Isolation, Identification And Breeding Of L-malic Acid Yielding Rhizopus Delemar Strain

L-malic acid (L-malic acid, referred to as LMA) is the intermediate metaboli-te in the tricarboxylic acid cycle of cells. It is mainly used in food additives. As the third-largest common food acid in the whole world, L-malic acid is widely used in medicine, cosmetics and other drugs that treat liver dysfunction. In this paper, a large amount of screening work was carried out for suitable strains in preliminary experiments. Based on the obtained strains with certain acid production, researchers started to analyze and optimize the fermentation process, then studied on the metabolic pathway, related enzymes, metabolic flux, and screened new strains by using mutation breeding which can wake the ways of generating by-products in the metabolic process so that to improve the synthetic metabolism effect.The main results are as follows:(1 preliminary screened strain with certain ability of producing malic acid in the factory fermentation wastewater and further confirmed by physiological characteristics was named as Rhizopus delemar HF-119 after determining the homology. Then 22.52 g/L of L-malic acid was yielded based on the screened strain, which proved that the strain has certain ability to produce L-malic acid.(2)On the basis of the single factor experiment results, the culture medium of L-malic acid fermentation by Rhizopus delemar strain was optimized by response surface methodology. The optimal medium components are as follows:glucose 100 g/L, ammonium sulfate 4.0 g/L, magnesium sulfate 0.30 g/L, ferrous sulfate 0.025 g/L, dipotassium hydrogen phosphate 0.5 g/L, zinc sulfate 0.1 g/L and calcium carbonate 80 g/L. In this experiment, the yield of L-malic acid was 34.87 g/L.(3)Stirred tank was employed to improve the experimental process. The optimum fermentation condition was obtained by single factor experiment: temperature of 32℃, stirrer tank ventilation of 3.0 L/m, rotation rate of 500 rpm, spore suspension incubated for 48 hours, continuous synthesis carried out after 48 hours during the fermentation process and incubation time of 72hours and added 25 g/L xylose inco-ferment 6C and 5C sugars. In this experiment, the yield of L-malic acid was 61.71 g/L.(4) UV radiation combined with spreading onto the plate medium which contained high concentrations of allyl alcohol can reduce by-product ethanol. The strain was named as Rhizopus delemar HF-120. The analysis of the malic acid flux showed the malic acid flux of Rhizopus delemar HF-120 was 1.389 mmol/(g DW h), and the malic acid flux of Rhizopus delemar HF-119 was 1.057 mmol/(g DW h). UV radiation combined with spreading onto the plate medium which contained high concentrations of fluoroacetate for reducing by-products of fumaric acid and succinate, and elected the most energetic strain as mutant strain which was named as Rhizopus delemar HF-121. The malic acid flux of Rhizopus delemar HF-121 was 2.163 mmol/(g DW h).