Mutagenic Breeding Of High Oil Yielding And Heterotrophic Chlorella ARTP And The Reuse Of The Supernatant Of Its Fermentation Broth

In this study,Atmospheric Room Temperature Plasma?ARTP?mutagenesis technique was used to mutate and breed the Heterotrophic Chlorella,in order to obtain the excellent algae with high oil production.The supernatant of its fermentation broth can be used as mixture water to recycle the nutrients in supernatant,which can effectively reduce the cost and environmental pollution.Therefore,a systematic study was carried out on Chlorella choosing,recycling of the supernatant and the fermentation process optimization,which was divided into the following parts:?1?The strains with high genetic stability and oil content were screened by ARTP mutagenesis;?2?The best harvest time was determined;?3?The recycling percentage of the supernatant is studied;?4?The nutrient composition of Yeast Powder,K₂HPO₄,MgSO₄ and microelement was optimized;?5?The fermentation process parameters of pH value,temperature,loading volume,and illumination intensity were optimized;?6?And the results were verified by a 50 L fermenter amplifying test.The ARTP mutagenesis method was used to mutate the heterotrophic Chlorella.The optimal mutagenic time was 50 s with the irradiation time as the independent variable and the fatality rate of 90%as the threshold.In the plate culture medium,the color of the bacterial colony was used as the initial screening index.Finally,the oil in the algal cells was extracted at the end of the shaking bottle fermentation stage and the concentration was measured.The candidate algae strain was used as the starting algae strain to continue mutagenesis screening.The results showed that under the condition of ARTP mutagenesis,the screening rate of the mutant was 0.8%,and the oil content of the mutant was 5.4%higher than that of the original strain.It was also determined that the optimal collection period for the fermentation of heterotrophic chlorella in the shaking table was 108 h in the stable period,and the unit bacterial mass could reach the maximum value of 11.2 g/L.The selected target algae strains were used as the source of algae species,and the supernatant of fermentation broth was used as the water for ingredients.The fermentation formula was optimized through the reuse ratio test,the single-factor test and the L₉?3⁴?orthogonal test.The single factor experiment was used to optimize the fermentation parameters,and the feasibility of culturing heterotrophic chlorella with supernatant was investigated.The results showed that the fermented supernatant recycling proportional accounted for 30%of total fermented liquid when processing effect is best.On this basis the fermentation medium and fermentation process parameter were optimized.The results were as follows:2 g/L yeast powder,0.3 g/L K₂HPO₄,0.2 g/L MgSO₄,0.5 mL mother liquor of trace element,pH 7,fermentation temperature 30?,50%loading volume,light intensity is 7000 lx.Took the selected target algae strains as sources of algae filament.Fermented supernatant recycling proportional is 30%.With 2 g/L yeast powder,0.3 g/L K₂HPO₄,0.2g/LMgSO₄,0.5 mL mother liquor of trace element,pH 7,fermentation temperature 30?,50%loading volume and 7000 lx light intensity,50 L fermenter amplifying test was carried out.After 240 h fermentation,accumulation of oil content was about 5.4%higher than control group.And the concentration of first recycling supernatant fluid was 71%higher than the control group.The accumulated concentration in the secondary and tertiary recycling decreased successively,but was still higher than that in the control group.According to the calculation,the recycling of supernatant could save 30%of water consumption,40%of nutrients consumption and reduce environmental pollution.