Biotechnology Letters, Vol 19, No 3, March 1997, pp.

237–240

1
By-product formation in
cell-recycled continuous culture
of Lactobacillus casei
Ik-Keun Yoo, Ho Nam Chang*, Eun Gyo Lee, Yong Keun Chang
1 and Seung-Hyeon Moon1
Department of Chemical Engineering and BioProcess Engineering Research Center, KAIST University, Taejon
305Ð701, Korea. 1Department of Environmental Science and Engineering, Kwangju Institute of Science and
Technology, Kwangju 506Ð303, Korea

While the volumetric productivity of lactic acid increased in continuous culture of Lactobacillus casei with cell recycle,
enhanced formation of by-products such as acetate, formate, ethanol, and D-lactate was observed in the cell-recycled
fermenter compared with a simple chemostat at a similar range of dilution rate. The increased formation of by-product
which was signiÞcantly dependent on substrate limitation resulted from a lower dilution rate rather than a high cell
1 concentration in the cell-recycled fermenter.

24 pts min base to base from Key words to line 1 of text

Introduction product quality as well as the productivity is of

Lactic acid is a possible monomeric material for making
biodegradable polylactic acid polymer (Datta et al.,
1995). Although there are numerous reports on lactic
acid fermentation, few groups have considered the
production of lactic acid as a monomer for polymer
1 synthesis. Physical properties of this polymer, such as
crystallinity and degradability, can be adjusted by
manipulating the D/L isomeric ratio of lactic acid
(Lipinsky and Sinclair, 1986). Ohara (1994) noted that
the formation of by-product requiring removal during
purification step should be minimized in the produc-
tion of monomeric lactic acid for polylactic acid
polymer. Continuous culture with cell recycle has been
extensively studied since a high cell density can provide
a much higher volumetric productivity in lactic acid
1 fermentation (Ohleyer et al., 1985; Mehaia and Cheryan,
1987). On the other hand, changes in the balance of
fermentation products were observed during the period
of glucose limitation in continuous cultures with cell
recycle (Borch et al., 1991; Hjorleifsdottir et al., 1991;
Major and Bull, 1989). However, since all of these
studies were performed with a stringent glucose limi-
tation, the effect of cell concentration rather than the
effect of substrate limitation on by-product formation
was not exactly determined in the cell-recycled
1 fermenter. The aim of this study was to investigate
whether the high cell concentration in cell-recycled
culture would affect by-product formation in compar-
ison with a chemostat without cell recycle, because the
increasing interest.
Materials and methods
Microorganism and medium preparation
Lactobacillus casei ssp. rhamnosus (ATCC 10863) was
grown in a medium containing (g/L): glucose, 40 or 80;
yeast extract, 15; sodium-acetate.3H2O, 1; K2HPO4,
0.5; KH2PO4, 0.5; MgSO4.7H2O, 0.2; MnSO4.H2O,
0.03; and FeSO4.7H2O, 0.03. An antifoam (Antifoam
289, Sigma) was added to the medium at 0.1 ml/L.
Fermentation conditions
The cultures were grown in a jar fermenter (2.5 L: Korea
Fermenter Co., Korea) at 42°C. The pH was maintained
at 6.0 by 10 M NaOH and nitrogen gas was sparged
into the fermenter. Constant stirring (200 rpm) was
maintained during the cultivation. The cells were re-
cycled through a hollow fiber ultrafilter (M.W. cut-off
of 100,000, Amicon H1P 100–43). The hollow fiber
filter unit and associated tubings were sterilized by
circulation of a 0.3% formaldehyde and 200 ppm
sodium hypochlorite mixture overnight and rinsed with
20 L sterile water before each fermentation run. The
operating volume of the fermentation system was main-
tained at 900 ml, consisting of about 840 ml in the
reactor and 60 ml in the recycle loop. The culture circu-
lation rate through the filter was 1.5 L/min. A bleed
from the broth was withdrawn directly from the fer-
menter by using a peristaltic pump. A feed pump was

© 1997 Chapman & Hall Biotechnology Letters · Vol 19 · No 3 · 1997 237

 I-K. Yoo et al.

