Actinobacillus succinogenes를 이용한 숙신산 생산

김문일, 임성춘, 이우기*, 장호남

대전광역시 유성구 구성동 373-1 한국과학기술원 화학공학과

대전광역시 유성구 화암동 62-1 첨단기술사업화센터 5302호 이원바이오텍(주)*

Succinic acid production by Actinobacillus succinogenes

Moon Il Kim, Seong Chun Yim, Woo Gi Lee*, and Ho Nam Chang

Department of Chemical Engineering, KAIST 373-1, Kusong-dong, Yusong-gu, Taejon, Korea, 305-701

EI biotech Co. Ltd., HTC #5302, 62-1 Whaam-dong, Yusong-gu, Taejon 305-348, Korea*

Introduction

Succinic acid is a C4-dicarboxylic acid and an intermediate of the tricarboxylic acid(TCA) cycle. It has been mainly produced by chemical process. But recently, Much effort has been exerted in producing succinic acid by fermentation using microbe, because the method of using microbial fermentation fixes green-house gas, CO2.

Actinobacillus succinogenes sp. 130Z is a gram-negative facultatively anaerobe, ferments glucose to succinic acid as a major product and acetate, formate, lactate, and ethanol are minor products. This strain can endure high glucose osmotic pressure. Succinic acid production by this strain was much higher than by E. coli and reached higher concentration than A, succiniciporducens

(Samuelov et al. 1991; Guettler et al, 1996).

In this study, we carried out fermentation of glucose to succinic acid by 100 ml flask fermentation with different initial glucose concentration and pH. We also carried out succinic acid production with changing MgCO3 concentration and air condition.

Materials and methods

Organisms  and culture conditions

Actinobacillus succinogenes (ATCC 55618) was obtained from the ATCC (Rockville, Md., USA). The growth medium contained per liter : 5.0 g yeast extract, 9.6 g NaH2PO4·2H2O, 10.0 g NaHCO3, 15.5 g K2HPO4. Separately autoclaved solutions of glucose were added to the medium after autoclaving. Na2S·9H2O (final concentration 0.02 %) was added in order to make strict anaerobic conditions. CO2 gas purged into the flask for about 1 minute in order to make CO2 100% condition. Media were inoculated with 1% (by volume) samples of cultures grown in th same medium and incubated at 37℃.

Analytical method

Growth of cell mass was measured by optical density (OD660) using a spectrophotometer (Pharmacia Biotech, Ultrospec 3000, Sweden). Fermentation products and residual glucose concentration were determined by HPLC (Hitachi chromatography system, Tokyo, Japan) equipped with Ion exchange column (Aminex HPX-87H, 300 mm × 7.8 mm, Hercules, CA) using 0.01 N H2SO4 as a mobile phase and a refractive index detector (L-3300, Hitachi chromatography system).

Results and discussion

Effect of carbon dioxide in air on cell growth and succinic acid production

Cell growth and succinic acid production by Actinobacillus succinogenes are affected by the level of carbon dioxide during the culture, because succinic acid production requires additional carbon dioxide.

In this study, cell growth and succinic acid concentration in 100 % CO2 condition showed higher cell growth and succinic acid concentration than simple air condition (Fig 1). The difference in final succinic acid concentration between CO2 100 % gas and air was about 1 g/L. Because CO2 didn't supply continuously in a flask culture, succinic acid production was limited.

Effect of MgCO3 in a medium on cell growth and succinic acid production

MgCO3 palyed an important role of pH maintenance and supplying CO2 gas. In this study, cell growth and succinic acid production with MgCO3 showed higher results than without MgCO3. But the results of 10 g/L MgCO3 didn't show large difference with 5 g/L MgCO3 (Fig 2). Final succinic acid concentration with 10g/L MgCO3 reached over 8 g/L and yield reached over 60 %.

Effect of initial glucose concentration on cell growth and succinic acid production

Because Actinobacillus succinogenes can endure high osmotic pressure, we could use high concentration of glucose. In cases of 20 g/L and 40g/L glucose concentration, cells grew up to near 3.5 of OD660 and succinic acid concentration approached to about 8g/L (Fig 3). In the case of 10g/L glucose, glucose was fully consumed, and final succinic acid concentration did not show high results because of insufficiency of glucose. In the case of 80 g/L glucose, cells grew up to 3.26 of OD660. But cells in 80 g/L glucose concentration grew slower and lower than the cells in other glucose concentration because of substrate inhibition.

Effect of initial pH on cell growth and succinic acid production

Actinobacillus succinogenes could grow between pH 6 and pH 7.5. But in this study, the range between pH 7 and pH 7.5 was the best pH condition on cell growth and succinic acid production. In the range between pH 6 and pH 6.5, cells grew up to from 1.65 to 2.63 of OD660, lower than the results between pH 7 and pH 7.5. But the succinic acid yield between pH 6 and pH 6.5 showed higher results than the data between pH 7 and pH 7.5 (Table 1). when we fermented glucose to succinic acid in pH 6.5, final succinic acid concentration was 5.7 g/L, but yield was about 87 %. In pH 7.5, final succinic acid concentration reached 9.2 g/L, and yield was 81% (Fig 4).

Acknowledgements

This work was partially supported by Ministry of Science ＊ Technology, EIbiotech Co. Ltd. and the Brain Korea 21 Project.

References

Michael V. Guettler, Denise Rumler and Mahendra K. Jain : Actinobacillus succinogenes sp.nov., a novel succinic-acid-producing strain from the bovine rumen. International Journal of Systematic Bacteriology 49: 207-216(1999)

Mariet J. Van der Werf, Michael V. Guettler, Mahendra K. Jain, J. Gregory Zeikus : Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z, Arch Microbiol 167: 332-342(1997)

J. G. Zeikus, M. K. Jain, P. Elankovan : Biotechnology of succinic acid production and markets for derived industrial products, Appl Microbiol Biotechnol 51: 545-552(1999)

D.H. Park, M. Laivenieks, M. V. Guettler, M. K. Jain and J. G. Zeikus : Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production, Applied and environmental microbiology, p2912-2917(1999)

pH variable	maximum OD660	final succinic acid concentration (g/L)	yield (%)
pH6	1.65	3.2	89.7
pH6.5	2.63	5.7	87.1
pH7	3.40	8.5	80.1
pH7.5	3.55	9.2	81.0