1076 He et al.

/ J Zhejiang Univ SCI 2005 6B(11):1076-1080

Journal of Zhejiang University SCIENCE

ISSN 1009-3095
http://www.zju.edu.cn/jzus
E-mail: [email protected]

Batch and fed-batch production of butyric acid

by Clostridium butyricum ZJUCB

HE Guo-qing (何国庆)†, KONG Qing (孔 青)†‡, CHEN Qi-he (陈启和), RUAN Hui (阮 晖)
(Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China)
†
E-mail: [email protected]; [email protected]
Received July 13, 2005; revision accepted Setp. 10, 2005

Abstract: The production of butyric acid by Clostridium butyricum ZJUCB at various pH values was investigated. In order to
study the effect of pH on cell growth, butyric acid biosynthesis and reducing sugar consumption, different cultivation pH values
ranging from 6.0 to 7.5 were evaluated in 5-L bioreactor. In controlled pH batch fermentation, the optimum pH for cell growth and
butyric acid production was 6.5 with a cell yield of 3.65 g/L and butyric acid yield of 12.25 g/L. Based on these results, this study
then compared batch and fed-batch fermentation of butyric acid production at pH 6.5. Maximum value (16.74 g/L) of butyric acid
concentration was obtained in fed-batch fermentation compared to 12.25 g/L in batch fermentation. It was concluded that culti-
vation under fed-batch fermentation mode could enhance butyric acid production significantly (P<0.01) by C. butyricum ZJUCB.

Key words: Clostridium butyricum, Batch fermentation, Fed-batch fermentation, pH, Butyric acid production, Acetic acid
doi:10.1631/jzus.2005.B1076 Document code: A CLC number: Q939

INTRODUCTION preferably for butyric acid production because they

have many advantages over other species due to their
Butyric acid has several potential applications simple medium requirement for cell growth and rela-
in industry. Its applications in the foodstuffs and tively high product yield (Michel-Savin et al., 1990a;
beverage industries are widespread. It may be used 1990b). Also, these strains can be readily isolated
as the pure acid in the dairy industry, or in the form from soil, wastewater, animal digestion systems,
of esters as a food additive to increase fruit fragrance. contaminated dairy products, etc. Zigová et al.(1999)
Furthermore, butyric acid could also have some reported that the optimal cultivation conditions for
important physiological functions. For example, butyric acid production by Clostridium butyricum
butyric acid esters are the character-impact flavors in were as follows: temperature of 35~37 °C and at-
tropic fruits and dairy products (Centeno et al., 2002; mosphere of pure CO2 or pure N2 or N2 and CO2 in
Watson et al., 2002). Butyrate is also believed to ratio of 1:9. Utilizable and/or suitable common car-
have therapeutic effect against cancer cells bon sources include glucose, as well as lactose from
(Beyer-Sehlmeyer et al., 2003; Lupton, 2004). whey, sucrose from molasses, starch, potato wastes,
Though butyric acid can be conventionally produced wheat flour, cellulose or dextrose.
by oxidation of butyraldehyde, there has been in- Clostridium butyricum is a typical butyric acid
creasing interest in the bioproduction of butyric acid bacterium found in soil and intestines of healthy
via fermentation (Zigová et al., 1999). Several an- animals and humans (Murray et al., 1984). One strain
aerobic bacteria can produce butyric acid as the of C. butyricum preserved in our lab was screened and
major end product during fermentation of sugars. identified as a probiotic bacterium (Kong et al., 2004).
Among them, Clostridium species have been used For butyric acid fermentation, batch, fed-batch,
continuous and cell-recycle-fermentations are most
‡
Corresponding author frequently used. Results of such experiments offer
 He et al. / J Zhejiang Univ SCI 2005 6B(11):1076-1080 1077

