Actinobacillus succinogenes sp. 130Z의 성장과 숙신산 생산

김문일, 장용근, 장호남

한국과학기술원 생명화학공학과

Growth and succinic acid production of Actinobacillus succinogenes sp. 130Z

Moon Il Kim, Yong Keun Chang, and Ho Nam Chang

Department of Chemical and Biomolecular Engineering, KAIST 373-1, Guseong-dong, Yuseong-gu, Daejeon, Republic of Korea, 305-701

Introduction

Anaerobic bacteria can produce a variety of organic acids as end products of glucose metabolism (Zeikus, 1980). Recently, the fermentative production of succinic acid from carbohydrates by anaerobic bacteria has become of applied significance as a renewable resource-based route for C4 chemical feedstocks. Although the industrial potential for succinic acid fermentations was recognized as early as 1980 (Zeikus, 1980), recently interest has focused on the biological production of succinic acid as an important industrial fermentation process.

Green technology is becoming a driving force in the chemical industry because of the current need to decrease pollution caused by petrochemical processes. While petrochemical processes often use fixed, non-renewable sources and cause global warming by increasing the total amount of carbon in the environment, biobased carbon processes use abundant carbon sources which are continuously recycled because plants or some microorganisms capture CO2 in the atmosphere and convert it into their biomass. Consequently, biobased processes have net-zero impact on greenhouse gas emissions. So hydrocarbon, petrochemical based economy needs to be replaced with biobased carbohydrate economy, which is renewable, environmentally reasonable.

To date, succinic acid has been produced commercially by chemical processes. Recently, however, fermentative production of succinic acid from renewable biomass by anaerobic bacteria has become of great interest (Landucci et al., 1994). Although many different kinds of bacteria produce succinic acid as a fermentation end product (O'Herrin et al., 1993), few species can produce it with high concentration and yield. A newly discovered facultative anaerobic bacterium Actinobacillus succinogenes is considered as the best succinic acid producer because it can endure high glucose osmotic pressure and produce significant amount of succinic acid with a high productivity.

In this study, we examined the effects of several medium components on the growth of Actinobacillus succinogenes and succinic acid production. We also carried out fed-batch and continuous cultures to investigate the time profile of growth and succinic acid production.

Materials and methods

Organisms and culture conditions

Actinobacillus succinogenes sp. 130Z (ATCC 55618) was obtained from the American Type Culture Collection (Rockville, MD). The growth medium for seed culture contained per liter : 9.6 g NaH2PO4․2H2O, 10.0 g NaHCO3, 15.5 g K2HPO4, 5.0 g yeast extract. Separately autoclaved solutions of glucose were added to the medium after autoclaving. Na2S․9H2O (final concentration 0.2 g/L) was added to ensure strict anaerobic conditions.

Fed-batch cultures were performed in a 2.5L jar fermenter at 38℃ while the pH was adjusted at 7.0 using 5N NaOH solution. Feeding solution was prepared with 300 g/L glucose and 100 g/L yeast extract. The feeding solution was added to 1L of the initial culture medium with the constant feeding and intermittent feeding.

Continuous cultures were carried out with working volume of 500 ml in a 2.5L jar fermenter. Feeding solution contained per liter :  4.8 g NaH2PO4․2H2O, 10.0 g NaHCO3, 7.75 g K2HPO4, 5.0 g yeast extract, 20 g glucose. The feeding solutions were purged with oxygen-free CO2 gas for 24 h to establish anaerobic conditions before uses. After 12-h of batch operation, the feeding solution was added at various dilution rates while an equal volume of spent medium was removed from the fermenter. The new steady states were confirmed by the constant levels of cells, glucose, and organic acids in the medium for three consecutive samples taken at 3-8 hour intervals after at least 5 turnovers.

