Anaerobiospirillum succiniciproducens에 의한 숙신산의 생산

이 평천, 이 우기, 권선훈, 이 상엽, 장 호남

한국과학기술원, 생물공정연구센터/화학공학과

Production of succinic acid by Anaerobiospirillum succiniciproducens

Pyung Cheon Lee, Woo Gi Lee, Sunhoon Kwon, Sang Yup Lee, Ho Nam Chang

Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology

Introduction

Succinic acid is a dicarboxylic acid produced as an intermediate of the tricarboxylic acid cycle and also as one of the fermentation products of anaerobic metabolism. It can be used in a wide range of applications such as in the manufacture of synthetic resins and biodegradable polymers, and as the intermediate for chemical synthesis [1]. 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 [2]. Although many different kinds of bacteria produce succinic acid as a fermentation end product, few species can produce it as the major product with high yield. An anaerobic bacterium Anaerobiospirillum succiniciproducens has been considered as the best succinic acid producer because it can produce a significant amount of succinic acid from glucose [3]. Previous studies showed that A. succiniciproducens produces succinic acid and acetic acid as the major end products and lactic acid and ethanol as minor ones depending on culture conditions. In particular, the levels of CO2 and culture pH were reported to be the most critical factors affecting both cell growth and succinic acid production [4]. In contrast, Nghiem et al. [5] reported that an external supply of CO2 was not necessary for cell growth and succinic acid production when Na2CO3 was initially present in the medium. In the above study, however, CO2 sparging studies were carried out at the fixed pH of 6.0. Since culture pH can affect the solubility of CO2, it is necessary to study the effect of CO2 supply at different pH values. In addition to CO2, an external electron source such as H2 can affect cell growth and distribution of fermentation products. Electrons from H2may be incorporated into the fermentation products resulting in the increase of succinic acid yield. Additional electrons may also accelerate metabolism resulting in the enhancement of cell growth. Initial glucose concentration can affect cell viability and distribution of fermentation products as well as the level of CO2. Higher concentration of glucose can result in long lag time and inhibition of cell growth. These aspects have not been systematically studied.

In this study we report on the effect of external H2 supply on cell growth and succinic acid production. We also report on the effect of CO2 at various pH values and initial glucose concentration on cell growth and succinic acid production.

Materials and methods

Organism and growth conditions

Anaerobiospirillum succiniciproducens (ATCC 29305) was obtained from the American Type Culture Collection (Rockville, MD.). Cells were grown in sealed anaerobic bottles containing 100 mL medium with CO2 as the gas phase, unless stated otherwise. The growth medium contained per liter: 10 g glucose, 10 g polypeptone, 5 g yeast extract, 3 g K2HPO4, 1 g NaCl, 1 g (NH4)2SO4, 0.2 g CaCl2□2H2O, 0.2 g MgCl2□6H2O, and 1 g Na2CO3.

For the batch cultivation, cells were grown in a jar fermenter (2.5 L, Korea fermenter Company, Incheon, Korea) containing 1 L of a different medium containing per liter: 20 g glucose, 10 g polypeptone, 5 g yeast extract, 3 g K2HPO4, 1 g NaCl, 5 g (NH4)2SO4, 0.2 g CaCl2□2H2O, 0.2 g MgCl2□6H2O, 5 mg FeSO4□7H2O, and 5 g Na2CO3. Temperature and pH were maintained at 39 oC and 6.5 using 1.5 M Na2CO3, respectively. Foam was controlled by adding Antifoam 289 (Sigma Chemical Co., St. Louis, MO). Adjusting the aeration rate (0.025 vvm) and agitation speed (200 rpm) maintained CO2 concentration in fermenter. Gas mixture (H2/CO2) was supplied into fermenter by gas mixer (New Brunswick Scientific Co., NJ.).

Analytical method

Glucose and fermentation products such as succinic acid and acetic acid were analyzed by high-performance liquid chromatography equipped with an ion exchange column (Aminex HPX-87H, 300 mm x 7.8 mm, Hercules, CA) using 0.012 N H2SO4 as a mobile phase. Cell growth was monitored by measuring optical density at 660 nm (OD660) using a spectrophotometer (Ultrospec3000, Pharmacia Biotech, Sweden).

Chemicals and gases

All chemicals used were of reagent grade and were obtained from either Junsei Chemical Co. (Tokyo, Japan) or Sigma Chemical Co. (St. Louis, MO). Gases were scrubbed free of oxygen by passage over gas purifier (P.J. Cobert Associates, Inc., St. Louis, MO).

