Medium-chain-length polyhydroxyalkanoate 고농도 생산을 위한 Pseudomonas 균주의 유가식 배양기술 개발

이승환, 이상엽

대전광역시 유성구 구성동 373-1 한국과학기술원 화학공학과 및 생물공정연구센터

Development of fed-batch culture for the production of Medium-chain-length polyhydroxyalkanoate by Pseudomonas

Seung Hwan Lee, Sang Yup Lee

Department of Chemical Engineering and BioProcess Engineering Research Center, KAIST 373-1 Kusong-dong, Yusong-gu, Taejon 305-701

Introduction

Polyhydroxyalkanoates(PHAs) are energy and/or reducing power storage materials accumulated intracellularly by numerous bacteria under nutrient such as nitrogen, phosphorus, sulfur, oxygen or magnesium limited condition in the presence of excess carbon source.

It has been reported that an unbalanced growth condition with excess carbon source and simultaneous limitation of nutrients such as oxygen, nitrogen, sulfur, magnesium or phosphorus is favorable for PHA accumulation in many wild-type bacteria. In the production of poly(3-hydroxybutyrate) [P(3HB)] by Ralstonia eutropha, it has been reported that the initial phosphate concentration in the culture medium was important for increasing the final P(3HB) productivity and concentration.

The aim of this paper was to develop a fermentation strategy for the high level production of Medium-chain-length(MCL)-PHAs by employing Pseudomonas putida from oleic acid as a carbon source, to isolate new MCL-PHA producing bacteria and to produce MCL-PHA using newly screened strain. We describe first a detailed fermentation strategy coupled with phosphorus limitation to achieve high cell concentration of P. putida with high PHA content. Next, the isolation of new bacteria and its fermentation will be discussed.

Materials and methods

P. KT2442 and newly screened bacteria were used in all experiments. Pseudomonas sp. KR-22 and other bacteria were isolated from the soil and raw sewage samples.

In all experiments, three different media were used: two chemically defined media, MR-1 medium and MR-2 medium were used in the fed-batch cultures and LB medium and MR-2 medium were used for two stage flask cultures. The MR-1 medium contains per liter: 22 g KH2PO4, 3.0 g (NH4)2HPO4, 1.4 g MgSO4˛7H2O, 0.8 g citric acid, 10 ml trace metal solution. The MR-2 medium contains per liter: 6.67 g KH2PO4, 4 g (NH4)2HPO4, 0.8 g MgSO4˛7H2O, 0.8 g citric acid, 5 mL trace metal solution.

PHA concentration was determined by gas chromatography (HP5890, Hewlett-Packed, Wilmington, DE) using n-butyric acid as an internal standard. The PHA standards were prepared by solvent extraction. The composition of the standards was determined by 13C and 1H nuclear magnetic resonance spectroscopy (AMX 500, Bruker, Germany). Residual cell concentration was defined as cell concentration minus PHA concentration. Phosphate concentration was determined by ion chromatography (Waters 432, Milford, MA) using KH2PO4 as a standard.

Results and discussion

Production of MCL-PHA by fed-batch culture of P. putida

When cells were cultivated in MR-1 medium (fed-batch A), the final cell concentration, PHA concentration and PHA content obtained in 28.7 h were 173 g/L, 32.3 g/L and 18.7 wt%, respectively, resulting in the productivity of 1.13 g PHA/L-h. Residual cell concentration did not increase after reaching 140 g/L. After cell growth stopped, PHA concentration and PHA content increased from 9.8 g/L to 32.3 g/L and from 6.4 wt% to 18.7 wt%, respectively. Even though high cell concentration could be obtained by this feeding strategy in MR-1 medium, PHA concentration and PHA content obtained were rather low. From the previous studies on the production of PHA by fed-batch culture, it was found that the nutrient limitation resulted in the increase of the PHA content. It was also found that the time point of applying nutrient limitation had a significant effect on PHA production. When P. putida was cultivated under various nutrient limiting conditions (N, P, S or O), the highest PHA content was obtained by phosphorus limitation among the tested. In order to apply the phosphorus limitation, fed-batch cultures were carried out with different initial concentrations of phosphate.

