Study On Breeding High - Yielding Grease And Carotenoid Strains By Protoplast Fusion In

Oil producted by microbial is an important source of biodiesel. In our laboratory we pre-screened a strain of red yeast Rhodotorula glutinis with high content of oil and carotenoid. To further improve the content of oil, we focus on fusion breeding within species and further optimizing the fermentation conditions. Firstly, this experiment is to optimize the factors, in which R. glutinis protoplast formed and regenerated, and breed the strain with high content of oil through intraspecies protoplast fusion technology. The optimum conditions for R. glutinis forming and regenerating protoplast were as follows:10h culture strains,0.7 mol/L NaCl osmotic stabilizers with pH value 6.0 washing twice for regenerating protoplast,2% snailase and 1.0% cellulase as enzyme solution incubating 35℃for 30 min at rotation speed 120 r/min,0.6 mol/L mannitol osmotic stabilizers with pH value 6.0 washing twice.Through optimizing the factors,R. glutinis protoplast formation rate and regeneration rate reached 83.34% and 78.12%,respectively.Under the optimizing conditions,R. glutinis protoplasts were fused with 40% PEG(MW=6000).A fused strain No.7 strain was screened which contained high oils and carotenoids, the lipid content of which accounted dry cell weight for 68.42% increased 7.42% compared with the parent strain. While the biomass of the No.7 strain increased 1.04 g/L than the parent strain, the carotenoid content of the No.7 strain was 1.65 times compared with the parent strain.The features of highly producing oils and carotenoids were stable.However, the contents of oil and carotenoid of the No.7 strain need to be further improved by optimizing the fermentation conditions.The best culture conditions for producing oil by the No.7 fused strain as the start strain were studied using uniform design principles and single factor experiments. The optimal conditions were as follows:sucrose as the optimal carbon source at the concentration of 40g/L,12g/L yeast extract and 3 g/L peptone as the optimal nitrogen source, temperature 28℃, pH value of culture medium 6.0,5% inoculum,rotation speed at 120 r/min, incubation for 3 days.Then the strain was cultured in 5L fermentor.Then the largest oil yield of the strain got to 11.28 g/L (6d).The maximum biomass and lipid content of the strain reached to 16.80 g/L and 69.18%,which was increased by 19.00% and 8.18% than the parent strain, respectively.After optimizing the fermentation conditions,the biomass of the No.7 strain was increased by 10.96% than the start strain.While the No.7 strain highly produced oil, it was found that the carotenoid of the No.7 strain was also increased.The effects of culture conditions on the carotenoid yield production were investigated using uniform design principles and single factor experiments. The optimal conditions were as follows:sucrose as the optimal carbon source at the concentration of 40 g/L,12 g/L yeast extract and 3 g/L peptone as the optimal carbon source, temperature 28℃,pH value of culture medium 6.0, inoculation 30%,rotation speed at 150r/min,incubation for 3 days. Then the strain was cultured in 5L fermentor. The No.7 strains reached the maximum carotenoid yield of 8.09 mg/L (5d). Thus after optimizing, the carotenoid yield is 56.24% more than the start strain.