湖泊红球藻等离子诱变及其高产虾青素藻株培养条件的优化

Plasma Mutagenesis of Haematococcus lacustris and Optimization of Culture Conditions for High-yield Astaxanthin Algae Strains

为进一步提高湖泊红球藻(Haematococcus lacustris)的工业利用价值,本研究使用常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变仪对湖泊红球藻进行等离子诱变。以藻细胞致死率为指标确定等离子诱变的适宜输入功率和诱变时间。诱变之后通过固体平板培养初筛和液体培养复筛获得高产虾青素的突变藻株。再以藻细胞密度为指标采用单因素实验及正交试验对高产藻株的营养生长阶段的培养条件进行优化,并筛选虾青素诱导阶段适宜虾青素积累的高光照条件。在优化的培养条件下多次继代后观察高产突变藻株的遗传稳定性。结果表明,湖泊红球藻等离子诱变的适宜条件是功率240 W、诱变时间150 s或功率400 W、诱变时间120 s。通过初筛和复筛获得生长快、虾青素产量高的突变藻株有11株,其中编号为HP3的突变藻株生长最快、虾青素产量最高,培养后藻细胞密度和虾青素产量较出发株分别提高了25.5%和61.6%。经过两阶段培养条件的优化,HP3的藻细胞密度和虾青素产量较优化前分别提高了14.3%和19.3%,达7.2×10^(5)cell/mL和31.264 mg/L。湖泊红球藻高产突变藻株HP3多次继代后藻细胞生长良好,遗传稳定,培养后藻细胞密度和虾青素产量均与初代培养相近。研究结果对湖泊红球藻产虾青素工业藻株的选育具有实际意义。

To further enhance the industrial utilization value of Haematococcus lacustris,the plasma mutagenesis of Haematococcus lacustris was carried out by an atmospheric and room temperature plasma(ARTP)mutagenesis equipment.The optimum input power and mutagenesis time for plasma mutagenesis were determined with lethal rate of algal cells as the index.After mutagenesis,high-yield astaxanthin mutant algae strains were obtained through primary screening of solid plate culture and secondary screening of liquid culture.Then,the culture conditions of high yield algal plants at vegetative growth stage were optimized by single-factor and orthogonal experiment with algae cell density as the index,and the suitable high light conditions for astaxanthin accumulation during astaxanthin induction stage were selected.The genetic stability of the high yielding mutant algae strains was observed after multiple subcultures under the optimized culture conditions.The results showed that the optimum conditions for plasma mutagenesis of Haematococcus lacustris were 240 W for 150 s or 400 W for 120 s.11 Mutant alga strains with fast growth and high astaxanthin yield were obtained through primary screening and rescreening,wherein the strain HP3 grew fastest and had the highest astaxanthin yield.After culture,its cell density and astaxanthin yield were increased by 25.5%and 61.6%respectively compared with the original strain.After two-stage optimization,the cell density and astaxanthin yield of HP3 increased by 14.3%and 19.3%respectively,reaching 7.2×10^(5) cell/mL and 31.264 mg/L.HP3 showed good growth and stable heredity.Its cell density and astaxanthin yield were similar to those of primary culture.The results have practical significance for the breeding of industrial algal strains producing astaxanthin from Haematococcus lacustris.