deeper insight into strain physiology and behaviour 0.3~0.4 L per minute. The pH was controlled to dif-
(Zigová and Šturdik, 2000). In order to obtain a ferent required pH values in the range of 6.0~7.5.
higher quantity of butyric acid, there is one important
parameter which should be investigated, the pH. The Fed-batch fermentation in bioreactor
most effective pH is that at which only undissociated Fermentation was initiated as batch fermentation.
acid is present. This value depends on the dissociation The pH followed the optimum pH acquired from
constant of the acid and for butyric acid it is 4.63. On batch fermentation. After 12 h, it was converted to
the other hand, too low a pH is not suitable for acid fed-batch fermentation by adding concentrated glu-
production. With C. acetobutyricum the optimal pH cose feed (glucose concentration=200 g/L) at a con-
for butyrate production (acidogenesis) is 6~6.5 and stant feed rate of 0.025 L/h. Feeding was stopped after
for butanol (solventogenesis) is 4.5~4.8 (Vandák et 32 h and residual glucose in the fermentation broth
al., 1997). was allowed to ferment by batch fermentation (Lang
This study aimed at investigating the effect of et al., 1997; Shoemaker and Wright, 2003).
pH on cell growth and butyric acid formation by
Clostridium butyricum ZJUCB. In order to find the Assays
desired pH cultivation value we first compared dif- The cell growth was measured as the dry cell
ferent pH cultivation conditions for batch fermenta- weight. The culture sample (10 ml) was centrifuged at
tion in a 5-L bioreactor, and then studied the butyric 3000 r/min for 10 min, and the cell pellet was washed
acid production in fed-batch fermentation. twice with distilled water, dried to constant weight at
80 °C, and then weighed (Chen et al., 2004).
The glucose concentration in the medium was
MATERIALS AND METHODS measured using the DNS method (Miller, 1959).
Quantitative analysis of butyric acid and acetic
Microorganism and culture media acid was performed by gas chromatography under the
The bacterial strain (C. butyricum ZJUCB) used following conditions: glass column packed with Po-
in this study was preserved in the Department of Food rapak Q (1.5 m×1.5 mm), N2 as the carrier gas, tem-
Science and Nutrition, Zhejiang University, Hang- peratures of the flame ionization detector (FID) and
zhou, China. The Hungate Anaerobic Culture Tech- injection port set at 260 °C, and column temperature
nique was used to isolate, purify, identify and incu- at 215 °C. The concentration of butyric acid was de-
bate the strain of C. butyricum (Chung and Bryant, termined according to a standard calibration curve.
1997; Min, 1999). The fermentation medium was
composed of 20 g glucose, 3.7 g corn steep flour, 1 g Statistical analysis
(NH4)2SO4, 1.24 g NaHCO3, 0.2 g MnSO4·H2O, 0.2 g Each experiment was performed in triplicate.
MgSO4·7H2O, 0.02 g CaCl2 per liter at pH 7.5. The Data of interest were analyzed with t-test.
medium was autoclaved at 121 °C for 20 min, and
cooled to room temperature prior to use.
RESULTS AND DISCUSSION
Batch fermentation in bioreactor
A 24-h preculture was used to inoculate the cells Batch fermentation
in a 5-L bioreactor (with 4 L working volume) at 5% 1. Butyric acid production at different pH values
level. The monitoring and/or control of various pa- In order to study the influence of pH on C. bu-
rameters, such as temperature and pH, were per- tyricum ZJUCB cell growth and butyric acid produc-
formed in a PID (Proportional-Integral-Derivative) tion, four different pH (pH 6.0, pH 6.5, pH 7.0 and pH
control unit (B. Braun, Diessel, Germany). The bio- 7.5) experiments were performed in the bioreactor.
reactor was operated at 37 °C. Anaerobiosis was en- The time-course of butyric acid batch fermentation at
sured by flushing oxygen-free nitrogen gas through the different pH is shown in Fig.1. With rising oper-
the medium. Oxygen-free nitrogen gas was also ating pH, the maximum biomass concentration
passed through the culture fluid at a rate of about (Fig.1a) and maximum butyric acid (Fig.1b) value
1078 He et al. / J Zhejiang Univ SCI 2005 6B(11):1076-1080

