Biotechnology Letters 24: 1987–1991, 2002.

© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

1987

A shaking bioreactor equipped with twin ceramic membranes for acetic

acid production using Acetobacter pasteurianus

J. Horiuchi,1,∗ , M. Narumi1 , K. Tada1 , M. Kobayashi1, T. Kanno1 & T. Suzuki2

1 Department of Chemical System Engineering, Kitami Institute of Technology, 165 Koen-cho, Kitami, Hokkaido
090-8507, Japan
2 Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba

278-8510, Japan
∗ Author for correspondence (Fax: 81-157-26-9415; E-mail: horiuchi@betta.chem.kitami-it.ac.jp)

Received 22 July 2002; Revisions requested 21 August 2002; Revisions received 30 September 2002; Accepted 1 October 2002

Key words: acetic acid fermentation, Acetobacter pasteurianus ceramic membrane, purfusion culture, shaking
bioreactor

Abstract
A shaking bioreactor system with twin internal ceramic membranes was developed for effective perfusion culture
and applied to the continuous production of acetic acid using Acetobacter pasteurianus. The system makes it
possible to carry out the back-washing of the membrane without stopping the continuous operation because one
membrane can be washed by medium feed flow while another membrane provides filtration of the broth by the
simple switching of the medium and the broth flow direction. The medium flow through the membrane could
successfully wash the surface of the membrane thereby effectively maintaining the filtration ability. By using the
system, continuous operation of more than 800 h was achieved and the maximum acetic acid productivity reached
13.4 g l−1 h−1 using air enriched with 40% O2 .

Introduction back-washing. Therefore, if sufficient back-washing

Shake-flask culture is widely employed as the ba-
sic technique for obtaining cell mass or the product
of microorganisms in the fermentation and biotech-
nology fields (Büchs 2001). In order to apply the
shake-flask culture for the effective production of bio-
logical material, we developed a shake-flask internally
equipped with a ceramic membrane (SCM flask), in
which microbial cells were retained at high concen-
tration (Suzuki et al. 1997). This had several advan-
tages including rapid start-up, no cell washout and a
high productivity and led to the successful applica-
tion of the system to various fermentation processes
(Kamoshita et al. 1997, Ohashi et al. 1998). However,
similar to other perfusion cultures, it requires periodic
back-washing of the ceramic membrane to prevent it
from clogging or decreasing the filtration rate. The
necessity of the back-washing results in a decreased
productivity because the operation is stopped during
of the membrane were possible without affecting the
operation of the culture, it would greatly improve the
performance of the system. If the culture medium,
which is continuously fed to a bioreactor, could be
used for the back-washing, it would be possible to op-
erate the perfusion culture without interfering with the
operation and the availability of the membrane reactor.
In this study, we report the development of a shak-
ing bioreactor equipped with a twin ceramic mem-
branes (SBTCM) system and its use in continuous
acetic acid production. The system makes it possible
to carry out the back-washing of the membrane us-
ing the medium without stopping substrate feeding by
switching the medium and filtrate flow direction.
1988
Table 1. Steady state analysis of acetic acid fermentation by shaking bioreactor system equipped with twin ceramic membranes
under various conditions.

Dilution Aeration PO2 Shaking Outlet (g l−1 ) Productivity Cell conc. Filtration flux
rate (1/h) rate (vvm) (atm) rate (rpm) Ethanol Acetic acid (g l−1 h−1 ) (g l−1 ) (ml h−1 cm−2 )

0.093 0.5 0.21 130 4.53 31 2.88 0.82 0.22

0.2 0.5 0.21 130 18.5 13.3 2.66 0.93 0.8
0.2 1 0.21 130 15.8 15.4 3.08 1.24 0.8
0.2 1.5 0.21 130 14.4 17.2 3.44 1.66 0.8
0.2 1.75 0.21 130 14.4 17 3.40 1.81 0.8
0.2 1.75 0.21 230 1.97 34.1 6.82 2.38 0.8
0.375 1.75 0.21 230 14.5 18.5 6.94 3.22 1.5
0.375 2 0.3 230 7.22 28.5 10.7 4.32 1.5
0.375 2 0.4 230 3.18 34.1 12.8 2.31 1.5
0.481 2 0.4 230 8.01 27.9 13.4 3.33 1.92

