A shaking bioreactor equipped with twin ceramic membranes for acetic acid production using Acetobacter pasteurianus

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1 Biotechnology Letters 24: , 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.Narumi 1,K.Tada 1, M. Kobayashi 1, T. Kanno 1 & T. Suzuki 2 1 Department of Chemical System Engineering, Kitami Institute of Technology, 165 Koen-cho, Kitami, Hokkaido , Japan 2 Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba , Japan Author for correspondence (Fax: ; 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% O 2. Introduction Shake-flask culture is widely employed as the basic technique for obtaining cell mass or the product of microorganisms in the fermentation and biotechnology fields (Büchs 2001). In order to apply the shake-flask culture for the effective production of biological material, we developed a shake-flask internally equipped with a ceramic membrane (SCM flask), in which microbial cells were retained at high concentration (Suzuki et al. 1997). This had several advantages including rapid start-up, no cell washout and a high productivity and led to the successful application 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 back-washing. Therefore, if sufficient back-washing 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 operate the perfusion culture without interfering with the operation and the availability of the membrane reactor. In this study, we report the development of a shaking bioreactor equipped with a twin ceramic membranes (SBTCM) system and its use in continuous acetic acid production. The system makes it possible to carry out the back-washing of the membrane using the medium without stopping substrate feeding by switching the medium and filtrate flow direction.

2 1988 Table 1. Steady state analysis of acetic acid fermentation by shaking bioreactor system equipped with twin ceramic membranes under various conditions. Dilution Aeration PO 2 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 ) (mlh 1 cm 2 ) Materials and methods Bacterial strain, medium and analytical procedure Acetobacter pasteurianus, which was kindly provided 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, Polypepton 5. Ethanol was aseptically added to the autoclaved medium. A sample from the SBTCM system was centrifuged 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 OD 660 corresponded to about 0.4 g dry cells l 1. The CO 2 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 filters (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 Al 2 O 3 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 cm 2 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 ceramic filters of the SBTCM system. When valve 1 is open and valve 2 is closed, the filter B is used for substrate 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 repeating 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 between 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 supplied from the top; the supply rate was modified in proportion to the medium feed rate so as to maintain an aerobic condition. O 2 -enriched air with a 40% O 2 content was also used. The culture ph was not controlled. The volumetric O 2 transfer rate (OTR, mg O 2 l 1 h 1 ) was stoichiometrically calculated (1 mol O 2 required for 1 mol acetic acid production by Ace-

3 1989 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, noco 2 production, trace by-products, and low biomass production) based on the acetic acid productivity (P). OTR = P (32/60) The overall O 2 transfer coefficient, k α L (h 1 ), was estimated based on the following equation, assuming a steady state: kl α = O 2 consumed/do gradient = OTR/(DO DO r ). DO and DO r denote the saturated dissolved O 2 concentration at 30 CandtheO 2 concentration in the culture broth, respectively. 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 h 1 with a reciprocation rate of 130 rpm. The interval for switching the flow direction was 12 h in this run based on repeated trials so as to maintain the membrane filtration ability. After inoculation, the acetic acid concentration linearly increased and no cells were observed in the product after filtration. After confirming the establishment of a steady state, the dilution rate was stepwise increased as shown in Figure 3. The aeration rate was increased along with the increase in the dilution rate.

