Screening Of Heat-resistent Rhizopus Oryzae Strain Producing L-lactic Acid And Optimization Of Fermentation Conditions

Rhizopus oryzae As3.819 was mutagenized with Co⁶⁰γ-irradiation, after domesticated in seed medium by temperature gradient, and then first screened with bromocresol green plate, second screened with fermentation. Mut-5 was screened out, which was resistant to high temperature of 38℃. And its L-lactic acid yield was 91.20 g/L, increased 24.15 % compared with parent strain. Hereditary stability test demonstrated that the productivities of mut-5 from 1^st to 7^th serial passage were stable.By unifactor and orthogonal design of experiment, the fermentation conditions of the mut-5 were optimized with flask. The results showed that the optimum fermentation medium were glucose 120 g/L, (NH₄)₂SO₄ g/L, KH₂PO₄ 0.3 g/L, ZnSO₄·7H₂O 0.2 g/L, MgSO_４·7H₂O 0.4 g/L, CaCO₃ 60 g/L. Under the condition that the fermentation temperature was 38℃, the optimum culture conditions were inoculums volume 10 %, liquid volume 20 %, rotating rate 200 r/min. The L-lactic acid yield of mut-5 reached 92.84 g/L.In order to preliminary study energy metabolism of lactic acid biosynthesis, the research was focused on effects of temperature on the F₁Fo-ATPase activity, intracellular energy charge (EC) and oxidation-reduction state of mut-5 and parent strain respectively. The results showed that energy charge and the FiFo-ATPase activity was a positive correlation, and oxidation-reduction state and energy charge was a negative correlation. In 38℃, the F₁Fo-ATPase activity of mut-5 was increased 42.37 % compared with parent strain; and higher obviously than the F₁Fo-ATPase activity of parent strain in 32℃.N,N'-dicyclohexylcarbodiimide (DCCD), which is the specific inhibitor of F₁Fo-ATPase, had effects on F₁Fo-ATPase activity and L-lactic acid producing. F₁Fo-ATPase activity was researched in fermented 48 h and it was found that F₁Fo-ATPase activity of mut-5 without DCCD was higher than parent strain without DCCD in 38℃, when the DCCD was added, the F₁Fo-ATPase activity of mut-5 was decreased by 20.84 %. The concentration of DCCD had effects on mut-5 fermentation producing lactic acid. The L-lactic acid yield of mut-5, which added the concentration of DCCD was 0.8 mmol/L, was 15.59 % lower than the L-lactic acid yield of mut-5 without DCCD. Therefore, with the enhancement of F₁Fo-ATPase activity of mut-5, the amount of intracellular ATP increased, and the resistance of cell on external environment enhanced, resulting in improving the tolerance of mut-5 to temperature, and guaranteeing the L-lactic acid production.