Materials and methods
Bacterial strain, medium and analytical procedure
Acetobacter pasteurianus, which was kindly pro-
vided by the Hokkaido Industrial Technology Center,
was used for the acetic acid fermentation (Miyazaki
et al. 1996). It was grown on medium with the
following composition: (g l−1 demineralized water)
ethanol 31.6, glucose 5, yeast extract 2.5, Polypep-
ton 5. Ethanol was aseptically added to the autoclaved
medium.
A sample from the SBTCM system was cen-
trifuged at 4 ◦ C, 7000 × g, for 6 min. The supernatant
was used to determine the glucose, ethanol and acetic
acid concentrations by HPLC. The cell concentration
was determined turbidomedically at 660 nm; one unit
of OD660 corresponded to about 0.4 g dry cells l−1 .
The CO2 content of the exhaust gas was analyzed
by a gas chromatograph equipped with a thermal
conductivity detector.
Shaking bioreactor with a twin ceramic membranes
(SBTCM) system
Figure 1 is a schematic outline of the experimental
SBTCM system. Two cylindrical alumina ceramic fil-
ters (type MF-0.2 µm, NGK Co., Ltd, Nagoya, Japan)
were fitted in parallel at the shoulder of a 500 ml glass
shaking flask. The working volume was 200 ml. The
ceramic filter is made of Al2 O3 with a mean pore size
of 0.2 µm and 0.2 mm at the outer and inner surfaces,
respectively. The inner and outer diameters, and the
length were 8 mm, 11 mm and 150 mm, respectively.
The effective surface area was 50 cm2 for each filter.
The medium feed line and product line were linked to
the flask from pump 1 and pump 2 via valve 1 and
valve 2.
Figure 2 shows the line arrangement of twin ce-
ramic filters of the SBTCM system. When valve 1 is
open and valve 2 is closed, the filter B is used for sub-
strate feeding (back-washing) and the filter A works
for broth filtration. When the medium flows from the
inside to the outside of the ceramic filter, it also works
to back-wash the filter surface. When valve 1 is closed
and valve 2 is opened, filter B is used for filtration and
filter A is for medium feed (back-washing). By repeat-
ing this operation at certain intervals, simultaneous
substrate feed and filter back-washing is possible. The
filtration flux has to be the same as the back-washing
flux of membrane because the medium feed rate is the
same as the broth withdrawal rate. The valve operation
was conducted manually.
The flask was shaken on a reciprocal shaker be-
tween 130 and 230 rpm. The SBTCM system was
autoclaved for 30 min at 120 ◦ C and then used for the
experiments. The culture temperature was maintained
at 30 ◦ C and the complex medium was continuously
fed at various feed rates. Aseptic air was also sup-
plied from the top; the supply rate was modified in
proportion to the medium feed rate so as to main-
tain an aerobic condition. O2 -enriched air with a 40%
O2 content was also used. The culture pH was not
controlled.
The volumetric O2 transfer rate (OTR, mg
O2 l−1 h−1 ) was stoichiometrically calculated (1 mol
O2 required for 1 mol acetic acid production by Ace-

Fig. 1. Schematic diagram of a shaking bioreactor system equipped with twin ceramic membranes.
Fig. 2. Line arrangement of twin ceramic filters in a shaking bioreactor system equipped with twin ceramic membranes.
tobacter, no CO2 production, trace by-products, and
low biomass production) based on the acetic acid
productivity (P).
OTR = P × (32/60) × 103 .
The overall O2 transfer coefficient, kαL (h−1 ), was es-
timated based on the following equation, assuming a
steady state:
kLα = O2 consumed/DO gradient = OTR/(DO∗ –DOr ).
DO∗ and DOr denote the saturated dissolved O2 con-
centration at 30 ◦ C and the O2 concentration in the
culture broth, respectively.
1989
Results and discussion
First, the performance and operational stability during
the long-term operation of the SBTCM system were
examined. Figure 3 shows the operating results of the
SBTCM system. Fermentation was commenced at a
dilution rate of 0.093 h−1 with a reciprocation rate
of 130 rpm. The interval for switching the flow di-
rection was 12 h in this run based on repeated trials
so as to maintain the membrane filtration ability. Af-
ter inoculation, the acetic acid concentration linearly
increased and no cells were observed in the prod-
uct after filtration. After confirming the establishment
of a steady state, the dilution rate was stepwise in-
creased as shown in Figure 3. The aeration rate was
increased along with the increase in the dilution rate.
1990