For the fed-batch culture started with the initial KH2PO4 concentration of 7.5 g/L (fed-batch B), final cell concentration, PHA concentration and PHA content obtained in 34 h were 168 g/L, 60.7 g/L and 36.1 wt%, respectively, resulting in the productivity of 1.79 g PHA/L-h. Even though final cell concentration obtained in fed-batch B was slightly lower than that in fed-batch A, the PHA content, PHA concentration and PHA productivity were all considerably higher in fed-batch B. After the limitation of phosphorus, PHA concentration and PHA content increased from 5.9 g/L to 60.7 g/L and 4.9 wt% to 36.1 wt%.

In fed-batch C, the initial KH2PO4 concentration in MR-1 medium was further reduced to 4 g/L. The cell concentration, PHA concentration and PHA content obtained in 38 h were 141 g/L, 72.6 g/L and 51.4 wt%, respectively, resulting in the productivity of 1.91 g PHA/L-h (Fig. 1). PHA concentration and PHA content significantly increased from 5.9 g/L to 72.6 g/L and from 7.5 wt% to 51.4 wt% after phosphorus limitation. This is the highest productivity reported to date for MCL-PHAs.

Further reduction of the initial phosphate concentration resulted in a similar PHA content, but lower PHA concentration and lower PHA productivity were obtained.

Production of MCL-PHA by newly screened bacteria, Pseudomonas sp. KR-22

We screened the bacteria which have a capability to utilize oleic acid from soil and raw sewage samples. This strain was isolated Pseudomonas sp. KR-22. To find optimal PHA accumulating condition for Pseudomonas sp. KR-22, various nutrient limited conditions (nitrogen, sulfur, phosphorus) were examined using oleic acid as a sole carbon source. PHA content was 15 wt% under phosphorus limited condition. In other nutrient limited and no limited condition, PHA contents were lower than 1 wt%.

Based on the results of flask cultures, fed-batch culture was carried out for production of MCL-PHA. Because culture condition of this strain was similar to P. putida, the same feeding strategy was applied first. However, the optimal pH of culture broth and the response of DOC and pH after carbon source depletion were different to that of P. putida. Therefore, new feeding strategy for this strain was developed.

Since phosphorus limitation was the best condition for PHA accumulation, phosphorus limited MR-2 medium (4 g/L of KH2PO4) was used for fed-batch culture. The amount of phosphorus was a little higher than that of medium used P. putida culture. pH was set at 6.5 which was the optimal condition for cell growth.

Time profiles of DCW, PHA content, and phosphate concentration were shown in Fig. 2. Final DCW, PHA content, and PHA concentration obtained in 41 h was 82 g/L, 28.1 wt%, and 23 g/L, respectively. Residual cell concentration was obtained 90 g/L in 30 h, and phosphorus limitation was applied after that. PHA content was increased from 12.2 wt% to 28.1wt% during phosphorus limiting period.

Acknowledgement

This work was supported by the BioProcess Engineering Research Center, by the Ministery of Science and Technology, and by the First Young Scientists Award to Sang Yup Lee by the President of Korea.

Referances

1. Anderson, A. J. and Dawes, E. A. Microbiol. Rev., 54, 450(1990).

2. Huijberts, G. N. M. and Eggink, G. Appl. Microbiol. Biotechnol., 46, 233(1996).

3. Lee, S. Y. Biotechnol. Bioeng., 49, 1(1996).

4. Lee, S. Y. Trends Biotechnol., 14, 431(1996).

5. Steinb□chel, A., and F□chtenbusch, B. Trends Biotechnol., 16, 419(1998).

Fig. 1 Time profiles during fed-batch culture when the initial KH2PO4 concentaion in the MR-1 medium was 4 g/L

Fig. 2  Time profiles during fed-batch culture of Pseudomonas sp. KR-22