1 used to supply the fresh medium at a rate which was
determined from the rate of total outflow minus the
addition rate of a 10 M NaOH solution.
Analytical methods
Cell concentration was determined optically at 620 nm
and then calibrated into dry cell weight. Glucose
concentration was measured enzymatically by using the
Glucose-E kits (Youngdong Pharm. Co., Korea). Total
1 lactic acid and acetate were measured by HPLC
equipped with an RI detector (Hitachi L-6000, Japan)
using an ion-exchange Aminex HPX-87H column (Bio-
Rad) at 50°C. The flow rate was 0.6 ml/min and the
mobile phase was 0.005 M H2SO4. Acetate, D-lactate,
formate, and ethanol concentrations were determined by
standard enzyme test kits (Boehringer Mannheim,
Germany).
Results and discussion
1 Table 1 shows the steady state concentrations of cells
and lactic acid at different combinations of dilution rate
(the ratio of the total outflow rate to the fermentation
volume) and bleed ratio (the ratio of the cell-containing
flow rate leaving the fermenter to the total outflow rate).
The concentration of glucose used in the feed was
40 g/L. Cell concentrations in cell recycle cultures were
considerably higher than those at similar dilution rates
in the chemostat. Similar observations have been made
in other studies on lactic acid fermentation with cell
recycle. Lactic acid production was also enhanced under
a cell recycle condition at D = 0.23 h–1 compared with
the chemostat at D = 0.21 h–1 due to the complete
utilization of glucose. This implies that a high cell
density is desirable for increasing the productivity of
lactic acid at a higher dilution rate.
The mean concentrations of by-products are shown in
Table 2 for each steady state of cell recycle and chemo-
stat cultures. A significant increase in the concentra-
tions of secondary products such as acetate, formate, and
ethanol was observed in cell recycle cultures in compar-
ison with the chemostat at a similar range of dilution
rate, although some differences in steady state data
(± 5%) were observed at a same dilution rate. A similar
increase, though smaller, was found for the percentage
of D-lactate. Ethanol and acetate were produced in
similar molar quantities, and formate production was
much higher than ethanol or acetate. The specific
productivities of these secondary products decreased
with increasing the cell concentration in cell recycle

Table 1 Comparison of steady state concentrations of cell, total lactic acid, and glucose between chemostat and
cell recycle cultures
1
1 D (hÐ1) and 2B Cell (g/L) Glucose (g/L) Lactic acid (g/L)

Chemostat D = 0.09 5.8 ± 0.3 < 0.05 32.5 ± 0.8

D = 0.21 4.8 ± 0.2 10.5 ± 1.3 25.6 ± 0.7
Cell recycle D = 0.11, B = 0.49 11.1 ± 0.4 < 0.05 31.9 ± 1.0
D = 0.11, B = 0.21 27.9 ± 2.8 < 0.05 30.5 ± 1.8
D = 0.23, B = 0.22 26.7 ± 0.9 < 0.05 31.8 ± 0.7
1
D (dilution rate): the ratio of the total outßow rate to the fermentation volume.
2
B (bleed ratio): the ratio of the cell-containing ßow rate to the total outßow rate.

Table 2 Steady state concentrations and speciÞc productivities of acetate, formate, ethanol, and percentage of
1 D-lactate in total lactic acid
1
D (hÐ1) and 2B % D-lactate Acetate Formate Ethanol
(mmol/L) (mmol/L) (mmol/L)

Chemostat D = 0.09 3.4 7.4 14.4 6.2

*<0.11> <0.22> <0.10>
D = 0.21 2.7 2.8 5.3 2.9
<0.12> <0.23> <0.13>
Cell recycle D = 0.11, B = 0.49 3.9 10.8 23.5 9.8
<0.11> <0.23> <0.10>
D = 0.11, B = 0.21 4.6 15.0 32.6 14.1
1 <0.06> <0.13> <0.06>
D = 0.23, B = 0.22 4.1 8.1 16.2 6.5
<0.07> <0.14> <0.06>

*<> speciÞc productivity, mmol/g cell h.

