Studies on the production of poly(3-hydroxybutyric acid) [P(3HB)] and poly(3-hydroxybutyric-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)] by high cell density culture of Alcaligenes eutrophus and recombinant Escherichia coli were carried out to improve the fermentation productivity.
Performance characteristics of P(3HB) production by A. eutrophus in a CSTR, two-stage CSTR, membrance cell recycle reactor(MCR), and two-stage MCR-CSTR were simulated. The kinetic model and parameter values of batch cultures of A. eutrophus suggested by Heinzle and Lafferty(1980) were used to describe the reactor performances. The steady state concentrations of cell, P(3HB), residual biomass and limiting substrate($NH_4^+$) were calculated as a function of dilution rate and bleed ratio when the feed concentration of $(NH_4)_2SO_4$ was 2.3 g/L. The maximum productivity of CSTR was 0.19 g/L/h at the dilution rate of 0.05 $h^{-1}$. In membrance cell recycle reactor, the concentration of cells and P(3HB) contents decreased with increasing bleed ratio and dilution rate. At the bleed ratio of 0.1, the maximum productivity was 2 g/L/h, which was 10 times more productive than the CSTR. The maximum productivities of two stage CSTR and two-stage MCR-CSTR were 0.23 and 2.3 g/L/h, which were 20% and 15% higher than those of one-stage system, respectively.
A recombinant E. coli strain XL1-Blue ($KS^-$) harboring the P(3HB) biosynthesis genes from A. eutrophus was used to produce P(3HB) by pH-stat fed-batch culture in complex medium. Initial glucose concentration for optimal growth was found to be 20 g/L from a series of flask culture. A final P(3HB) concentration of 89 g/L could be obtained after 42 h of cultivation. The pH-stat fed-batch culture of recombinant E. coli K12(pSYL 104) using defined medium was not suitable for efficient production of P(3HB) due to the formation of acetic acid. Exponential feeding fed-batch culture of E. coli K12(pSYL 104)achieved high cell concentration (101 g/L) by controlling the specific growth rate as low as 0.1 $h^{-1}$. However, the P(3HB) content was very ow because of the different conditions for cell growth and P(3HB) production.
A eutrophus NCIMB 11599 was cultivated to produce P(3HB) from glucose by two-stage fed-batch culture technique. The glucose concentration of the culture broth was controlled at 10-20 g/L by two methods : using exit gas data obtained from a mass spectrometer and using an on-line glucose analyzer. The effect of ammonium limitation on P(3HB) formation at different culture phases was studied. The final cell concentration, the P(3HB) concentration, and the P(3HB) productivity increased as ammonia feeding was stopped at a higher cell concentration. High concentrations of P(3HB) (121 g/L) and total cells (164 g/L) were obtained in 50 h when ammonia feeding was stopped at the cell concentration of 70 g/L. The maximum P(3HB) content reached 76% of dry cell weight and the productivity was 2.42 g/L/h with the yield of 0.3 g P(3HB)/g glucose.
The metabolic pathway of P(3HB-co-3HV) synthesis from propionic acid was theoretically analyzed. The biochemical yield of P(3HB-co-3HV) from propionic acid and glucose/propionic acid and glucose/propionic acid were 0.41 and 0.43 (g PHA/g substrate), respectively. The 3-hydroxyvaleric acid (3HV) fraction in the copolymer was predicted with the ratio of propionic acid to glucose (P/G ratio) in medium.
P(3HB-co-3HV) was produced from glucose and propionic acid by fed-batch culture of A. eutrophus. The glucose concentration of the culture broth was controlled at 10-20 g/L using an on-line glucose analyzer during the whole culture period. Nitrogen limitation was applied at the cell concentration of 60-70 g/L, when the glucose feed was replaced by the mixture of glucose and propionic acid. The effect of the P/G ratio in the feeding solution on the copolymer production was examined. The final copolymer concentration of 117, 74 and 64 g/L, polymer content of 74, 57, and 56.5% of dry cell weight, and productivity of 2.55, 1.67, and 1.64 g/L/h were obtained when the P/G ratio in the feed was 0.17, 0.35, and 0.52 (mol propionic acid/mol glucose), respectively, showing decreasing tendency with increasing P/G ratio. However, the 3HV fraction in the copolymer increased with increasing P/G ratio, resulting in 14.3 mol % of 3HV at the P/G ratio of 0.52(mol/mol). The concentration of propionic acid in the culture broth was maintained below 1.3 g/L when the P/G ratio in the feed was 0.17 or 0.35(mol/mol), while it increased gradually when using the ratio of 0.52(mol/mol).
