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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.
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
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)
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
Tween 80 (as surfactant to increase transfer of sugar and studies: the same maximum specific growth rate (0.29 h21)
lactic acid through membrane cells). The effect of the was obtained in both studies (Batch and Chemostat).
bacterial extract was tested to evaluate the feasibility of (Figure 7).
substituting yeast extract. High sugar concentration was The mean kinetics of the three optimum experiments
added to demonstrate sucrose inhibition. Further small (n°15A, B, and C) were calculated and summarized in Table
variations in pH and temperature were tested to determine 9. The results obtained with these three experiments show
the process stability with a view to industrial scaleup. that error does not exceed 10% for all the parameters
The overall results obtained under the 15 different considered; moreover, the results of this culture could be
experimental conditions were represented in Table 8. interpreted after global realization of experimental design.
Under condition n°15A, biomass (3.1 g l21) and lactic Errors were determined with statistic studies given by
acid (55 g l21) were obtained without accumulation of experimental design.18 The effect of each parameter tested
fructose (Figures 4 and 5). is summarized in Table 10.
Analysis errors were estimated for lactic acid and su-
crose concentration at 5% (HPLC, enzymatic technique, and
Discussion
conductivity) and 10% (HPLC), respectively. The total
concentration of the by-products did not exceed 1 g l21 with B. coagulans TB/04 is an atypical strain for lactic acid
a very low accumulation of acetate and ethanol (Figure 6). production. The optimum temperature for growth and lactic
These results agree with the results obtained in chemostatic acid production was around 52°C as with other thermophilic
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
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 (%)
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 fermentation with high concentrations of pure sucrose gave
useful in the industrial production of lactic acid because it the same results (data not shown); moreover, under stressed
will reduce the cost by avoiding sterile conditions. Contin- conditions (high pH, high dilution rate, high temperature),
uous fermentation studies were less affected by pH and cells accumulate low amounts of fructose. The fructose
temperature variations; thus, handling in industrial pro- assimilation pathway is probably a limiting stage for B.
cesses becomes easy. coagulans TB/04. This result confirms studies of Heriban et
Ammonium salt addition into culture medium was rec- al.11 which have shown that the fructose hydrolysis pathway
ommended by many authors,3,11 but their effects were not is indicative in lactic bacteria.
proven in our experiments. In the same way, Tween 80 as The most important parameter for lactic acid production
surfactant had no significant effect which was contrary to by B. coagulans TB/04 is the nitrogen source. Yeast extract
the Lactobacillus sp. The reason is probably due to the is essential for a good fermentation performance. It is
different membrane structure of the Bacillus strain.19 assimilated as the nitrogen source and contains vitamins and
Biomass increased under aerobic conditions, but lactic cofactors for growth.15 The maximum biomass, specific
acid concentration dramatically decreased. This distinctive growth rate, final concentration of lactic acid, specific lactic
feature was described for B.sp.SHO-1 by Ohara and Ya- acid production rate, and specific sugar consumption rate
hata10 in 1996 and was also confirmed by this experiment. were increased with yeast extract supplementation. Liquid
Experimental design indicates that high concentrations
of molasses led to decreased lactic acid production, indicat-
ing the inhibitory effect of high sugar concentrations. A
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
Figure 6 The best fermentation obtained during the experi- Figure 7 The best fermentation obtained during the experi-
mental design near optimal conditions mental design near optimal conditions
and powdered yeast extract have the same effects on growth Conclusions
and the lactate production rate. Bacterial extract was pre-
pared from the Bacillus strain. This source is very promising A lactic acid producer was isolated and characterized as B.
as a partial substitute for yeast extract in order to reduce the coagulans TB/04 (industrial classification). The termophilic
costs of medium. The experimental design shows the character is ideal for industrial production of lactic acid. The
potentiality of bacterial extract to increase maximal specific specific lactic acid production rate is high in comparison
growth rate and the lactic acid production rate. The maxi- with other lactic acid bacteria due to limited growth;
mum biomass and product formed did not show significant however, the final concentration of lactic acid is low when
difference. This could be due to a defect in the preparation compared with L. rhamnosus cultures because the high
of the bacterial extract. initial concentration of sugar cannot be used with B.
The results obtained with a combination of the three coagulans TB/04. This limiting state, moreover, could be
types of nitrogen sources (batch 15 A, B, and C) are given considerably reduced using an adapted process: fedbatch or
in Table 11. Even though the sugar concentration used high cell-density reactor coupling fermentation and micro-
under our experimental conditions was lower than that used filtration module20,21 (Figure 8). Productivity and perfor-
for Lactobacillus rhammosus cultures, the mean specific mances could be increased if the biomass of B. coagulans is
lactic acid production rate (g) was higher for B. coagulans increased.
TB/04. This parameter corresponds to the capacity of the Ammonium supplementation is not a key parameter in
cells to produce lactic acid. contrast with the yeast extract. Medium costs could be
All the three lactic bacterial strains compared in Table 11 reduced by substituting yeast extract with bacterial ex-
are homofermentative. Sugar is only for growth and lactate tract or corn steep liquor. This work allows for the
production. Bacillus strains are thermophilic (contrary to consideration of B. coagulans for the industrial produc-
Lactobacillus) and are adapted for industrial production; tion of lactic acid.
moreover, high disparities exist in the same species of B.
coagulans and the fermentation conditions. Heriban et al.11
Acknowledgments
demonstrated the key role of fructophosphokinase and
pyruvate kinase in the glycolic flux of cells. A high We thank the European Union for financial support to the
concentration of yeast extract (15 g l21) and salt supple- AAIR PL94 –2285 project. We also thank all the members
mentation considerably increases biomass concentration,15 involved in this project and especially Brussels Biotech
but the specific production rate was fourfold less efficient S.A, the project coordinator, for supplying the bacterial
than B. coagulans TB/04. strain.
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
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 (%)
Bacillus
Lactobacillus Bacillus coagulans coagulans
rhamnosusa Heriban et al.11 TB/04
Figure 8 Scheme of lactic acid production process using high-cell density reactor