238 Biotechnology Letters · Vol 19 · No 3 · 1997

 By-product formation in cell-recycled continuos culture of Lactobacillus casei
Figure 1 Cell, glucose, and lactic acid concentrations in a
continuous culture with cell recycle: s, cell; u, glucose; n,
lactic acid (dilution rate, D = 0.23 hÐ1; bleed ratio, B = 0.04).
1 tration reached about 87 g/L after 130 h of culture. The
cultures at the same dilution rate of 0.11 h–1. On the
other hand, the increase of dilution rate in cell recycle
cultures with similar cell concentration (27.9 and 26.7
g/L) did not significantly affected the specific produc-
tivities. This implies that the increased concentrations
of by-product can be obtained at a lower dilution rate.
From this we could speculate that the substrate limita-
tion resulted from a lower dilution rate more signifi-
1 cantly affected the by-product formation rather than the
level of cell concentration in the fermenter. De Vries et
al. (1970) and Thomas et al. (1979) concluded that the
observed changes in yields and product profile during
glucose limitation represented a channelling of pyruvate
away from lactate dehydrogenase towards the ATP-
generating formation of acetate by acetate kinase
involving pyruvate formate-lyase in the initial reaction.
To clarify whether the increased formation of by-product
1 was caused by high levels of cell concentration or not,
a cell recycle culture without substrate limitation was
carried out. Figure 1 shows the time profile of contin-
uous culture operated at D = 0.23 h–1 and B = 0.04.
Figure 2 Time proÞle of by-products formation in a contin-
uous culture with cell recycle: s, acetate; u, % D-lactate;
n, formate; ,, ethanol.
Glucose fed to the fermenter to give 80 g/l was not
completely consumed during the culture. Cell concen-
maximum volumetric productivity of lactic acid was
12.0 g/L?h. Major and Bull (1985) reported a maximum
volumetric productivity for this strain of 8.93 g/L?h in
a continuous culture without cell recycle at a dilution
rate of 0.35–0.4 h–1 when 50 g/l of glucose was used in
the feed. Figure 2 shows the profile of by-product forma-
tion during the continuous culture shown in Figure 1.
While cell concentration increased with time, there was
no remarkable change in by-product concentrations.
Thus, the specific rates of by-product formation was
continually declined at increasing cell concentration.
After 130 h of culture, the specific productivities of
acetate, formate, and ethanol were 0.014, 0.041, and
0.018 mmol/g cell?h, respectively.
The concentrations of these by-products were much
lower than those in the continuous cultures with strin-
gent glucose limitation shown in Table 2, although
there is a risk of insufficient nutrient supply to the cells
at a high concentration. Since low residual sugar concen-
trations are desirable for saving raw material and
product recovery costs, the dilution rate needs to be
optimized in continuous cultures. In conclusion, the
Biotechnology Letters · Vol 19 · No 3 · 1997 239
 I-K. Yoo et al.
1 dilution rate in cell-recycled fermenter must be care-
fully adjusted in addition to the bleed ratio for control-
ling cell concentration to avoid a stringent sugar
limitation, at the same time to keep the glucose concen-
tration as low as possible.
References
Borch, E., Berg, H., and Holst, O. (1991). J. Appl. Bacteriol. 71,
265–269.
Datta, R., Tsai, S.P., Bonsignore, P., Moon, S.-H., and Frank,
1 J.R. (1995). FEMS Microbiol. Rev. 16, 221–231.
De Vries, W., Kapteijn, W.M.C., VanDer Beek, E.G., and
Stouthamer, A.H. (1970). J. Gen. Microbiol. 63, 333–345.
240 Biotechnology Letters · Vol 19 · No 3 · 1997
Hjorleifsdottir, S., Holst, O., and Mattiasson, B. (1991). Bioprocess
Eng. 6, 29–34.
Lipinsky, E.S. and Sinclair, R.G. (1986). Chem. Eng. Progress 82,
26–32.
Major, N.C. and Bull, A.T. (1985). Biotechnol. Lett. 7, 401–405.
Major, N.C. and Bull, A.T. (1989). Biotechnol. Bioeng. 34,
592–599.
Mehaia, M.A. and Cheryan, M. (1987). Process Biochem. 22,
185–188.
Ohara, H. (1994). Bioprocess and Industry 52, 642–644.
Ohleyer, E., Blanch, H.W., and Wilke, C.R. (1985). Appl.
Biochem. Biotechnol. 11, 317–332.
Thomas, T.D., Ellwood, D.C., and Longyear, V.M.C. (1979). J.
Bacteriol. 138, 109–117.
Received as Revised 28 January 1997