Alcaligenes eutrophus와 재조합 Escherichia coli의 고농도 배양을 통해 P(3HB) 및 P(3HB-co-3HV)의 발효 생산성을 향상시키기 위한 연구를 수행하였다. 연속 반응기 (CSTR), 2단 연속 반응기, 막재순환 반응기 (MCR), 2단 막재순환-연속 (MCR-CSTR)에서 A. eutrophus 의 P(3HB) 생산 성능을 해석하였다. Heinzle와 Lafferty (1980) 가 제안한 속도식을 사용하여, 제한기질인 $(NH_4)_2SO_4$의 공급 농도가 2.3 g/L일 때 정상상태의 균체 농도, P(3HB) 농도, 잔여균체 농도, $NH_4^+$ 농도를 희석율과 블리드비의 함수로 계산하였다. CSTR의 최대생산성을 희석율 0.05 $h^{-1}$ 에서 0.19 g/L/h였다. 막재순환 반응기에서의 균체농도와 P(3HB) 함량은 블리드비와 희석율이 증가할수록 감소하였으며, 블리드비 0.1에서 CSTR 보다 10배 향상된 2 g/L/h의 최대 상산성을 얻을 수 있었다. 2단 CSTR과 2단 MCR-CSTR의 최대 생산성은 1단 CSTR, 1단 MCR보다 각각 20 %,15 % 향상된 0.23 g/L/h와 2.3 g/L/h였다.
A. eutrophus의 p(3HB) 생합성 유전자를 포함하는 재조합 E. coli XL1-Blue $(KS^-)$의 pH-stat 유가식 배양을 complex 배지를 사용하여 수행하였다. 균체성장에 최적인 초기포도당 농도는 20 g/L임을 FLASK 배양을 통해 알 수 있었으며, 이룰 토대로 유가식 배양을 실시하여 42 시간에 89G/l의 p(3HB) 농도를 얻었다. Defined 배지를 이용한 재조합 E. coli K12 (pSYL 104)의 pH-stat 유가식 배양은 아세트산의 생성으로 인해 P(3HB) 생성에 적합하지 않았다. 비성장 속도를 0.1 $h^{-1}$로 낮게 제어한 E. coli K12 (pSYL 104)의 exponential feeding 유가식 배양을 통해 101g/L의 고농도 균체를 얻을 수 있었으나 P(3HB) 함량은 매우 낮았다.
고농도의 P(3HB)를 생산하기 위해 a. eutrophus NCIMB 11599의 2단 유가식 배양을 수행하였다. 배양액중의 포도당 농도를 균체성장과 P(3HB) 합성에 최적인 10-20 g/L로 제어하기 위해 mass spectrometer의 CER data를 이용하는 방법과 pn-line 포도당 분석기를 이용하는 방법을 제안하였다. 높은 균체 농도에서 질소원을 제한할수록 최종 균체 농도, P(3HB)생산성과 수율은 각각 2.42g/L/h와 0.3 (g(3HB)/g포도당)이었다.
프로피온산으로부터 P(3HB-co-3HV)가 합성되는 대사 경로를 이론적으로 해석하였다. 프로피온산과 포도당/프로피온산 혼합 기질로부터 생성되는 P(3HB-co-3HV)의 생화학적 수율은 가각 0.41과 0.43(g PHA/g 기질)이었다. 생성되는 P(3HB-co-3hv)의 3HV 함량을 배지중의 초기 프로피온산과 포도당의 비 (P/G비)로부터 예측할 수 있었다.
포도당과 프로피온산으로부터 P(3HB-co-3HV)를 생산하기 위해 on-line 포도당 분석기를 이용한 A. eutrophous의 유가식 배양을 수행하였다. 배양액중의 포도당 농도는 on-line 포도당 분석기를 이용하여 전시간에 걸쳐 10-20 g/L로 유지하였다. 균체농도가 60-70 g/L에 이를 때까지 포도당만 공급하다가, 이후 질소원을 제한하면서 포도당과 프로피온산의 혼합 용액을 공급하여 3HV 생성을 유도하였다. 공급 기질중의 P/G비에 따른 P(3HB-co-3HV) 생성 효과를 조사하였다. P/G 비가 각각 0.17, 0.35, 0.52 (mol 프로피온산/mol 포도당)일때 최종 P(3HB-c0-3HV)농도 117, 74, 64 g/L, P(3HB-co-3HV)가 합량 74,57,56.5 %, 생산성 2.55, 1.67, 1.64 g/L/h를 각각 얻었다. P/G비가 증가할수록 3HV 함량은 14.3 mol %까지 직선적으로 증가 하였다. 배양액중의 프로피온산 농도는 P/G 비가 0.17, 0.35 (mol/mol)일 때는 1.3 g/L 이하로 유지되었으나, P/G 비 0.52 (mol/mol)에서는 점점 증가하는 경향을 보였다.
