유청으로부터 재조합대장균의 유가식배양에 의한 poly(3-hydroxybutyrate)의 생산

안우석, 김범수, 임미애, 김태완, Brian K. O'Neill, 이상엽

한국과학기술원 화학공학과, 건양대학교 화학공학과, 아델라이드 대학교 화학공학과

Poly(3-hydroxybutyrate) production from whey by fed-batch cultivation of new recombinant Echerichia coli

Woo Suk Ahn, Beom Soo Kim*, Mi Ae Lim*, Taek Wan Kim*, Brian K. O′Neill**, and Sang Yup Lee

Department of Chemical Engineering, Korea Advance Institute of Science and Technology, Department of Chemical Engineering, Konyang University*, Department of Chemical Engineering, Univ. of Adelaide, Australia**

Polyhydroxyalkanoates (PHAs) are accumulated intracellularly by numerous microorganism. PHAs possess biodegradable property and physical properties similar to various petrochemical-based polymers. But it is difficult to commercialize PHAs because of the higher production cost of PHAs compared with the conventional polymers. Economic evaluation of the process for the production of PHB suggested that the cost of substrate contributes most significantly (up to 50%) of the overall production cost. Therefore, PHB will become economically more competitive if it can be produced from a cheap substrate such as whey. Whey (4.5% (w/v) lactose, 0.8% (w/v) protein, 1.0% (w/v) salts, and 0.1-0.8% (w/v) lactic acid) is the major by-product from the dairy industry. If this waste product is used, the production cost of PHAs can be much lowered.

This study describes the strategies for achieving higher cell yield to do the high cell density cultivation and controlling the timing of PHA synthesis to search the optimum PHB content in whey based medium.

Materials and Methods

Bacterial strains and plasmid DNA. E. coli CGSC 6576 and CGSC 4401 were used in this study. The plasmid pSYL107 containing the Ralstonia eutropha PHA biosynthesis operon and the E. coli ftsZ gene and pJC4 containing the Alcaligenes latus PHA biosynthesis operon have been described previously. All strains were cultured at 30 ℃ in R medium and 100 □/L ampicillin was added.

Pretreatment of whey solution. Bovine whey powder containing 11 % (w/w) of proteins and 65% (w/w) of lactose was purchased from Sigma (St. Louis, Mo.). Whey solution was prepared by dissolving 310 g whey powder and 0.36 g MgSO4·7H2O in 1 L distilled water. After autoclaved, the solution was centrifuged at 11000 g to remove precipitates. In fermentation B, to improve the purity of feeding solution, the solution in which whey was dissolved was adjusted to pH 4.5 with 37% HCl and was autoclaved at 121 ℃ for 15 min. Using the Watman No. 3 membrane and the diatomaceous earth, small protein particles were removed. Finally, the filtered solution was adjusted to pH 6.5 with 12N NaOH.

Culture condition. To investigate the timing of PHA biosynthesis, 2.5 L jar fermenter (Korea Fermenter Company, Incheon) was used. the initial medium and lactose concentration were 0.8 L R medium and 20 g/L. Fermentation A and B were carried out in a 6.6 L jar fermentor (Bioflo 3000, New Brunswick Scientific Co., Edison, NJ). The initial medium was 1.6 L R medium. Cells were cultured at 30 ℃ and pH was maintained at 6.95 by the addition of 28% (v/v) NH4OH. The dissolved oxygen level was maintained above 40% by the agitation speed to 1000 rpm and by the supplementation of pure oxygen. The nutrient feeding strategy was the pH-stat. In fermentation A, 7.4 L nutrient feeding solution which concentration was 210 g/L lactose was added. These feeding whey solution was made by the simple treatments of autoclaving and centrifugation. During the fermentation, 6.5 L culture broth was removed because of the limitation of the jar volume. In fermentation B, 2.7 L nutrient feeding solution with 280 g/L lactose was added without removing the culture broth. This feeding whey solution was made by acidification, sterilization, filtration and neutralization.

