Primary Study On Production Of L-Lactic Acid By High Cell Density And Semi-continuous Fermentation With Rhizopus Oryzae

High cell density and semi-continuous fermentation of microorganism can achieve such a result of shortening the fermentation time and reducing the cost by increasing the cell density and volumetric yields efficiency. By the study of high cell density culturing of Rhizopus oryzae and production of L-lactic acid by semi-continuous fermentation, the culture medium ingredient and fermentation condition were optimized, the optimum method and parameter of fed-batch culture were determinated, the fermentation strength and the kinetics were researched as well. The main results were as following:(1) The culture medium ingredient and fermentation condition were optimized by the combination of single factor experiments and orthogonal experiments. The results were as following: When the key parameters being glucose 160g/L, (NH₄)₂SO₄ 6g/L, NaH₂PO₄ 0.150g/L, KH₂PO₄ 0.150g/L, MgSO₄·7H₂O 0.25g/L , ZnSO₄·7H₂O 0.22g/L; Inoculum size was 5%, inoculums age was 24h, and work volume was 40%;the biomass of Rhizopus oryzae in the first batch of fermentation could achieve 12.503 g/L。(2) The biomass of Rhizopus oryzae were studied when culture medium were feeding in constant speed, in different speed and feedback by the concentration of glucose which was proved to be the best strategy. The parameters of feeding culture were optimized as follows: the glucose/ammonium sulfate ratio was 20:1(w/w), culture medium feeding speed was feedback by the concentration of glucose from 24h to 60h, the initial work volume was 7L in 15L bioreactor, temperature was 32℃, Rotation rate was 200 r/min, aeration velocity was 2 L/min per liter. The yield of L-lactic acid is 117 g/L; the biomass is 18.54 g/L, which increased by 91.9% over that of batch culture.(3) The ferment method and the culture medium ingredient in different phase were optimized. Adopt feedback-feeding culture to increase biomass in the first batch of fermentation. Culture mediumâ…¡was added to the reactor from 2^nd to 5^th batch of fermentation to increase biomass further which was composed of glucose100g/L, (NH₄)₂SO₄ 4g/L,KH₂PO₄ 0.150g/L,MgSO₄·7H₂O 0.22g/L and ZnSO₄·7H₂O 0.33g/L. Culture mediumâ… was added to the reactor from 6^th batch of fermentation to the production of L-lactic acid which was composed of glucose100g/L, (NH₄)₂SO₄ 2g/L, KH₂PO₄ 0.10g/L, MgSO₄·7H₂O 0.22g/L, ZnSO₄·7H₂O 0.33g/L. The fermentative strength is 3.045g/(L·h) in average.(4) Kinetic models of L-lactic acid production by high cell density culture of Rhizopus oryzae were established.The model of Rhizopus oryzae growth : (?)The model of L-lactic acid yield : (?)The results showed that the models could describe the process exactly.