Immobilized Fermentation Of L-lactic Acid With Rhizopus Oryzae

Lactic acid, as a kind of organic acid, has a wide range of applications in medicine, food, chemical engineering and so on. PLA made from lactic acid is biodegradable and environmental friendly, which can be used in medicine, tissue engineering, hospital and so on, meanwhile, PLA maybe solve the "white pollution" problem instead of traditional plastic in the future. Compared with the conventional batch fermentation, the immobilized fermentation to produce lactic acid by Rhizopus oryzae can provide many advantages, such as shorter fermentation period, higher lactic acid yield and reusable ability.A novel matrix was developed for fermentation to produce L-lactic acid by Rhizopus oryzae in shake-?asks. The matrices were made of fine mesh of stainless steel and cotton. Its spatial network structure had a good effect over Rhizopus oryzae. The author tested results of immobilized fermentation by the experiment using glucose, corn starch and cellulose as carbon source, respectively.First, the fermentation was carried out with glucose which was the conventional carbon source. During the seed culture, the concentrations of glucose and yeast extract were 50g/L and 2.5g/L, respectively. Biomass concentration was up to 6.8g/L after 24 hours. During the fermentation, the initial concentration of glucose was 80g/L, the optimized conditions of immobilized fermentation were as follows: using three matrices at 33℃, 180rpm, with volume ratio of 20%, and adding CaCO3 in time. The average L-lactic acid concentration of 50.00g/L, the yield of 65.0%, and the average productivity of 1.2g/ (L·h) were obtained after 48-hour fermentation. Compared with cell-free fermentation, the concentration of L-lactic acid, the yield and the productivity increased by 79%, 250% and 150%, respectively, in immobilized fermentation. However, the period decreased by 24 hours. The process of immobilized fermentation was in good with stability in seven repeated batch fermentation. A high lactic acid concentration of 87.30g/L was obtained from glucose in fed-batch fermentation after 140h.Second, the fermentation was carried out with corn starch as carbon source. The corn starch was hydrolyzed by two kinds of enzymes to be used in the fermentation process. During the fermentation, the concentration of the corn starch was 120g/L. The optimized conditions of immobilized fermentation were as follows: using three matrices at 33℃, 190rpm, with volume ratio of 20%, and adding CaCO3 in time. The average L-lactic acid concentration of 82.51g/L, the yield of 68.76%, and the average productivity of 1.4g/ (L·h) were obtained after 60-hour fermentation. Compared with cell-free fermentation, the concentration of L-lactic acid, the productivity and the yield increased by 76.18%, 111.40% and 76.19%, respectively, in immobilized fermentation. The process of immobilized fermentation was in good with stability in repeated batch fermentation. A high lactic acid concentration of 94.6g/L was obtained from corn starch in fed-batch fermentation after 180h.Then, the simultaneous saccharification and fermentation were carried out with cellulose as carbon source. 250mL triangle-bottle was chosed for reaction,48.8mL citrate buffer and 2g cellulose after retreated were added into the bottles,as well as KH2PO4 0.3g/L;MgSO4 .7H2O 0.25g/L;ZnSO4 .7H2O 0.08g/L,thenall of them were sterilized with 121℃for 20min,enzymatic hydrolysis carried with 1.2mL cellulase on 50℃for 24h.Then,put the biomass immobilized by two matrices into the former media. The optimized conditions of immobilized fermentation were as follows: 35℃, 200rpm, with solid-liquid ration of 0.04, and adding CaCO3 for adjusting pH. The average L-lactic acid concentration of 16g/L, the yield of 40%, and the average productivity of 0.11g/ (L·h) were obtained after 120-hour fermentation.At last, immobilized fermentation by matrices were carried out in a bubble-column bioreactor. After 6h , all of the spores were adsorbed to the matrices. Under the way of single factor, the optimization of the fermentation conditions were as follow: aeration was 0.5vvm, temperature was 32℃,adding CaCO3 after 4h.In the 9-batch fermentation, the average of lactic acid concentration, yield, productivity were 54.86g/L, 80%, 0.57 g/L.h, respectively. Compared with cell-free fermentation, L-lactic acid concentration, the yield increased by 78% and 78.2%, respectively, the time for fermentation extended by 36h.