4 1990 Fig. 3. Performance and long-term stability of a shaking bioreactor system equipped with twin ceramic membranes for acetic acid fermentation. (a) Aeration rate (vvm), (b) dilution rate (h 1 ), (c) cell conc. (mg l 1 ), (d) productivity (g l 1 h 1 ), (e) ethanol ( ) and acetic acid ( ) concentrations in filtrate. Operation was stable and no process malfunction occurred. The filtration rate was successfully maintained and no clogging of the ceramic membrane occurred during the operation. At around 500 h, since it was considered that the productivity was limited by O 2 transfer, the shaking rate was increased from 130 rpm to 230 rpm to enhance the O 2 supply, leading to a significant increase in the productivity. The maximum productivity finally reached about 10.7 g l 1 h 1 with a dilution rate of h 1 with 30% O 2 enriched air. The operation of the reactor continued for about 800 h. Cell concentration in the culture broth finally reached 5.2 g l 1. The total filtration volume during 800 h operation was approximately ml, which was 170-fold of the initial volume (200 ml). The acetic acid yield over the theoretical acetic acid concentration stoichiometorically calculated from the amount of ethanol consumed was about 0.85 (w/w). Product of the SBTCM system is cell free and no further treat- Fig. 4. Operating results of a shaking bioreactor system equipped with twin ceramic membranes under high dilution rate conditions. (a) Aeration rate (vvm), (b) dilution rate (h 1 ), (c) cell 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 of soluble products. Then, the process performance under high dilution rate conditions of the SBTCM system was investigated. Figure 4 shows the results. The shaking speed was 230 rpm and the interval for switching the flow direction was 12 h in this run. After seeding, as the dilution rate was raised, the residual ethanol concentration increased. The dilution rate was increased to h 1 at 87 h with the supply of O 2 -enriched air. Productivity was drastically increased to 12.8 g l 1 h 1 at a dilution rate of h 1 and 13.4 g l 1 h 1 at a dilution rate of h 1, respectively. An increase in the PO 2 clearly increased the productivity, which suggested that O 2 transfer is the rate-limiting step of acetic acid fermentation. Operation was also stable and cell concentration during cultivation was around 3.2 g l 1. These results indicate that the medium feeding flow through the membrane successfully wash the sur-

5 1991 face of the membrane and was effective to maintain the filtration ability of the membrane. About 2 h backwashing 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 summarized in Table 1. The overall O 2 transfer rate, k α L was estimated to be around 700 h 1 (PO 2 :0.21, shaking rate: 230 rpm, 1.75 vvm). The filtration fluxes, which are the same as the back-washing fluxes of the membranes, 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 permeation 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 experimental 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% O 2. 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, productivity 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 O 2 -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 periods of the system because they were terminated due to the experimental limitation by manual operation. Therefore, the operation period could be prolonged 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 application 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. References Büchs J (2001) Introduction to advantages and problems of shaken cultures. Biochem. Eng. J. 7: Ghommidh C, Navarro JM, Durand G (1982) A study of acetic acid production by immobilized Acetobacter cells: oxygen transfer. Biotechnol. Bioeng. 24: Horiuchi J, Tabata K, Kanno T, Kobayashi M (2000) Continuous acetic acid production by a packed bed bioreactor employing charcoal pellets derived from waste mushroom medium. J. Biosci. Bioeng. 89: Kamoshita Y, Suzuki T, Ohashi R (1997) A dense cell culture system for aerobic microorganisms using a shaken ceramic membrane flask with surface aeration. J. Ferment. Bioeng. 85: Kondo M, Suzuki Y, Kato H (1988) Vinegar production by Acetobacter cells immobilized on ceramic honeycomb monolith. Hakkokogaku 66: Miyazaki S, Otsubo M, Aoki H, Sawaya T (1996) Acetic acid fermentation with quince, asparagus using isolated acetic acid bacteria. Nihon Shokuhin Kagaku Kougaku Kaishi 43: Ohashi R, Mochizuki E, Suzuki T (1998) High-level expression of the methanol-inducible β-galactosidase gene by perfusion culture of recombinatant Pichia pastoris using a shaken ceramic membrane flask. J. Ferment. Bioeng. 86: Sueki M, Kobayashi N, Suzuki A (1991) Continuous acetic acid production by the bioreactor system loading a new ceramic carrier for microbial attachment. Biotechnol. Lett. 13: Suzuki T, Kamoshita Y, Ohashi R (1997) A dense cell culture system for microorganisms using a shake flask incorporating a porous ceramic filter. J. Ferment. Bioeng. 84: