microbial conversion of 4-oxoisophorone by thermomonospora curvata using an air-bubbling hollow...

�9 1988 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0273-2289/88/1902--0209502.40

Microbial Conversion of 4-Oxoisophorone

by Thermomonospora curvata Using an Air-Bubbling Hollow Fiber Reactor

KOJI SODE, 1 MINORU HONDA, 3 YOICH! MIKAMI, 3 TSUYOSH! YANAGIMOTO, 4 AND ISAO KARUBE 1'2'*

1Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, tVleguro-ku, Tokyo, 153; 2Research

Laboratory of Resources Utilization, Tokyo, Institute of Technology, 4259 Nagatsuta-cho,/Vlidori-ku, Yokohama, 227; 3Central Research Institute, Japan Tobacco Inc., 6-2 Umegaoka, Midori-ku, Yokohama, 227; and 4Research Department, Engineering Division, NOK Corp.,

4-3- I Tsujido-shinmachi, Fujisawa, 251, Japan

ABSTRACT

Microbial conversion of 4-oxoisophorone (OIP) by thermophilic bacterium Thermomonospora curvata was attempted in a continuous process.

The correlation between cell growth and microbial conversion was first examined in a batch culture. The results indicated that this microbial conversion was strongly dependent upon cell growth. In a continuous microbial conversion of OIP using a continuous stirred tank reactor, the cell density in the reactor seemed to be the limiting factor in the OIP conversion. Therefore, we developed an air- bubbling hollow fiber reactor to achieve a high density culture. By using this bioreactor, more than 3.3 times higher productivity was achieved. In addition, during the process, only a slight cell contamination to the product was observed. Therefore, this bioreactor is suitable for the continuous microbial conversion, considering further downstream processes and high productivity.

*Author to whom all correspondence and reprint requests should be addressed.

Applied Biochemistry and Biotechnology 209 Vol. 19, 1988

210 Sode et al.

Index Entries: Microbial conversion; thermophilic bacteria; hollow fiber reactor; Thermomonospora curvata; 4-oxoisophorone.

INTRODUCTION

Immobilization of microorganisms is widely accepted as a method having many advantages. Particularly, when keeping a high density of cells in a reactor, even at a high dilution rate to subsequently achieve a high productivity. Some microbial processes are known to be associating with cell growth. In such cases, if we apply the immobilization tech- niques for the processes, cell growing at the outside of the immobilizing gel beads will cause the contamination of the product. In addition, by using entrapment methods for microorganisms immobilization, cells im- mobilized inside of the gel beads will not contribute to the bioprocesses because of the large diffusion barrier.

The application of membrane technology to bioprocesses is increas- ing. The production of high density culture of microorganisms by using membrane reactors, like crossflow filtration systems, has been studying by several researchers (1-4). Utilizing membranes with appropriate molecular cutoff, microorganisms can be maintained in a reactor even at a high dilution rate, and, also, no diffusion barrier of entrapped cell exists.

The authors have been studying the microbial conversion of terpe- noids (5-8). The application of thermophilic bacteria to 6rganic com- pound synthesis has many advantages because of the high temperature employed (9-12). We previously reported on the continuous conversion of 2,6,6,-trimethyl-2-cyclohexene-l,4-dione (4-oxoisophorone, OIP) to ( 6R ) -2, 2, 6, - trimethyl-1,4-cycl ohexane-dione ( (3 R) - dihydro- 4 -oxoisophorone DOIP) by Thermomonospora curvata using several bioreactors (13). Utilizing hollow fiber type reactor, the contamination by cells in the product was reduced, however, the productivity was not satisfactory. This was because of low cell viability.

In this study, we demonstrate a novel bioreactor for microbial conversion using hollow fiber. First, we investigate the correlation of cell growth and microbial conversion of OIP, and also demonstrate a continuous microbial conversion utilizing conventional continuous stirred tank reactor. Then, we show a continuous process using an air-bubbling hollow fiber reactor.

MATERIALS AND METHODS

Chemicals

4-Oxoisophorone (OIP) and (3R)-dihydro-4-oxoisophorone (DOIP) were supplied by Japan Tobacco Inc. All other chemicals were of reagent grade.

Applied Biochemistry and Biotechnology Vol. 19, 1988

Microbial Conversion of 4-Oxoisophorone 211

JVlicroorganisms and Cultivation

Thermomonospora curvata JTS321 provided by Japan Tobacco Inc. was used in this study. The culture conditions are as reported in our previous study (13).

Nlicrobial Conversion of OIP

Batch Culture T. curvata was inoculated in a 500 mL flask containing 100 mL of me-

dium. After cell growth was observed, 300 mg of OIP was added in the medium, and cultivation was continued. Varifying the time of OIP addition, the effect of cell growth on the conversion rate was examined.

Continuous Conversion Using Continuous Stirred Tank Reactor (CSTR) A 1000 mL of CSTR (Marubushi Bioeng. Co. Ltd., Chiyoda-ku,

Tokyo, Japan) with 500 mL of working volume was used in this study. The operational condition is shown in each figure.

Continuous Conversion Using Air-Bubbling Hollow Fiber Reactor

The hollow fiber reactor was supplied by NOK Corp. (Fujisawa-shi, Kanagawa, Japan). The reactor was constructed from polyvinyldifluoride fibers (molecular cut off; 400,000, surface area; 326.7 cm2). T. curvata was inoculated in the extra capillary space (ECS; an internal space between packed hollow fibers and the shell of the reactor, working volume; 9.2 mL). The medium with 3 mg ml- 10IP was continuously fed at ECS, and the product was recovered through the hollow fiber.

Analytical Methods

The analytical methods are as described in our previous study (13).

RESULTS AND DISCUSSION

lVlicrobial Conversion of OIP in a Batch Culture

Figure 1 shows the time course of microbial conversion of OIP in a batch culture. Thirteen h after the addition of OIP, the conversion extent was 85%. At this point, the byproduct, cis-4-hydrooxy-3,3,5-trimethy|cy- clohexanone was less than 5% of total OIP derivatives. The highest cell density was achieved in 4 h after OIP addition. As can be seen from this figure, OIP conversion was mostly employed at the rapid growth phase. Therefore, this microbial conversion seems to be a growth associated type.


2 1 2 S o d e et al.

I

r~

1,ot . ...... . :

; r~

o ol/A - !

c 0 o

0 4 8 12 16 20 24 28

Time (h) Fig. 1. Time course of OIP conversion. (-O-); OIP concentration, (-Q-);

DOIP concentration, (--G--); cell concentration.

OIP was a d d e d at several points in the growth phase of T. curvata batch culture, and the correlat ion be tween cell growth and microbial convers ion was investigated. Figure 2 shows the time course of p roduct ion rate of DOIP per cell. The m a x i m u m value was observed in the early phase of the batch culture.

Figure 3 represents the correlation be tween specific g rowth rate and DOIP produc t ion rate per cell. A good linear correlation was observed in this figure. Thus, this microbial convers ion is strongly d e p e n d e n t on cell growth . This suggests that it is essential to keep a high cell g rowth rate to achieve a high produc t ion rate of DOIP in a cont inuous process.

Continuous/Vlicrobial Conversion of OIP Using CSTR

The con t inuous microbial convers ion of OIP was then a t t empted us ing CSTR.

Figure 4 shows the effect of di lut ion rate on the cell n u m b e r in a reactor. With the increase of di lut ion rate, cell number decreased. However , the d e p e n d e n c y of cell n u m b e r on dilution rate was not marked , as far as can be seen for the di lut ion rates we examined. It is apparent , that at the di lut ion rates we examined, no significant washou t of cell has occurred.


Microbial Conversion of 4-Oxoisophorone 2 1 3

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Time (h) Fig. 2. Time course of production rate of DOIP per ceil.

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Specific growth rate (h -1) Fig. 3. Correlation between specific growth rate and DOIP production


2 1 4 S o d e et al.

I

4

x 3

CD

-Q2 E

1

I:D I I I

0 0,2 0.4 0,6

Dilution rate (h -1) Fig. 4. Effect of dilution rate on cell concentration during OIP continuous

conversion using CSTR. Temperature; 50~ aeration; 500 mL min-1, OIP concentration; 3 mg mL-1.

The effect of d i lu t ion rate on OIP convers ion extent is s h o w n in Fig. 5. The m a x i m u m convers ion ex ten t was observed at d i lu t ion rate 0.1-0.2 h -1 (65%). At a h igher d i lu t ion rate than 0.2 h -1, the c o n v e r s i o n ex ten t decreased .

Figure 6 r ep resen t s the effect of di lut ion rate on DOIP p roduc t iv i ty . At a d i lu t ion rate 0.4 h - 1 , the h ighes t DOIP product iv i ty was ach i eved (430 m g h - 1 L - 1).

Therefore , the extent of convers ion and product iv i ty w e r e s t rong ly d e p e n d e n t o n the d i lu t ion rate. If we in tend the i m p r o v e m e n t of the produc t iv i ty of DOIP, the fol lowing two approaches migh t be a t t e m p t e d . First is to increase the catalytic activity of cells and second is to increase the a m o u n t of the cells in the reactor.

If we a t t e m p t to increase the catalytic activity of cells, we have to increase the specific g r o w t h rate according to the results f rom Fig. 3. H o w - ever, the specific g r o w t h rate in the reactor was d o m i n a t e d by the dilu- t ion rate. C o n s i d e r i n g the resul ts f rom Fig. 6, the increase in the d i lu t ion rate h ighe r t h a n 0.6 h -1 will cause the decrease in the p r o d u c t i v i t y of DOIP. It w o u l d be o w i n g to the re ten t ion t ime of OIP in the reactor.

C o n s i d e r i n g the resul ts f rom Fig. 4, no marked cell w a s h o u t was ob- se rved . Thus , cell concen t ra t ion in the reactor was in its m a x i m u m at the d i lu t ion rate we examined . This sugges t s that it w o u l d be diff icult to achieve the s ignif icant increase in the cell concentra t ion u s i n g this reactor.

Applied Biochernistry and Biotechnology Vol. 19, 1988

Microbial Conversion of 4.0xoisophorone

100

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Dilution rate (h -l) Fig. 5. Effect of dilution rate on OIP conversion extent. Operational con-

dition is shown in Fig. 4.

Therefore , it m i g h t be impossible to i m p r o v e the produc t iv i ty of DOIP as far as u s ing this reactor. In the o the r words , we have to deve lop a nove l bioreactor sy s t em to achieve a h i g h e r p roduc t iv i ty of DOIP.

Continuous IVJicrobial Conversion of OIP Using an Air-Bubbling Hollow Fiber Reactor

To achieve a h ighe r efficiency of OIP convers ion , we then investi- ga ted an air-bubbl ing ho l low fiber reactor. The schemat ic d iagram of this reactor is s h o w n in Fig. 7. The h igher cell dens i t y t han those ach ieved in a CSTR was expected , since the molecular cutoff of the m e m b r a n e u s e d in the ho l low fiber was small e n o u g h to k e e p cells in a reactor. TherPfnrp we a t t e m p t e d the h igher di lut ion rate t h a n we e x a m i n e d in a process us ing CSTR.


216 S o d e et al.

,--M !

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4.J

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0

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~" i00 0

I I I

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Dilution rate (h -1) Fig. 6. Effect of Dilution rate on DOIP productivity. Operational condi-

tion is shown in Fig. 4. DOIP productivity is expressed as the amount of DOIP produced in 1 h/1 L working volume reactor.

The effect of di lut ion rate on OIP convers ion extent is s h o w n in Fig. 8. The h ighes t convers ion extent (70%) was observed at di lut ion rate 0.24 h-1. With the increase of the dilution rate, the convers ion extent decreased. C o m p a r e d wi th the conversion extent d e p e n d e n c y on di lut ion rate of a process us ing CSTR, however , it was significantly d imin i shed by using hol low fiber reactor.

Figure 9 shows the effect of di lut ion rate on DOIP product ivi ty. As far as the di lu t ion rate we examined, no m a x i m u m productivi ty was observed. H o w e v e r , wi th the increase of di lut ion rate, the product iv i ty also increased. The h ighes t productivi ty (1400 m g h - 1 L - 1) was achieved at di lut ion rate 2.5 h -1. This productivi ty was approximately 3.3 t imes higher product iv i ty than the max imum product ivi ty achieved us in$ CSTR. Theoretically, us ing CSTR, an increased dilution rate up to 4 h - "



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218 S o d e et al.

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Dilution rate (h -1) Fig. 8. Effect of dilution rate on OIP conversion extent (air-bubbling hol-

low fiber reactor) Temperature; 50~ aeration; 25 mL min- ~, OIP concentration; 3 mg mL-1.

could be employed (Fig. 3). However~ as can be seen in Fig. 6, di lut ion rates h igher than 0.6 h - 1 will cause lower productivity. Since the p roduc t was recovered th rough the ho l low fiber, no cell washou t has occurred even at such a high di lut ion rate. Subsequently, a high product iv i ty was achieved by using air-bubbling hol low fiber reactor.

In addi t ion, the p roduc t wi thou t cell contaminat ion is h ighly desir- able, w h e n consider ing fur ther downs t ream processing. Dur ing cont inuous conversion, a small a m o u n t of cell leakage in the p roduc t was observed. This was because of the lack of mechanical sealing or some crack in the hol low fiber. However , it can be improved by fur ther opt imizat ion of the materials. Therefore, these types of reactors us ing ho l low fibers are suitable for con t inuous microbial conversion associating cell g rowth .

To improve productivi ty, one should optimize the microenviron- men t to maintain an ideal g rowth rate, since the cells' g rowth rate is con-



1400

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1200 I

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800 14-- 0

600 -r-~

4-J

-~ 400 0

200

0

I I I I I I

0,4 0,8 1,2 1,6 2,0 2,4

Dilution rate (h -I) Fig. 9. Effect of dilution rate on DOIP productivity. Operational condi-

tion is shown in Fig. 8. DOIP productivity is expressed as the amount of DOIP produced in 1 h/1 L working volume reactor.

trolled only by the changing dilution rate. However, the correlation between dilution rate and specific growth rate was applied for the continuous process where the mass balance is controlled by the dilution rate at the steady state. In the air-bubbling hollow fiber reactor, cells were maintained in the reactor throughout the process and never washed out. As far as judging from the results obtained in this study, the high density culture system caused a high efficiency of microbial conversion. Therefore, the microenvironment seemed to be suitable for cell growth.

By controlling and optimizing pH, cell density, and dissolved oxygen in the extracapiUary space of the reactor, higher productivity can be expected.


220 S o d e et al.

ACKNOWLEDGMENT

We thank Vincent M. McNeil for help in compiling in this paper .

REFERENCES

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4. Iijima, S., Kawai, S., Mizutani, S., Taniguchi, M., and Kobayashi, T. (1987). Appl. Microbiol. Biotechnol. 26, 542.

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6. Mikami, Y., Fukunaga, Y., Arita, M., and Kisaki, T. (1981), Appl. Environ. Microbiol. 41, 610.

7. Mikami, Y., Fukunaga, Y., Hieda, T., Obi, Y., and Kisaki, T. (1981), Agric. Biol. Chem. 45, 331.

8. Mikami, Y., Fukunaga, Y., Arita, M., Obi, Y., and Kisaki, T. (1981), Agric. Biol. Chem. 45, 791.

9. Hori, N., Hieda, T., and Mikami, Y. (1984), Agric. Biol. Chem. 48, 123. 10. Sonnleitner, B. and Fiechter, A. (1983), Trends in Biotechnol. 1, 74. 11. Sonnleitner, B., Giovanni, F., and Fiechter, A. (1985), J. Biotechnol. 3, 33. 12. Sonnleitner, B. and Fiechter, A. (1986), Appl. Microbiol. Biotechnol. 23, 424. 13. Sode, K., Kajiwara, K., Tamiya, E., Karube, I., Mikami, Y., Hori, N., and

Yanagimoto, T. (1987), Biocatalysis 1, 77.


microbial conversion of 4-oxoisophorone by thermomonospora curvata using an air-bubbling hollow...

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