High-efficient And Titer Lactic Acid Production From Corn Stover By Bacillus Coagulans LA204 Using Simultaneous Saccharification And Fermentation

Poly lactic acid(PLA), based on the polymerization of lactic acid, is one of the biodegradable and biocompatible polmers, and has significantly increased the global interest in lactic acid production for the green alternatives to petroleum-derived plastics. Lactic acid production from food crops would increase the conflict of the food safety, and the price of lactic acid. The lactic acid production from non-food substrate, such as agriculturaial straw, would decrease the price of lactic acid and open another way of high efficient utilization of the agriculturial straw instead of pollute the environment. In this study, we screening a Bacillus coagulans strain LA204 with high lactic acid titier and efficient from corn straw. The results of this study are showed as follows:1. A xylose homolactic fermentation microorganism was screened from soil by using bromocresol green. With the substrate of 50 g/L xylose, the lactic acid liter achieved 46 g/L with the yield of 0.92 g/g and L-lactic acid purity of 98.5%. BLAST analysis of the 16 S r DNA sequence of this microorganism against the NCBI database demonstrated 99% similarity to B. coagulans 36D1(DQ297926), thus, this strain was designated as B. coagulans LA204.2. The lactic acid production from five lignocellulosic materials in flask was studied. Miscanthus, corn stover, corn cob, wheat stover and rice stover were pretreated with 1 mol/L sodium hydroxide with a soild loading of 25% at 75℃ for 3 hs and then washed to p H lower than 8.0. The SSF(simultaneous saccharification and fermentation) prcess of five pretreated(with washed) stovers or non-pretreated stovers were carry out in 500 m L flask in 50 ℃ with 50 g/L pretreated and washed straw and 10 g/L yeast extract in an enzyme(Celic2) loading of 50 FPU/g substrate. Subsequent to fermentation, the lactic acid yield was 0.75 g/g to pretreated miscanthus as the highest and were 0.52 g/g, 0.44 g/g, 0.46g/g, and 0.49 g/g to pretreated corn stover, corn cob, wheat stover and rice stover, respectively. The lactic acid yield of non-pretreated stovers were all less than 0.3 g/g.The basic fermentation parameters of LA204 in flask were studied and the results show that,(Ⅰ) LA was able to utilize glucose, xylose and celluiose drived from lignocellulosic biomass hydrolysis;(Ⅱ) The effect of initial p H values on lactic acid production was examined and the results indicate that an initial p H of 6.0 is optimal for lactic acid fermentation by LA204;(Ⅲ) the effect of temperature on LA204 lactic acid production in both glucose and xylose media were examined and the results indicate that 50℃ was the optimum temperature;(Ⅳ) the results of the ability of LA204 lactic acid production based on different nitrogen sources showed that fermentation with yeast extract reach the highest yield.4. Lactic acid production with LA204 was examined in 50 g/L xylose and 50 g/L glucose media in a 5-L anaerobic bioreactor at a condition of 50 ℃ and p H 6.0 furtherly. The final lactic acid titer in glucose medium was 45.3 g/L with the yield and productivity were 0.9 g/g and 0.903 g/L/h respectively, and the lactic acid titer in 53 g/L xylose medium was 45.58 g/L, the yield and productivity were 0.86 g/g and 0.885 g/L/h which was little lower than in glucose.5. The initial simultaneous saccharification and fermentation experiment using 50 g/L Na OH-pretreated and washed corn stover or Na OH-pretreated and without washed corn stover as the carbon source and 10 g/L yeast extract as the nitrogen source was carried out in a a 5-L anaerobic bioreactor with the condition of 50 ℃, p H6.0 and 150 r/min and the Celic2 loading was 30 FPU/g stover. The LA titer from Na OH-pretreated and washed corn stover reached 30.41 g/L and the yield reached 0.60 g/g stover, and the average productivity was 0.496 g/L/h. But the lactic acid titer, yield and productivity from unwashed Na OH-pretreated corn stover were only 12.5 g/L, 0.25 g/g and 0.34 g/L/h respectively, which mean that the inhibitor in Na OH-pretreated corn stover had a great inhibition to the lactic acid fermentation.6. The fed-batch simultaneous saccharification and fermentation experiment of corn stover was also carry out in 5-L anaerobic bioreactor, with the fermentation condition of 50 ℃, p H 6.0 and 300 r/min. The basic substrate was 80 g/L Na OHpretreated corn stover with washed and 20 g/L yeast extract. After cultivated 18 hs, group1 feeding with Na OH-pretreated corn stover without washed, and group2 feeding with Na OH-pretreated and washed corn stover. Feeding with Na OH-pretreated corn stover without washed, the lactic acid production was inhibited, the lactic acid titer, yield and productivity were 57.3 g/L, 0.43 g/g and 0.85 g/L/h, the results showed that feed with Na OH-pretreated corn stover without washed could not derepression but only remission. On the contrary, feed with Na OH-pretreated and washed corn stover, the lactic acid titer reached a very high level in 97.6 g/L, the yield and productivity were 0.67 g/g and 1.63 g/L/h. The results indicated that fed-batch would increase the final lactic acid titer and productivity remarkable.The final concentration of L-LA in the fermentation of pretreated and washed corn stover was 98%.