You are on page 1of 5

Biotechnology Letters 24: 1987–1991, 2002.

© 2002 Kluwer Academic Publishers. Printed in the Netherlands.


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:

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

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

of the membrane were possible without affecting the
Shake-flask culture is widely employed as the ba- operation of the culture, it would greatly improve the
sic technique for obtaining cell mass or the product performance of the system. If the culture medium,
of microorganisms in the fermentation and biotech- which is continuously fed to a bioreactor, could be
nology fields (Büchs 2001). In order to apply the used for the back-washing, it would be possible to op-
shake-flask culture for the effective production of bio- erate the perfusion culture without interfering with the
logical material, we developed a shake-flask internally operation and the availability of the membrane reactor.
equipped with a ceramic membrane (SCM flask), in In this study, we report the development of a shak-
which microbial cells were retained at high concen- ing bioreactor equipped with a twin ceramic mem-
tration (Suzuki et al. 1997). This had several advan- branes (SBTCM) system and its use in continuous
tages including rapid start-up, no cell washout and a acetic acid production. The system makes it possible
high productivity and led to the successful applica- to carry out the back-washing of the membrane us-
tion of the system to various fermentation processes ing the medium without stopping substrate feeding by
(Kamoshita et al. 1997, Ohashi et al. 1998). However, switching the medium and filtrate flow direction.
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
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 The effective surface area was 50 cm2 for each filter.
The medium feed line and product line were linked to
Bacterial strain, medium and analytical procedure the flask from pump 1 and pump 2 via valve 1 and
valve 2.
Acetobacter pasteurianus, which was kindly pro- Figure 2 shows the line arrangement of twin ce-
vided by the Hokkaido Industrial Technology Center, ramic filters of the SBTCM system. When valve 1 is
was used for the acetic acid fermentation (Miyazaki open and valve 2 is closed, the filter B is used for sub-
et al. 1996). It was grown on medium with the strate feeding (back-washing) and the filter A works
following composition: (g l−1 demineralized water) for broth filtration. When the medium flows from the
ethanol 31.6, glucose 5, yeast extract 2.5, Polypep- inside to the outside of the ceramic filter, it also works
ton 5. Ethanol was aseptically added to the autoclaved to back-wash the filter surface. When valve 1 is closed
medium. and valve 2 is opened, filter B is used for filtration and
A sample from the SBTCM system was cen- filter A is for medium feed (back-washing). By repeat-
trifuged at 4 ◦ C, 7000 × g, for 6 min. The supernatant ing this operation at certain intervals, simultaneous
was used to determine the glucose, ethanol and acetic substrate feed and filter back-washing is possible. The
acid concentrations by HPLC. The cell concentration filtration flux has to be the same as the back-washing
was determined turbidomedically at 660 nm; one unit flux of membrane because the medium feed rate is the
of OD660 corresponded to about 0.4 g dry cells l−1 . same as the broth withdrawal rate. The valve operation
The CO2 content of the exhaust gas was analyzed was conducted manually.
by a gas chromatograph equipped with a thermal The flask was shaken on a reciprocal shaker be-
conductivity detector. tween 130 and 230 rpm. The SBTCM system was
autoclaved for 30 min at 120 ◦ C and then used for the
Shaking bioreactor with a twin ceramic membranes experiments. The culture temperature was maintained
(SBTCM) system at 30 ◦ C and the complex medium was continuously
fed at various feed rates. Aseptic air was also sup-
Figure 1 is a schematic outline of the experimental plied from the top; the supply rate was modified in
SBTCM system. Two cylindrical alumina ceramic fil- proportion to the medium feed rate so as to main-
ters (type MF-0.2 µm, NGK Co., Ltd, Nagoya, Japan) tain an aerobic condition. O2 -enriched air with a 40%
were fitted in parallel at the shoulder of a 500 ml glass O2 content was also used. The culture pH was not
shaking flask. The working volume was 200 ml. The controlled.
ceramic filter is made of Al2 O3 with a mean pore size The volumetric O2 transfer rate (OTR, mg
of 0.2 µm and 0.2 mm at the outer and inner surfaces, O2 l−1 h−1 ) was stoichiometrically calculated (1 mol
respectively. The inner and outer diameters, and the O2 required for 1 mol acetic acid production by Ace-
length were 8 mm, 11 mm and 150 mm, respectively.

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 Results and discussion

low biomass production) based on the acetic acid
productivity (P). First, the performance and operational stability during
the long-term operation of the SBTCM system were
OTR = P × (32/60) × 103 .
examined. Figure 3 shows the operating results of the
The overall O2 transfer coefficient, kαL (h−1 ), was es- SBTCM system. Fermentation was commenced at a
timated based on the following equation, assuming a dilution rate of 0.093 h−1 with a reciprocation rate
steady state: of 130 rpm. The interval for switching the flow di-
rection was 12 h in this run based on repeated trials
kLα = O2 consumed/DO gradient = OTR/(DO∗ –DOr ).
so as to maintain the membrane filtration ability. Af-
DO∗ and DOr denote the saturated dissolved O2 con- ter inoculation, the acetic acid concentration linearly
centration at 30 ◦ C and the O2 concentration in the increased and no cells were observed in the prod-
culture broth, respectively. 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.

