 120 g l21 60 g l21 (Table 4). Glucose and fructose were not detected for pH
2 Powder yeast extract 0 g l21 2 g l21
3 Liquid yeast extract 0 g l21 2 g l21 dw values between 6.2– 6.8, but a very low concentration of
4 Bacterial extract 0 g l21 2 g l21 dw fructose (,2 g l21) was detected for pH values between
5 (NH4)2 SO4 0 g l21 2 g l21 6.0 –7.0 (data not shown). Other by-products such as ace-
6 (NH4)2 HPO4 0 g l21 1 g l21 tate, pyruvate, ethanol, fumarate, formate, and citrate were
7 pH 6.1 6.4
8 Temperature 50°C 52°C detected at very low concentrations (total ,2 g l21 6 0.5).
9 Tween 80 0 ml l21 1 ml l21
Influence of aeration. This parameter was studied under
Nine parameters studied for two levels (21) (1). Dry weight, dw the same continuous culture described for the previous
a
Sucrose equivalent concentration study. For two different pH values, the culture medium was

Enzyme Microb. Technol., 1999, vol. 24, February 15/March 193

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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,
Dilution rate Biomass Lactic acid residual Glucose Fructose Yield By-productsa
(h21) (g l21) (g l21) (g l21) (g l21) (g l21) (%) (g l21)

0.03 0.7 20.1 77 0 0 93 1

0.05 0.9 19.5 78 0 0 92 1.1
0.1 0.9 19 79 0 0 93 0.9
0.125 1.1 18.5 79 0 0.2 90 0.9
0.15 1.1 18 79 0 0.5 89 1
0.175 1.05 17.6 79.5 0 0.6 90 1.2
0.185 1.02 16.4 80.5 0 0.7 88 1.3
0.2 1 14.9 81 0 0.9 87 1.4
0.225 0.95 13.7 83 0 1.7 88 1.3
0.25 0.92 12.5 83 0 2.4 86 1.4
0.275 0.88 12.3 83 0 2.9 84 1.6
0.3b 0.5 10 82 0.9 4.8 83 1.9

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

aerated (0.06 m3 h21). The biomass concentration increased salts, pH 6.5, and a temperature of 50°C. Two pulses of
under aerobic conditions (Table 5) with a decrease in lactic yeast extract (powder and liquid yeast extract, respectively)
acid production. were successively added into the fermentor. A first pulse of
powder yeast extract was added at steady state. After
Temperature effect. This continuous culture was identical waiting for the steady state to be reached again, a second
to that of previous studies (same medium composition and pulse of liquid yeast extract was then added. Figure 3 shows
dilution rate) with a pH regulation at 6.5. The temperature the influence of the two types of yeast extract on growth.
was tested between 50 –58°C. The optimal temperature for The single yeast extract concentration curve corresponds to
growth and lactic acid production was found between both types since they have the same dry weight. Yeast
50 –52°C. B. coagulans grew at 58°C. This shows its extracts considerably increase biomass (two times and three
thermophile character (Table 6). times, respectively, with powdered yeast extract and liquid
yeast extract). Liquid yeast extract is more efficient than the
Influence of yeast extract. A continuous culture was powder at the same dry weight. Supplementation of the
performed with a low concentration of nitrogen source: medium with liquid yeast extract and powdered yeast
sucrose, 60 g l21; yeast extract, 2 g l21; without ammonium extract increased the lactic acid production from about 9 to
19 g l21 and 13.5 g l21, respectively (data not shown).
Table 4 Influence of pH on the growth of and lactic acid Batch fermentation
production by B. coagulans TB/04 in continuous fermentation at
0.2 h21 dilution rate and 50°C Eighteen batch fermentations were performed to study the
influence of nine parameters at two different levels of
pH regulation Biomass Lactic acid Sucrose residual experimental design (Tables 2 and 7). Experiment n°15 was
(3 N ammonia) (g l21) (g l21) (g l21) repeated (15A, B, and C) for statistical evaluation of this
study. These experiments were performed to evaluate the
6.0 3.8 13 41.5 influence of a high concentration of molasses (diluted 5
6.2 3.9 17.5 37.6
6.4 3.9 19.5 35.5 times 5 12°Bricks 5 120 g l21 equivalent sucrose),
6.5 4 22.5 32.3 different nitrogen sources (yeast extract of liquid and
6.6 3.95 18.8 35.7 powdered types and bacterial extract), ammonium salts and
6.8 4 18.5 36.5
7.0 3.8 15.4 40.2
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
Temperature Biomass Lactic acid Sucrose
(°C) (g l21) (g l21) (g l21)
pH O2 Biomass(g l21) Lactic acid(g l21)
50 4.1 22 32.3
6.0 2 3.8 13 52 4.2 21.9 32.2
6.0 1 4.5 9 54 3.6 19 34.6
6.4 2 3.9 19.5 56 3.4 16.3 38.1
6.4 1 4.6 11 58 3 13 41.4