Results and discussion

Effect of CO2 and pH

Cell growth and succinic acid production by A. succiniciproducens are affected by the level of CO2 and pH during the culture. Therefore, the effects of external CO2 supply at different culture pH values on cell growth and succinic acid production were studied in pH-controlled batch fermentation using initial glucose concentration of 40 g/L (Fig.1). During the fermentation without the supply of CO2, maximum cell concentrations obtained at both pH 6.2 and pH 6.5 were 5.3 and 5.6 of OD660, respectively. However, when supply of CO2 was carried out, cell growth was severely suppressed at both pH 6.2 and pH 6.5 resulting in 3.6 and 3.4 of OD660, respectively, and did not grow after. In contrast to the negative effect of CO2 on cell growth, succinic acid yield was positively affected at both pH 6.2 and pH 6.5. At pH 6.2, succinic acid yields were similar in the absence or presence of CO2 supply; 82 and 83%, respectively. However, at pH 6.5, the supply of CO2 had a significant effect on succinic acid yield; 84 or 88% in the absence or presence of CO2 supply, respectively.

Use of H2 as electron donors

Succinic acid is a highly reduced fermentation product over glucose. Therefore, the effect of external supply of H2, a potential electron donor, on cell growth and succinic acid production was studied with various H2/CO2 ratios (Fig.2). At the ranges between 0 and 10% H2 with base gas of CO2 when 20 g/L glucose was used, the external supply of H2 had an effect on cell growth and succinic acid yield. However, there existed optimum ratio of H2 to CO2, 5/95. Maximum cell concentration and succinic acid yield, obtained in the presence of supply of 5% H2, were 3.7 of OD660 and 91%, respectively, which were higher than 2.9 of OD660 and 86% in the absence of H2. Furthermore, supply of 5% H2 shorten the fermentation time resulting in increase of succinic acid productivity (1.7 g/L□h) which is the highest to date. However, the concentration of acetic acid produced as a byproduct during the culture was range between 20 and 21% and was did not influenced by the ratio of H2/CO2.

Effect of initial glucose concentration

Initial glucose concentration can influence cell viability and distribution of fermentation products as well as succinic acid yield. Therefore, to study the effect of initial glucose concentration, at different initial concentrations of glucose of 20, 40 and 80 g/L, cells were grown under same conditions. Cells grew continuously until glucose was depleted and maximum cell concentration was 3.0 of OD660 when 20 g/L glucose was used. However, when 40 g/L glucose was used, cells grew up to 3.4 of OD660 and did not grew anymore due to the inhibition even though remained glucose was all consumed for succinic acid production. Moreover, at an initial glucose of 80 g/L, maximum cell concentration was 4.0 of OD660 but cell growth curve declined abruptly after consumption of 40 g/L glucose. Succinic acid yield in this medium was also much lower, reaching a yield of 79% after 31 h as compared with 88 and 86% in medium containing initial glucose concentration of 20 and 40 g/L, respectively. However, distribution of fermentation products was similar under all initial glucose concentration tested.

In conclusion, the findings of this study, such as H2, CO2, and initial glucose concentrations, should help design a better strategy for the production of succinic acid by high cell density culture of A. succiniciproducens.

Acknowledgments

This research was supported by a grant from the Ministry of Science and Technology, Korea.

References

1. Zeikus J.G.: Annu Rev Microbiol, 34, 423(1980).

2. Landucci R., et al.: Appl. Biochem. Biotechnol., 45-46, 678(1994).

3. Laivenieks, M., et al.: Appl. Environ. Microbiol., 63, 2273(1997).

4. Samuelov, N. S., et al.: Appl. Environ. Microbiol., 57, 3013(1991).

5. Nghiem, N. P., et al.: Appl. Biochem. Biotechnol., 63-65, 565(1997).

Figure 1. Effect of CO2 at different culture pH on the cell growth and fermentation

products (succinic acid and acetic acid) in batch experiments. Cells were grown anaerobically in a jar fermenter with 40 g/L of glucose at the culture pH of 6.2 and

6.5 in the presence/absence of CO2. Panel A, pH 6.2 without CO2; B, pH 6.2 with CO2; C, pH 6.5 without CO2; D, pH 6.5 with CO2. For each panel, the OD660(▲), glucose (■), succinic acid (○), and acetic acid (◇) were measured as described in Materials and methods.

Figure 2. Effect of H2/CO2 ratio on the cell growth and succinic acid yield in batch experiments. Maximum cell concentration (  ) and succinic acid yield (  ) were measured as described in Materials and methods.