were higher than those at pH=6.0, and the highest decreasing pH. Thus lower operating pH would de-
biomass concentration and butyric acid value oc- crease butyric acid production and prolong the re-
curred at pH=6.5 with cell yield of 3.65 g/L and bu- quired fermentation time as well. In addition, it is not
tyric acid yield of 12.25 g/L. During the first 20 h, easy to control industry-scale fermentation when the
faster cell growth was observed at higher pH values pH is too low or too high. Based on the discussion
(pH 6.5, pH 7.0 and pH 7.5). After 20 h cultivation, above, pH 6.5 was considered to be beneficial for C.
the biomass declined. In general, the butyric acid butyricum cell growth and butyric acid production.
biosynthesis lagged cell growth by about 8 h and then 2. Effect of pH on specific growth rate
increased obviously. Acetic acid biosynthesis in- Fig.2 shows the results of µcell, Pbutyric acid and
creased simultaneously with cell growth (Fig.1c). At specific reducing sugar consumption rate at pH 6.0, pH
the former stage, the butyric acid concentrations at 6.5, pH 7.0 and pH 7.5 in 5-L bioreactor. Specific
different pH values were almost the same, but during growth rate (µcell) at the four pH values showed similar
mid- and later-stage the butyric acid concentrations of tendency before 24 h cultivation, although the highest
cells cultivated at pH 6.5, pH 7.0 and pH 7.5 were µcell occurred at pH 6.0; after 24 h, when µcell decreased
higher than those cultivated at pH 6.0. The consump- to minus and showed obviously different behavior. The
tion of reducing sugar at pH 7.0 was the fastest and curve fluctuated, especially during the later stage of
the lowest at pH 6.0 throughout the process (Fig.1d). experiment. It was thought that the microorganism
High pH was beneficial for cell growth and bu- autolyzed or reused the by-products in the broth and
tyric acid biosynthesis. But sometimes cell autolyza- grew again during the later stage of cultivation.
tion and enzyme inactivity are also accelerated when 3. Effect of pH on the specific butyric acid pro-
pH is too high, so targetted yield is negatively affected. duction rate
However, the operating pH must not be too low, as Fig.2b shows that during the period of 0 h to 24 h,
biochemical reaction rate typically decreases with specific butyric acid formation rate (Pbutyric acid) at pH

pH 6.0
3.5 12
pH 6.5
Biomass concentration (g/L)

pH 7.0
3.0 pH 7.5
10
Butyric acid (g/L)

2.5 8

2.0 6
pH 6.0
pH 6.5
1.5 4 pH 7.0
pH 7.5
1.0 2

0.5 0
0 4 8 12 16 20 24 28 32 36 40 44 48 0 4 8 12 16 20 24 28 32 36 40 44 48
Time (h) Time (h)
(a) (b)
16
5
14
Glucose concentration (g/L)

4 12
Acetic acid (g/L)

pH 6.0
10 pH 6.5
3 pH 7.0
pH 6.0 8 pH 7.5
2 pH 6.5 6
pH 7.0
pH 7.5 4
1
2
0 0
0 4 8 12 16 20 24 28 32 36 40 44 48 0 4 8 12 16 20 24 28 32 36 40 44 48
Time (h) Time (h)
(c) (d)
Fig.1 Time-course of batch fermentation at pH 6.0, pH 6.5, pH 7.0 and pH 7.5 (a) Biomass concentration;
(b) Butyric acid concentration; (c) Acetic acid concentration; (d) Glucose concentration
 He et al. / J Zhejiang Univ SCI 2005 6B(11):1076-1080 1079

6.0 was the fastest because the biomass concentration other metabolites, so butyric acid concentration
was the lowest at pH 6.0. After cultivation for 12 h, (9.717 g/L) was not high. This finding implied that
the maximum value of Pbutyric acid [2.128 g/(h·g bio- the substrate metabolites may have a role in contrib-
mass)] occurred at pH 6.0. uting to butyric acid production.