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 periods of the system because they were terminated
the filtration ability of the membrane. About 2 h back- due to the experimental limitation by manual opera-
washing process using pure water was required every tion. Therefore, the operation period could be pro-
24 h to avoid the clogging of the membrane in a longed by operational improvements. However, the
shaking bioreactor with a single ceramic membrane; operation of the shaking bioreactor system is more
this contributes to the improvement of total process complicated compared with that of the packed bed
performance. bioreactor. Automatic control system will be effective
The steady-state analysis for both runs is summa- and essential for longer operation and practical ap-
rized in Table 1. The overall O2 transfer rate, kαL was plication of the system. In particular, DO control for
estimated to be around 700 h−1 (PO2 :0.21, shaking optimal aeration, periodical medium flow change and
rate: 230 rpm, 1.75 vvm). The filtration fluxes, which automatic excessive cell withdrawal for prevention of
are the same as the back-washing fluxes of the mem- membrane clogging will contribute to improving its
branes, were between 0.22 to 1.92 (ml h−1 cm−2 ). process performance. Further investigation is required
These were similar values in previous studies and to clarify these points.
sufficient for successful recovery of membrane perme-
ation ability.
In our previous study (Horiuchi et al. 2000), we References
reported the performance of a packed bed bioreactor
using charcoal pellets for the acetic acid production Büchs J (2001) Introduction to advantages and problems of shaken
cultures. Biochem. Eng. J. 7: 91–98.
using the same bacterial strain under similar experi- Ghommidh C, Navarro JM, Durand G (1982) A study of acetic acid
mental conditions, in which the maximum acetic acid production by immobilized Acetobacter cells: oxygen transfer.
productivity was about 3.9 g l−1 h−1 using normal Biotechnol. Bioeng. 24: 605–617.
aeration and 6.5 g l−1 h−1 using air enriched with 40% Horiuchi J, Tabata K, Kanno T, Kobayashi M (2000) Continuous
acetic acid production by a packed bed bioreactor employ-
O2 . The maximum productivity obtained in this study ing charcoal pellets derived from waste mushroom medium. J.
was almost double compared with the results in our Biosci. Bioeng. 89: 126–130.
previous study. Kamoshita Y, Suzuki T, Ohashi R (1997) A dense cell culture system
Ghommidh et al. (1982) employed a ceramic for aerobic microorganisms using a shaken ceramic membrane
flask with surface aeration. J. Ferment. Bioeng. 85: 218–222.
monolith in a packed bed bioreactor and obtained a Kondo M, Suzuki Y, Kato H (1988) Vinegar production by Ace-
productivity of 10.4 g l−1 h−1 with 20 g acetic acid l−1 tobacter cells immobilized on ceramic honeycomb monolith.
by supplying pure oxygen. Kondo et al. (1988) also Hakkokogaku 66: 393–399.
used a ceramic monolith in a packed bed bioreactor Miyazaki S, Otsubo M, Aoki H, Sawaya T (1996) Acetic acid
fermentation with quince, asparagus using isolated acetic acid
and obtained a productivity of 4.37 g l−1 h−1 with bacteria. Nihon Shokuhin Kagaku Kougaku Kaishi 43: 858–865.
39 g acetic acid l−1 . Sueki et al. (1991) examined Ohashi R, Mochizuki E, Suzuki T (1998) High-level expression of
the use of Aphrocell (a porous ceramics) as a packing the methanol-inducible β-galactosidase gene by perfusion cul-
ture of recombinatant Pichia pastoris using a shaken ceramic
material for acetic acid production. They obtained a membrane flask. J. Ferment. Bioeng. 86: 44–49.
productivity of 6.5 g l−1 h−1 of productivity with 53 g Sueki M, Kobayashi N, Suzuki A (1991) Continuous acetic acid pro-
acetic acid l−1 under 90% oxygen-enriched air supply. duction by the bioreactor system loading a new ceramic carrier
When compared with the results described above, pro- for microbial attachment. Biotechnol. Lett. 13: 185–190.
ductivity of 12.8 g l−1 h−1 with 34.1 g acetic acid l−1 Suzuki T, Kamoshita Y, Ohashi R (1997) A dense cell culture system
for microorganisms using a shake flask incorporating a porous
and 13.4 g l−1 h−1 with 27.9 g acetic acid l−1 under ceramic filter. J. Ferment. Bioeng. 84: 133–137.
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