bioreactor cu membrane ceramice

8/7/2019 bioreactor cu membrane ceramice

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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 l1) Productivity Cell conc. Filtration flux

rate (1/h) rate (vvm) (atm) rate (rpm) Ethanol Acetic acid (g l1 h1) (g l1) (ml h1 cm2)

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 l1 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 aceticacid concentrations by HPLC. The cell concentration

was determined turbidomedically at 660 nm; one unit

of OD660 corresponded to about 0.4 g dry cells l1.

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 m, 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 Al2O3 with a mean pore size

of 0.2 m 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 tothe 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 l1 h1) was stoichiometrically calculated (1 mol

O2 required for 1 mol acetic acid production by Ace-


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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, 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, kL (h

1), was es-

timated based on the following equation, assuming a

steady state:

k

L=

O2 consumed/DO gradient=

OTR/(DO

DO

r

).DO and DOr denote the saturated dissolved O2 con-

centration at 30 C and the O2 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 0.093 h1 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.


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1990

Fig. 3. Performance and long-term stability of a shaking bioreactor

system equipped with twin ceramic membranes for acetic acid fer-

mentation. (a) Aeration rate (vvm), (b) dilution rate (h1), (c) cell

conc. (mg l1), (d) productivity (g l1 h1), (e) ethanol () and

acetic acid () concentrations in filtrate.

Operation was stable and no process malfunction oc-

curred. 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 O2transfer, the shaking rate was increased from 130 rpm

to 230 rpm to enhance the O2 supply, leading to a

significant increase in the productivity. The maximum

productivity finally reached about 10.7 g l1 h1 with

a dilution rate of 0.375 h1 with 30% O2 enriched

air. The operation of the reactor continued for about

800 h. Cell concentration in the culture broth finally

reached 5.2 g l1. The total filtration volume during800 h operation was approximately 33 700 ml, which

was 170-fold of the initial volume (200 ml). The acetic

acid yield over the theoretical acetic acid concentra-

tion 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 (h1), (c) cell conc.

(mg l1), (d) productivity (g l1 h1), (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 productionof soluble products.

Then, the process performance under high dilu-

tion rate conditions of the SBTCM system was in-

vestigated. 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 in-

creased to 0.375 h1 at 87 h with the supply of

O2-enriched air. Productivity was drastically increased

to 12.8 g l1 h1 at a dilution rate of 0.375 h1 and

13.4 g l

1

h

1

at a dilution rate of 0.481 h

1

, re-spectively. An increase in the PO2 clearly increased

the productivity, which suggested that O2 transfer is

the rate-limiting step of acetic acid fermentation. Op-

eration was also stable and cell concentration during

cultivation was around 3.2 g l1.

These results indicate that the medium feeding

flow through the membrane successfully wash the sur-


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1991

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, kL was

estimated to be around 700 h1 (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 h1 cm2).

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 l1 h1 using normal

aeration and 6.5 g l1 h1 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 l1 h1 with 20 g acetic acid l1

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 l1

h1

with39 g acetic acid l1. 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 l1 h1 of productivity with 53 g

acetic acid l1 under 90% oxygen-enriched air supply.

When compared with the results described above, pro-

ductivity of 12.8 g l1 h1 with 34.1 g acetic acid l1

and 13.4 g l1 h1 with 27.9 g acetic acid l1 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

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 effectiveand 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.

References

Bchs J (2001) Introduction to advantages and problems of shaken

cultures. Biochem. Eng. J. 7: 9198.Ghommidh C, Navarro JM, Durand G (1982) A study of acetic acid

production by immobilized Acetobacter cells: oxygen transfer.

Biotechnol. Bioeng. 24: 605617.

Horiuchi J, Tabata K, Kanno T, Kobayashi M (2000) Continuous

acetic acid production by a packed bed bioreactor employ-

ing charcoal pellets derived from waste mushroom medium. J.

Biosci. Bioeng. 89: 126130.

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: 218222.

Kondo M, Suzuki Y, Kato H (1988) Vinegar production by Ace-

tobacter cells immobilized on ceramic honeycomb monolith.

Hakkokogaku 66: 393399.

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: 858865.Ohashi R, Mochizuki E, Suzuki T (1998) High-level expression of

the methanol-inducible -galactosidase gene by perfusion cul-

ture of recombinatant Pichia pastoris using a shaken ceramic

membrane flask. J. Ferment. Bioeng. 86: 4449.

Sueki M, Kobayashi N, Suzuki A (1991) Continuous acetic acid pro-

duction by the bioreactor system loading a new ceramic carrier

for microbial attachment. Biotechnol. Lett. 13: 185190.

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: 133137.

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