| L-isoleucine (ILE) is one of the eight essential amino acids for humans. With the increasing demand of L-isoleucine market, it is very crucial to improve ILE productivity effectively. In this thesis, Corynebacterium glutamicum YILW, which could accumlate extracellular L-isoleucine, was used for L-isoleucine fermentation. A series of feasible strategies were carried out to improve L-isoleucine yield, substrate conversion ratio, and productivity. The main research and results are as follows:Response surface methodology was applied to optimize the culture composition for ILE fermentation by C. glutamicum YILW, based on the level of shake flask. It showed that three factors played the decisive roles in the medium, including corn steep liquor, soybean protein hydrolysate and biotin. The optimal concentrations of the variables were determined as:corn steep liquor 34.0 mL/L, soybean protein hydrolysate 8.5 mL/L, biotin 6.0 mg/L. Based on the 5 L fermentor fed-batch cultivation, the ILE production (8.98 g/L) was increased by 11.83%.The effects of different organic nitrogen sources for the fermentation of ILE were studied based on the level of 5 L fermentor. It was found that different organic nitrogen sources in seed medium had significant impact on ILE synthesis. And ILE production could reach the highest production about 19.12 g/L when corn steep liquor and yeast were selected as the organic nitrogen source in seed medium. Corn steep liquor was selected as the appropriate nitrogen source instead of soybean hydrolysates to cut down the cost for fermentation medium with appropriate concertration of biotin in the process of fermentation. Compared with the original conditions, the biomass and ILE production increased by 20.68% and 35.41% respectively.Fed-batch culture was performed to study the influence for fermentation with an exponential feeding strategies of the growth limited nutrients. Based on the analysis of the curve about the whole process of fermentation, a performance of fedding substrate including core steep liquor and phosphate, combined with the two-stage dissolved oxygen control strategy was carried on. With dual exponential feeding combining with two-stage dissolved oxygen control strategy, the biomass and maximum ILE production reached 32.29 g/L and 31.32 g/L after 60h, increased by 20.68% and 35.41% respectively. Compared to the control, the enhancements of ILE production was 20.97%. The results proved that fedding core steep liquor and phosphate in the growth phase could stimulate the growth and extend the phase to produce ILE. A higher level of dissolved oxygen was benefit for cell growth and a lower level of dissolved oxygen was in favor of producing ILE.An investigation about the effects of two types of surfactants, Tween-60 and Tween-80 were carried out growth and L-isoleucine (ILE) production by Corynebacterium glutamicum YILW. Shaking flask tests showed Tween-80 played a key role in cell growth and ILE production. The suitable concertration of Tween-80 was 2.0 g/L with adding it at 18h. As a result, L-isoleucine yield on cell was increased by 13.3%. Furthermore, the effect of Tween-80 on cell growth and ILE production were also investigated in a 5 L fermentor. The concentration of ILE increased from 23.89 g/L to 26.43 g/L by adding Tween-80 at 18 h. Those results indicated that Tween-80 enhanced ILE production, and it was good for branched chain amino acid fermentation.ILE fermentation with an combination of the above optimization conditions showed ILE production increased to 35.66 g/L with a biomass of 38.15 g/L. Compared to optimal batch culture, ILE production and biomass were enhanced by 440.08% and 367.88%, respectively. This result indicated the unification of high ILE production, high carbon transformation rate and high productivity could be achieved by optimize the fermentation conditions of ILE. |
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