194 Enzyme Microb. Technol., 1999, vol. 24, February 15/March

 Lactic acid production by B. coagulans: T. Payot et al.

Figure 3 Pulses of yeast extract. Effect on growth:

yeast extract theoretical concentration given by Eq. (1)
(■); concentration of biomass after the pulse of powder
yeast extract (E); and concentration of biomass after the
pulse of liquid yeast extract (h) Y 5 Y 0 (2 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 n°15A, biomass (3.1 g l21) and lactic
acid (55 g l21) were obtained without accumulation of
fructose (Figures 4 and 5).
Analysis errors were estimated for lactic acid and su-
crose concentration at 5% (HPLC, enzymatic technique, and
conductivity) and 10% (HPLC), respectively. The total
concentration of the by-products did not exceed 1 g l21 with
a very low accumulation of acetate and ethanol (Figure 6).
These results agree with the results obtained in chemostatic
studies: the same maximum specific growth rate (0.29 h21)
was obtained in both studies (Batch and Chemostat).
(Figure 7).
The mean kinetics of the three optimum experiments
(n°15A, 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 52°C as with other thermophilic

Table 7 Factorial fractional experimental design

Powder Liquid Bacterial

Sucrose Y.E. Y.E. extract (NH4)2SO4 (NH4)2HPO4 pH Temperature Tween 80

1 21 21 21 21 21 21 21 21 1
2 1 21 21 21 1 1 1 21 21
3 21 1 21 21 1 1 21 1 21
4 1 1 21 21 21 21 1 1 1
5 21 21 1 21 1 21 1 1 21
6 1 21 1 21 21 1 21 1 1
7 21 1 1 21 21 1 1 21 1
8 1 1 1 21 1 21 21 21 21
9 21 21 21 1 21 1 1 1 21
10 1 21 21 1 1 21 21 1 1
11 21 1 21 1 1 21 1 21 1
12 1 1 21 1 21 1 21 21 21
13 21 21 1 1 1 1 21 21 1
14 1 21 1 1 21 21 1 21 21
15A 21 1 1 1 21 21 21 1 21
15B 21 1 1 1 21 21 21 1 21
15C 21 1 1 1 21 21 21 1 21
16 1 1 1 1 1 1 1 1 1

Part of planning chosen for experiments, 1–16 experimental design; A–C, replicate experiments

Enzyme Microb. Technol., 1999, vol. 24, February 15/March 195

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Table 8 Comparative table of parameters obtained for each experiment planned

Experiment n° mmax (h21) R -p / X (g/l z h/g/l) R -s / X (g/l z h/g/l) X max (g/l21) P max (g l21) Y p/s (%)

