Lactic Aicd Production By Bacillus Coagulans And The Study Of Its Cellobiose Degradation Mechamism

The utilization of lignocellulosic biomass, an economical and renewable feedstock, could efficiently reduce the production cost of current industrial lactic acid production. In the present study, the enzymatic hydrolysis effeciency of bagasse sulfite pulp(BSP) was studied. Then Bacillus coagulans strain CC17 was employed to investigate the optimum condition of simultaneous saccharification and fermentation(SSF), and the SSF process was optimized and magnified at high substrate loading. As it was firstly found that no ?-glucosidase is needed in SSF with strain CC17, the cellobiose metabolic mechanism was preliminarily discussed in this thesis. It laid a foundation of the efficient utilization of lignocellulosic rawmaterials.The BSP contained 73.84% cellulose, 14.33% hemicellulose and 5.93% lignin. When the celluclast 1.5L and Novozyme 188 loading were 15 FPIU/g cellulose and 7.5 pNPGU/g cellulose,50 g/L glucose and 9.46 g/L xylose was obtained from 80 g/L BSP by 72 h hydrolysis. The enzymatic hydrolysis yield was 61.45%. Optimization of SSF by strain CC17 showed that the optimum fermentation medium contained YE 2.5 g/L, corn steep powder 1.2 g/L and(NH4)2SO4 5 g/L. Moreover, no ?-glucosidase was needed in SSF. Meanwhile, the addition of xylanase contributed to lactic acid production. 60 g/L BSP could produce 56 g/L lactic acid with 10 FPIU/g cellulose of cellulase and 120 U/g hemicellulose of xylanase after 72 h fermentation.Under high substrate loading, fed-batch process could effectively relieve the difficult of liquefaction. The fed-batch SSF experiment was started with 60 g/L cellulose. And additional 6 g cellulose was added at 36 h, giving a final cellulose concentration of 98.9 g/L. The fermentation conditions, enzyme loadings, and calcium carbonate were the same as in the batch experiments and they were added along with cellulose adding. After 144 h fermentation, the lactic acid production reached 69.2 g/L. Similiar lactic acid production was produced with cellulase from Youteer company instead of celluclast 1.5L. Based on the initial cellulose concentration of 30 g/L, when additional 3 g cellulose was added for 7 times during the first 48 h and all the enzymes and calcium carbonate were added initially, the lactic acid production in shake flask could reach 110 g/L while the yield reached 0.72 g/g cellulose. The scale-up SSF was conducted in 3 L NBS fermentor and the maximum production was 102.8 g/L. Meanwhile,the yield reached 0.80 g/g cellulose?Bacillus coagulans strain CC17 could directly metabolize cellobiose to produce lactic acid. 40 g/L cellobiose can produce 35 g/L lactic acid and the sugar-acid conversion reached 87.5% by 30 h fermentation. Enzymes which can cut off ?-1,4-glucosidic-bond must existed in B.coagulans CC17. The studies on degradation mechamism of cellobiose in B.coagulans CC17 demonstrated that no ?-glucosidase was discovered whether in vivo or vitro. But a hypothetical protein BCBGL1 from B.coagulans CC17 seemed to have an activity of 6-phospho-?-glucosidase. At least two potential cellobiose operons were found in the genome-wide of strain CC17. One of them contained the whole 6-phospho-?-glucosidase and PTS translocator. Moreover, it could be highly expressed with the induction of cellobiose. Thereby, we think an assumed pathway of cellobiose degradation, containing the phosphotransferase system(PTS) cellobiose uptake system and a phospho-?-glucosidase(catalyzing the hydrolysis of cellobiose-6-P into glucose and glucose-6-P), was firstly discovered in B.coagulans CC17.