Preliminary Studies On Mutation Breeding Of Chlorella Pyrenoidosa For High Lipid Productivity By ⁶⁰Co-? Irradiation

With the depletion of energy and environmental pollution,biofuels gradually get into our sight.With the characteristic of high lipid productivity,biofule from microalgae has been one of the hotspots of bioenergy researches,and mutation breeding of microalgae for lipid-producing has become a hotspot research direction.In this paper,the two-time mutation breeding of a commercial strain Chlorella Pyrenoidosa by 60Co-?was studied.Based on microalgae culture methods,high throughput screening method was successfully applied to the mutation breeding.In addition,in order to preserve the good characters of microalgae strains,a cryopreservation method for microalgae was studied,which would provide the experimental basis for microalgae preservation in the future.First,a method of secondary mutagenesis of Chlorella Pyrenoidosa using 60Co-? was established,and 5000 mutant strains were obtained.Second,the high-throughput screening method was established for Chlorella Pyrenoidosa.In the 96-well black plate,100?L of the original culture was added to each well,and then 97 ?L of 100%DMSO was added.After 25 minutes,3 ?L of 30 mg/L Nile Red dye stained for 5 minutes.The microplate was readed under 37? and shocked for 3min.The fluorescence intensity was detected under excitation wavelength of 485nm and the emission wavelength of 535nm,the starting algae as a contrast simultaneously.Third,four mutants with high lipid content were obtained by screening from 500060Co-? mutagenesis strains of Chlorella Pyrenoidosa using the established high-throughput screening method.Finally,the four mutants were preliminary cultured under HS-PC model and PS-PC model in 1L bubbling column reactor.Two of the best algae strains(CP-232 and CP-269)which are the highest lipid content of the four mutants were further validated in 3L and 15L bubbling flatplate bioreactors.The results showed that the lipid produtivity of two mutants(CP-232 and CP-269)were increased by 19.8%,23.8%,21.6%and 13.8%,respectively,in 3L and 15L bubbling flatplate bioreactor compare with the initial strain.In addition,the effects of DMSO and glycerol on the cryopreservation of algal cells were studied.The results show that the higher the concentration of cryoprotectant,the greater the cell damage in cryopreservation.In one-year perservation period,the longer the conservation time,the more serious cell damage.DMSO cryopreservation is better than glycerol,and the concentration of 5%of the DMSO cryopreservation is the best in 5%,7%,10%DMSO and 14%,20%,27%glycerol six cryopreservation methods with the smallest cell damage.