1 0.15 2.5 3.9 1.3 18 109

2 0.16 2.5 3.5 1.4 24 94
3 0.27 3 3.1 1.65 41 99
4 0.25 2.6 6.6 2.35 33 98
5 0.26 2.6 5.8 2.55 32 80
6 0.3 2.3 5.2 1.35 24 80
7 0.26 2.3 4.7 2.75 50 102
8 0.32 2.4 2.9 2.2 31 97
9 0.31 1.9 4.9 1.5 20 87
10 0.19 1.8 3.8 0.8 17 80
11 0.32 2.9 5.9 2.0 25 96
12 0.32 2.6 5.1 1.2 20 91
13 0.29 3.4 5.4 2.8 48 104
14 0.26 3.8 5.2 2.6 38 95
15A 0.28 6.1 9.5 3.1 55 92
15B 0.28 6.3 9.2 3 56 94
15C 0.29 5.8 9.2 3.2 53 91
16 0.29 5.6 7.6 2.5 55 92
Average 0.265 3 5.2 2 33.2 93.5
Repetitive Error % 5 5 5 5–10 5–10 . 10

Sixteen experiments (1–16) and three experiments of replicates (A–C). 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. Contin-
uous fermentation studies were less affected by pH and
temperature variations; thus, handling in industrial pro-
cesses becomes easy.
Ammonium salt addition into culture medium was rec-
ommended 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 Ya-
hata10 in 1996 and was also confirmed by this experiment.
Experimental design indicates that high concentrations
of molasses led to decreased lactic acid production, indicat-
ing 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 experi- Figure 5 The best fermentation obtained during the experi-
mental design near optimal conditions mental design near optimal conditions

196 Enzyme Microb. Technol., 1999, vol. 24, February 15/March

 Lactic acid production by B. coagulans: T. Payot et al.
Figure 6 The best fermentation obtained during the experi-
mental design near optimal conditions
and powdered yeast extract have the same effects on growth
and the lactate production rate. Bacterial extract was pre-
pared 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 maxi-
mum 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 (g) 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 l21) and salt supple-
mentation considerably increases biomass concentration,15
but the specific production rate was fourfold less efficient
than B. coagulans TB/04.
Table 92Summary 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)
Enzyme Microb. Technol., 1999, vol. 24, February 15/March
Figure 7 The best fermentation obtained during the experi-
mental design near optimal conditions
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 micro-
filtration module20,21 (Figure 8). Productivity and perfor-
mances 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 ex-
tract or corn steep liquor. This work allows for the
consideration of B. coagulans for the industrial produc-
tion 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.
R -p / X 6.1 6 0.3 h21
mmax 0.28 6 0.02 h21
R -s / X 9.5 6 0.5 h21
X max 3.1 6 0.2 gl21
P max 55 6 2.5 gl21
Y p/s 92 6 9%
197
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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 Y.E. Y.E. Bacterial

error Sucrose powder liquid extract (NH4)2SO4 (NH4)2HPO4 pH Temperature Tween 80

X max 10% — 11 11 NS NS NS 1 NS NS
P max 10% — 11 11 NS NS NS NS NS NS
mmax 5% NS 1 1 1 NS NS NS NS NS
R p- / X 10% NS 11 11 11 NS NS NS 1 NS
R -s / X 10% NS 1 1 11 — — 1 1 NS
Y p/s 10% NS NS NS NS NS NS NS NS NS

Very positive (11, effect was superior than twice as global error), positive (1, effect superior than global error), negative (2, 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

Bacillus
Lactobacillus Bacillus coagulans coagulans
rhamnosusa Heriban et al.11 TB/04

Growth temperature 38°C 55°C 52°C

Nitrogen source (g l21) Y.E.bb Y.E.15 Y.E.4 1 B.E.d2
Sugar (g l21) Whey permeate 100c Sucrose 80 Molasses 60e
mmax (h21) 0.45 ND 0.28
X max (g/l21) 8 14.8 3
P max (g/l21) 80 65 55
g (g/l z h/g/l cell) 0.286 0.098 0.382

Not determined, ND Mean specific lactic acid production rate, g

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

198 Enzyme Microb. Technol., 1999, vol. 24, February 15/March

 Lactic acid production by B. coagulans: T. Payot et al.
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