Breeding, Fermentation Condition Optimization And Metabolic Flux Analysis Of D-lactic Acid Over-production Strain

The lactic acid is one of the most important three organic acid in the world at present, it exists in many kinds of food, and it can be produced by a lot of organisms. There are two kinds of isomers of the lactic acid, they are D-lactic acid and L-lactic acid. D-lactic acid is considered to be a very important chemical compound as a chiral material or intermediate in organic synthesis, D-lactic acid is a prosooma of many chiral compounds with applications not only in the pharmaceutical and cosmetic industry but also in agriculture, for example, in highly efficient and lowly toxic pesticide and herbicide.In this paper, the strain SP-BME126 was treated with ultraviolet radiation for three rounds, and a mutant named SP-MD1 was selected with the bromocresol purple of 0.015%, and the production of D-lactic acid reached 78.1g/L, which is higher 7.1% than that of SP-BME126. The mutant is genetically stable.The effects of fermentation conditions on D-lactic acid formation were investigated by single factor experiments, including carbon source, nitrogen source, calcium carbonate, seed age, inoculum concentrationm, medium volume and temperature. Then we evaluated the effects of 7 factors during fermentation by Plackett-Burman design and picked out three most important ones that affected the D-lactic acid productivity the most, which were further optimized followed by steepest ascent and response surface methodology(RSM). The obtained optimal condition was: glucose 95g/L, peptone 25g/L, calcium carbonate 4g/100mL, seed age 24h, inoculum concentrationm 5%, medium volume 112mL/500mL, and temperature 35℃. Under this condition, strain SP-MD1 could produce D-lactic acid 80.5g/L, and the amount of glucose being transformed into D-lactic acid being 84.7%.In this paper, we used metabolic flux analysis to probe the character of SP-BME126 and SP-MD1 at different time of fermentation. We found the fluxes of acetate acid decreased and the decreasing range reached 70%, the fluxes of lactic acid increased greatly and more glucoses were used to transformed into D-lactic acid. The pyruvate was the node in the lactic acid fermentation metabolic pathway. This is very important for the further research work.