Analytical methods

Growth of the microorganism was monitored by measuring the optical density of the culture broth at 660 nm (OD660) using a spectrophotometer (Ultraspec 3000, Pharmacia Biotech, Sweden). The concentrations of glucose and succinic acid, acetic acid, formic acid, and lactic acid were measured by high-performance liquid chromatography (Hitachi L-3300 RI monitor, L-4200 UV-VIS detector, D2500 chromato-integrator, Tokyo, Japan) equipped with an ion exchange column (Aminex HPX-87H, 300 mm × 7.8 mm, Hercules, CA) using 0.012N H2SO4 as a mobile phase. The flow rate of the mobile phase was 0.6 ml/min, and the operating temperature was 50℃. Succinic acid yield was expressed as the amount of succinic acid produced from one gram glucose.

Results and discussion

In the request for developing an economic fermentation process of succinic acid, there has been much effort to utilize cheaper medium components. Among various components of culture medium, complex nitrogen sources and carbohydrates are important for determining the price of products. Therefore, eight nitrogen sources and ten carbohydrates were examined for their effects on cell growth and succinic acid production in flask cultures. For nitrogen sources, except yeast extract, only soytone was efficiently utilized, however, casamino acid, tryptone, tryptone peptone, peptone, meat extract, and soybean were not (Fig. 1). For carbohydrates, except glucose, fructose, mannose, mannitol, sucrose, maltose, and lactose were efficiently utilized, but arabinose, inositol, and glycerol were not (Fig. 2). Lactose is a main carbohydrate component in whey which is a waste of cheese industry. Since Actinobacillus succinogenes can use lactose as a carbon source, it can produce succinic acid from the waste product whey.

Fed-batch and continuous cultures were carried out to investigate the inclinations of cell growth and end product formation. Fed-batch culture using constant feeding strategy was examined to obtain high cell concentration and productivity of succinic acid. The feeding solution was constantly added by the ratio of 2.7 g/L/h. The maximum cell concentration reached at the end of fermentation was an OD660 of 6.1 (3.66 g DCW/L). Compared with that obtained by batch cultures, cell concentration was not enhanced and succinic acid yield was slightly decreased. Therefore, we examined if the intermittent feeding affected cell growth and succinic acid production. After the depletion of glucose in the medium, glucose was intermittently added. The maximum cell concentration was only an OD660 of 3.8 and succinic acid yield was not enhanced as formerly. These results indicated that there was a limitation of cell growth and succinic acid production in fed-batch cultures.

Anaerobic continuous culture was carried out at various dilution rates to investigate the time profile of cell growth and end product formation (Fig. 3). The steady state cell concentration was maintained an OD660 of 2.0-3.5 until a dilution rate reached 0.5 h-1. With increasing a dilution rate, glucose accumulated in the medium and its concentration reached 12.8 g/L at the dilution rate of 0.5 h-1. The steady state concentration of cells reached a peak value at a dilution rate of 0.2 h-1. After that, the cell concentration decreased with increasing a dilution rate. The steady state concentration of succinic acid decreased with increasing a dilution rate. Succinic acid yield (0.64-0.52) decreased with increasing a dilution rate. Because succinic acid was a growth-associated product, volumetric productivity of succinic acid increased with increasing a dilution rate. The maximum volumetric productivity of succinic acid of 2.12 g/L/h was obtained at a dilution rate of 0.4 h-1.

Acknowledgements

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

References

Guettler, M. V., D. Rumler, and M. K. Jain. 1999. Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen. Int. J. Syst. Bacteriol. 49: 207-216.

Landucci, R., B. Goodman, and C. Wyman. 1994. Methology for evaluating the economics of biologically producing chemicals and materials from alternative feedstocks. Appl. Biochem. Biotechnol. 45-46: 678-696.

O'Herrin, S. M., and W. R. Kenealy. 1993. Glucose and carbon dioxide metabolism by Succinivibrio dextrinosovens. Appl. Environ. Microbiol. 59: 748-755.

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

Zeikus, J. G. 1980. Chemical and fuel production by anaerobic bacteria. Ann. Rev. Microbiol. 34: 423-464.

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

Fig 2. Effect of carbohydrates on cell growth and succinic, lactic,

and acetic acid production

Fig 1. Effect of nitrogen sources on cell growth and succinic, lactic, and acetic acid production

Fig 3. Steady state values of continuous fermentation of Actinobacillus succinogenes sp. 130Z at various dilution rates