Analytical procedures. Cell growth was monitored by measuring the absorbance at 600 nm (DU□ Series 600 Spectrophotometer, Beckman, Fullerton, CA). PHB concentration was determined by gas chromatography (DS6200, Donam Co., Seoul). The PHA content (wt%) was defined as the percentage of the ratio of PHA concentration to cell concentration.  To estimate the concentration of lactose, high-performance liquid chromatography (Hitachi L-3300 RI monitor, L-4200 UV-Vis detector, L-600 pump, D-2500 chromato-integrator, Tokyo, Japan) equipped with an ion exchange column (Aminex HPX-87H, 300 mm × 7.8 mm, Hercules, CA) using 0.01 N H2SO4 as a mobile phase.

Results

Controlling the timing of PHB biosynthesis by DO limitation. The timing of PHB biosynthesis was controlled using agitation speed of the stirred tank fermentor with the recombinant E. coli CGSC 6576 harboring a high copy number plasmid, pSYL 107 containing R. eutropha PHA biosynthesis by the pH-stat fed-batch fermentation. In case of increasing the agitation speed to 1300 rpm, the final PHB content and dry cell weight were 56.9% and 55.5 g/L. To investigate the effect of DO limitation to recombinant E. coli for the production of PHB in whey based medium, various agitation speeds were applied. the final PHB content and dry cell weight were 70.1% and 15.2 g/L at 300 rpm and 80.2% and 31.2 g/L at 500 rpm (Table 1.). These results suggested that high PHB content could be achieved using the DO limitation by controlling agitation speed.

Achieving the higher cell yield with the pretreatment of whey. higher cell yield was achieved with the pretreatment of whey solution. The pretreatment of whey solution was performed with the process of acidification, sterilization, and filtration to remove the insolube particles. New recombinant E. coli harboring a high copy number plasmid containing A. latus PHA biosynthesis genes, CGSC 4401 (pJC4) was used in this pH-stat fed-batch fermentation. Using the pretreated whey solution containing 280 g/L lactose, pH-stat fed-batch fermentation produced the PHB content and cellular dry weight of 58% and 102.9 g/L without removing the culture broth during the fermentation. At the same time(Figure 1.), Cell yield was increased 0.51 g/g lactose higher than 0.42 g/g lactose in fermentation A.

With these strategies of the DO limitation and the pretreatment of whey solution, higher PHB content and cellular dry weight can be achieved without removing the culture broth.

Table 1. Effect of DO limitation using the agitation speed

Maximum agitation speed (rpm)	Culture time (h)	Dry cell weight (g/L)	PHB concent. (g/L)	PHB content (%)
1300 (pure O2 supplied)	35	55.5	31.6	56.9
300	70	15.2	10.6	70.1
500	52	31.2	25.1	80.2

(A)                                   (B)

Figure 1. Time profiles of dry cell weight, PHB concentration, PHB content and residual cell mass in case of (A) fermentation A and (B) fermentation B

Acknowledgements

This work was supported by the Korea-Australia International Cooperative Project from Ministry of Science and Technology.

References

Lee, S.Y. 1996. Plastic bacteria?: Progress and prospects for polyhydroxyalkanoate production in bacteria. Trends Biotechnol. 14:431-438

Choi, J. I., and S. Y. Lee. 1997. Process analysis and economic evaluation for poly(3-hydroxybutyrate) production by fermentation. Bioprocess Eng. 17:335-342

Lee, S. Y., A. P. J. Middelberg, and Y. K. Lee 1997. Poly(3-hydroxybutyrate) production from whey using recombinant Escherichia coli. Biotechnol. Lett. 19:1033-1035

Wong, H. H., and S. Y. Lee 1998. Poly(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli, Appl. Microbiol. Biotechnol. 50:30-33

Choi, J. I., S. Y. Lee, and K. Han 1998. Cloning of the Alcaligenes latus Polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of poly(3-hydroxybutyrate) in Escherichia coli, Appl. Environ. Microbiol. 64:4897-4903

Wang, F. and Lee, S. Y. 1997. Production of Poly(3-Hydroxybutyrate) by Fed-batch Culture of Filamentation-Suppressed Recombinant Escherichia coli. Appl. Environ. Microbiol. 63:4765-4769

Wang, F. and Lee, S.Y. 1998. High cell density culture of metabolically engineered Escherichia coli for the production of poly(3-hydroxybutyrate) in a defined medium. Biotechnol. Bioeng. 58:325-328

Choi, J. and Lee, S.Y. 1999. Factors affecting the economics of polyhydroxyalkanoates production by bacterial fermentation. Appl. Microbiol. Biotechnol. 51:13-21