Fig. 4. Operating results of a shaking bioreactor system equipped

with twin ceramic membranes under high dilution rate conditions.
Fig. 3. Performance and long-term stability of a shaking bioreactor (a) Aeration rate (vvm), (b) dilution rate (h−1 ), (c) cell conc.
system equipped with twin ceramic membranes for acetic acid fer- (mg l−1 ), (d) productivity (g l−1 h−1 ), (e) ethanol (
) and acetic
mentation. (a) Aeration rate (vvm), (b) dilution rate (h−1 ), (c) cell acid (
) concentrations in filtrate.
conc. (mg l−1 ), (d) productivity (g l−1 h−1 ), (e) ethanol (
) and
acetic acid (
) concentrations in filtrate.
ment is required to remove cells of the culture broth.
This will be beneficial for the commercial production
Operation was stable and no process malfunction oc-
of soluble products.
curred. The filtration rate was successfully maintained
Then, the process performance under high dilu-
and no clogging of the ceramic membrane occurred
tion rate conditions of the SBTCM system was in-
during the operation. At around 500 h, since it was
vestigated. Figure 4 shows the results. The shaking
considered that the productivity was limited by O2
speed was 230 rpm and the interval for switching the
transfer, the shaking rate was increased from 130 rpm
flow direction was 12 h in this run. After seeding,
to 230 rpm to enhance the O2 supply, leading to a
as the dilution rate was raised, the residual ethanol
significant increase in the productivity. The maximum
concentration increased. The dilution rate was in-
productivity finally reached about 10.7 g l−1 h−1 with
creased to 0.375 h−1 at 87 h with the supply of
a dilution rate of 0.375 h−1 with 30% O2 enriched
O2 -enriched air. Productivity was drastically increased
air. The operation of the reactor continued for about
to 12.8 g l−1 h−1 at a dilution rate of 0.375 h−1 and
800 h. Cell concentration in the culture broth finally
13.4 g l−1 h−1 at a dilution rate of 0.481 h−1 , re-
reached 5.2 g l−1 . The total filtration volume during
spectively. An increase in the PO2 clearly increased
800 h operation was approximately 33 700 ml, which
the productivity, which suggested that O2 transfer is
was 170-fold of the initial volume (200 ml). The acetic
the rate-limiting step of acetic acid fermentation. Op-
acid yield over the theoretical acetic acid concentra-
eration was also stable and cell concentration during
tion stoichiometorically calculated from the amount of
cultivation was around 3.2 g l−1 .
ethanol consumed was about 0.85 (w/w). Product of
These results indicate that the medium feeding
the SBTCM system is cell free and no further treat-
flow through the membrane successfully wash the sur-

face of the membrane and was effective to maintain
the filtration ability of the membrane. About 2 h back-
washing process using pure water was required every
24 h to avoid the clogging of the membrane in a
shaking bioreactor with a single ceramic membrane;
this contributes to the improvement of total process
performance.
The steady-state analysis for both runs is summa-
rized in Table 1. The overall O2 transfer rate, kαL was
estimated to be around 700 h−1 (PO2 :0.21, shaking
rate: 230 rpm, 1.75 vvm). The filtration fluxes, which
are the same as the back-washing fluxes of the mem-
branes, were between 0.22 to 1.92 (ml h−1 cm−2 ).
These were similar values in previous studies and
sufficient for successful recovery of membrane perme-
ation ability.
In our previous study (Horiuchi et al. 2000), we
reported the performance of a packed bed bioreactor
using charcoal pellets for the acetic acid production
using the same bacterial strain under similar experi-
mental conditions, in which the maximum acetic acid
productivity was about 3.9 g l−1 h−1 using normal
aeration and 6.5 g l−1 h−1 using air enriched with 40%
O2 . The maximum productivity obtained in this study
was almost double compared with the results in our
previous study.
Ghommidh et al. (1982) employed a ceramic
monolith in a packed bed bioreactor and obtained a
productivity of 10.4 g l−1 h−1 with 20 g acetic acid l−1
by supplying pure oxygen. Kondo et al. (1988) also
used a ceramic monolith in a packed bed bioreactor
and obtained a productivity of 4.37 g l−1 h−1 with
39 g acetic acid l−1 . Sueki et al. (1991) examined
the use of Aphrocell (a porous ceramics) as a packing
material for acetic acid production. They obtained a
productivity of 6.5 g l−1 h−1 of productivity with 53 g
acetic acid l−1 under 90% oxygen-enriched air supply.
When compared with the results described above, pro-
ductivity of 12.8 g l−1 h−1 with 34.1 g acetic acid l−1
and 13.4 g l−1 h−1 with 27.9 g acetic acid l−1 under
the supply of O2 -enriched (40%) air in our study are
considered to be quite competitive.
The operation periods used in this study do
not necessarily mean they are maximum operation
1991
periods of the system because they were terminated
due to the experimental limitation by manual opera-
tion. Therefore, the operation period could be pro-
longed by operational improvements. However, the
operation of the shaking bioreactor system is more
complicated compared with that of the packed bed
bioreactor. Automatic control system will be effective
and essential for longer operation and practical ap-
plication of the system. In particular, DO control for
optimal aeration, periodical medium flow change and
automatic excessive cell withdrawal for prevention of
membrane clogging will contribute to improving its
process performance. Further investigation is required
to clarify these points.
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