0.12
Fed-batch fermentation
pH 6.0
0.10 pH 6.5 According to effect of pH on butyric acid pro-
0.08 pH 7.0 duction shown above, we found the optimal cultiva-
pH 7.5
0.06 tion pH for cell growth and butyric acid production is
µcell (1/h)

0.04
0.02
6.5. In order to enhance the production of butyric acid,
0.00 then we adopted the fed-batch fermentation.
-0.02 Comparative profiles of butyric acid fermenta-
-0.04 tion under batch and fed-batch conditions are shown
-0.06
-0.08
in Fig.3. Before 20 h, the biomass of C. butyricum
0 4 8 12 16 20 24 28 32 36 40 44 48 was almost constant, and during the mid- and
Time (h) later-stages, the biomass in the fed-batch fermenta-
(a)
tion did not decrease. While for batch fermentation,
2.0
pH 6.0 after the biomass concentration peak, biomass rapidly
Pbutyric acid (g/(h·g biomass))

pH 6.5
pH 7.0 declined to lower than that of the fed-batch fermen-
pH 7.5
1.5 tation. For fed-batch fermentation, the cell growth
still remained at suitable rate after 20 h. As expected,
1.0
the fed-batch fermentation biomass was higher than
0.5
that of batch fermentation. Maximum biomass (about
3.81 g/L) was achieved in fed-batch fermentation.
0.0 These results indicated that fed-batch fermentation
0 4 8 12 16 20 24 28 32 36 40 44 48
strategy favors cell growth.
Time (h)
(b)
18 18
Specific reducing sugar consumption

Biomass concentration (g/L)

Glucose concentration (g/L)

pH 6.0 16 16
Acids concentration (g/L)

4 pH 6.5
14 14
rate (g/(h·g biomass))

pH 7.0
pH 7.5 12 12
3
10 10
8 8
2
6 6
4 4
1
2 2
0 0
0 0 4 8 12 16 20 24 28 32 36 40 44 48
0 4 8 12 16 20 24 28 32 36 40 44 48
Time (h)
Time (h)
(c) Fig.3 Comparison of batch and fed-batch fermen-
tation at pH 6.5
Fig.2 Batch fermentation at pH 6.0, pH 6.5, pH 7.0 ■ Biomass of batch; □ Biomass of fed-batch; ● Glucose of
and pH 7.5 carried out in 5-L bioreactor (a) µcell, (b) batch; ○ Glucose of fed-batch; ▲ Acetate of batch; ∆ Acetate
Pbutyric acid, (c) specific reducing sugar consumption of fed-batch; Butyrate of batch; Butyrate of fed-batch
rate

4. Effect of pH on specific reducing sugar con- Similar time-course profiles of butyric acid
sumption rate fermentation were observed among the two cultiva-
The consumption rate of reducing sugar at pH tion protocols as shown in Fig.3. The highest value
6.0 was faster than that at other pH values during the (12.25 g/L) was obtained after 32 h cultivation in
period of time=0 to 12 h. At pH 6.0 more substrate batch fermentation, while the maximum value (16.74
was contributed to cell growth and the production of g/L) of butyric acid concentration in fed-batch fer-
1080 He et al. / J Zhejiang Univ SCI 2005 6B(11):1076-1080

mentation was much higher. The differences of 90:1057-1070.

