Study On Preservation Conditions And Optimization Of Chlamydomonas Reinhardtii

Microalgae are raw materials for producing a variety of high value-added economic products,which are of great significance in solving the crisis of biological energy resources.Subculture is one of the most common algae preservation technologies.In recent years,cryopreservation technology has been widely used in plant germplasm preservation and detoxification.Cryopreservation of microalgae has also become an important research direction in the field of microalgae.In this paper,Chlamydomonas reinhardtii was used as the research object,and different preservation methods were selected.By analyzing the possible conditions of material pretreatment,preservation time,inoculation amount,hypertonic solution composition and dehydration protection,the most suitable preservation method of microalgae species and the best conditions for high survival rate were found.The experimental results are as follows:1.The survival rate of Chlamydomonas reinhardtii was tested by the subculture preservation method.Three methods were used for subculture preservation,and the optimal preservation conditions were obtained by single factor experiment.The results showed that the optimal preservation conditions were as follows:solid-liquid subculture preservation method,preservation time of 1 month,and inoculation amount of 10%.2.The gradient cooling was carried out on the basis of ultra-low temperature treatment with the compound protective agent,and the preservation conditions of Chlamydomonas reinhardtii were optimized by response surface methodology.The results showed that the content of compound protective agent was 30%sucrose,15%ethylene glycol,and 5%sucrose.Under this condition,the average survival rate of Chlamydomonas reinhardtii was44.30%.3.Methanol was used as an ultra-low temperature protection agent.The results of single factor experiments showed that the optimal preservation conditions of Chlamydomonas reinhardtii were as follows:methanol concentration of 4%,treatment time of 30 min at 7°C,thawing temperature of 35°C,and centrifugal speed of 1000 r/min.On the basis of single factor experiment,orthogonal experiment was carried out.The results showed that the factors affecting the survival rate were as follows:thawing temperature>treatment time>centrifugation speed>methanol concentration.The optimal combination of preservation conditions was A₁B₂C₂D₂,namely,methanol concentration of 4%,treatment time of 30 min,thawing temperature of 35 min,and centrifugation speed of 1000r/min.The survival rate of Chlamydomonas reinhardtii reached 69.66%.Based on the above experimental results,the survival rate of Chlamydomonas reinhardtii could reach the highest when the logarithmic growth phase of Chlamydomonas reinhardtii was treated with methanol as an ultra-low temperature protective agent,gradient cooling and then liquid nitrogen.