maximum butyric acid concentrations under the two Centeno, J.A., Tomillo, F.J., Fenández-García, E., Gaya, P.,
Nuňez, M., 2002. Effect of wild strains of Lactococcus
cultivation conditions were significant (P<0.01).
lactis on the volatile profile and the sensory characteris-
These results suggested that fed-batch fermentation tics of Ewes’ raw milk cheese. J. Dairy Sci.,
cultivation significantly improved butyric acid pro- 85:3164-3172.
duction as expected. Fed-batch fermentation cultiva- Chen, Q.H., He, G.Q., Schwarz, P., 2004. Studies on cultiva-
tion mode could also significantly improve acetic acid tion kinetics for elastase production by Bacillus sp.
production (P<0.01). EL31410. J. Agric. Food Chem., 52(11):3356-3359.
Chung, K.T., Bryant, M.P., 1997. Robert E. Hungate: Pioneer
During 12 h to 24 h of cultivation, the reducing of anaerobic microbial ecology. Anaerobe, 3:213-217.
sugar was consumed at a high rate under batch fer- Kong, Q., He, G.Q., Chen, Q.H., Chen, F., 2004. Optimization
mentation strategy. While under fed-batch fermenta- of medium composition for cultivating Clostridium bu-
tion strategy, the reducing sugar was consumed at a tyricum with response surface methodology. J. Food Sci.,
high rate during 12 h to 32 h of cultivation (from 12 h 69(7):163-168.
Lang, C., Gollnitz, C., Popovic, M., Stahl, U., 1997. Optimi-
to 32 h, constant addition of glucose at a rate of 5 g/h).
zation of fungal polygalacturonase synthesis by Sac-
charomyces cerevisiae in fed-batch culture. Chem. Eng.
J., 65:219-226.
CONCLUSION Lupton, J.R., 2004. Microbial degradation products influence
colon cancer risk: the butyrate controversy. J. Nutr.,
This study indicated that the influence of pH on 134:479-482.
Michel-Savin, D., Marchal, R., Vandecasteele, J.P., 1990a.
butyric acid fermentation was significant. Maximum
Control of the selectivity of butyric acid production of
butyric acid concentration (12.25 g/L) was achieved Clostridium tyrobutyricum. Appl. Microbiol. Biotechnol.,
when the cells were cultivated at pH 6.5 in batch 32:387-392.
fermentation. Comparison of the batch and fed-batch Michel-Savin, D., Marchal, R., Vandecasteele, J.P., 1990b.
fermentation showed that fed-batch fermentation Butyrate production in continuous culture of Clostridium
mode could significantly enhance butyric acid pro- tyrobutyricum: effect of end-product inhibition. Appl.
Microbiol. Biotechnol., 33:127-131.
duction (P<0.01). Maximum butyric acid concentra-
Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for
tion in fed-batch fermentation could reach 16.74 g/L determination of reducing sugar. Anal. Chem.,
at pH 6.5, representing a 36.65% increase in produc- 31:426-427.
tivity over the batch process. These results indicate Min, H., 1999. Microbial Research Techniques. Science Press,
that a fed-batch process at pH 6.5 yielded higher lev- Beijing.
Murray, R.G.E., Brenner, D.J., Bryant, M.P., Holt, J.G., Krieg,
els of cell growth and butyric acid concentration than
N.R., Moulder, J.W., Pfennig, N., Sneath, P.H.A., Staley,
batch process for butyric acid production from C. J.T., Williams, S.T., 1984. Bergey’s Manual of System-
butycium ZJUCB. Results of this study provide useful atic Bacteriology. Williams & Wilkins, Baltimore,
information to apply and industrial microbiologists, p.1160-1161.
and process engineers for designing scale-up strate- Shoemaker, S.P., Wright, L.L., 2003. Feedstock production,
gies for process optimization. genetic modification, and processing. Appl. Biochem.
Biotechnol., 105:3-4.
Vandák, D., Zigová, J., Šturdik, E., Schlosser, S., 1997.
Evaluation of solvent and pH for extractive fermentation
ACKNOWLEDGEMENT of butyric acid. Process Biochem., 32(3):245-251.
Watson, R., Wright, C.J., McBurney, T., Taylor, A.J., Linforth,
We deeply thank Dr. Y. Xu for her useful R.S.T., 2002. Influence of harvest data and light integral
comments during the preparation of this manuscript. on the development of strawberry flavor compounds. J.
Exp. Bot., 53(377):2121-2129.
Zigová, J., Šturdik, E., 2000. Advances in biotechnological
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