Studies On The Preservation Of Chlorella Vulgaris And Oocystis Borgei

Chlorella vulgaris and Oocystis borgei have been widely studied because they are unicellular eukaryotic green algae with the characteristics of high nutritional value and fast growth rate,In order to apply the excellent germplasm of C.vulgaris and O.borgei in scientific research and production,it is necessary to adopt appropriate preservation methods to keep them high biological activity.In this study,we preserved C.vulgaris and O.borgei by means of liquid subculture preservation,immobilization preservation,and cryopreservation,exploring the effects of temperature,light,algal cell density,and cryoprotectants on the preservation of these two green algae.The main research contents and results are as follows:1.C.vulgaris and O.borgei liquid subculture preservationIn order to explore the optimal liquid subculture preservation conditions for C.vulgaris and O.borgei.BG11 medium and SE medium was used to culture C.vulgaris and O.borgei until the exponential growth phase.Subsequently,we transferred the algal fluidto low temperature without light and low temperature with weak light for preservation,respectively,and analyzed the changes in algal cell density,chlorophyll content,and relative growth rate constant within four months.The results revealed that the number of algal cells of C.vulgaris and O.borgei were more stable under the condition of low temperature without light,and the growth performance in BG11 medium was better than that in SE medium.When cultured in BG11 medium and stored at low temperature without light,the changes in chlorophyll content of this two algal were both significantly slighter than those of other combinations（P<0.05）.Additionally,the relative growth rate of C.vulgaris and O.borgei in BG11 medium was significantly higher than that in SE medium（P<0.05）.There was no significant difference in the relative growth rate of C.vulgaris cultured in BG11 medium and preserved whether in low temperature without light or low temperature with weak light（P>0.05）,while the relative growth rate of O.borgei cultured in BG11 medium and preverved in low temperature without light was significantly higher than that in low temperature with weal light（P<0.05）.The results showed that the optimal liquid subsequent debpreservation conditions of C.vulgaris and O.borgei were as follows:cultured in BG11 medium,and preserved in low temperature without light.2.C.vulgaris and O.borgei immobilization preservationIn order to explore the best immobilization preservation conditions of C.vulgaris and O.borgei,we used agar-immobilized and sodium alginate-immobilized preservation methods to prevserve this two algal at low temperature without light（4℃,0 lx）.When exploring the optimal conditions in agar-immobilized preservation,we carried out the orthogonal experiments and single-factor experiments on the three factors of agar concentration,glucose content as well as algal cell density.The results showed that the optimal agar immobilized preservation conditions of C.vulgaris were 3%agar concentration,1%glucose content,and a algal cell density of 1.0×10⁷ind./m L,while the optimal conditions for O.borgei were 1%agar concentration,0.5%glucose content and a algal cell density of 5.0×10⁶ind./m L.The orthogonal experiments and single factor experiments were also carried out on the concentration of sodium alginate,Ca Cl₂concentration,as well as algal cell density to exploring the optimal conditions in sodium alginate-immobilized preservation.The results revealed that the optimal sodium alginate immobilization preservation conditions were 2%sodium alginate concentration,0.3mol/L Ca Cl₂concentration,a algal cell density of 5.0×10⁶ind./m L for C.vulgaris,and2%sodium alginate concentration,0.3 mol/L Ca Cl₂concentration,a algal cell density of1.0×10⁶ind./m L for O.borgei.Besides,we found that the effect of agar-immobilized preservation on C.vulgaris and O.borgei was better than that of sodium alginate-immobilized preservation when comparing the relative growth rate after resuscitation.C.vulgaris agar immobilized for 1 month had a relative growth rate of0.247 d^-1～0.275 d^-1,and 0.240 d^-1～0.268 d^-1for 3 months.O.borgei agar immobilized for1 month had a relative growth rate of 0.248 d^-1～0.272 d^-1,and 0.242 d^-1～0.270 d^-1for 3months.3.Cryopreservation of C.vulgaris and O.borgeiThe cryopreservation method was employed to preserve C.vulgaris and O.borgei in order to investigate the optimal cryopreservation conditions.Single factor experiments were used to evaluate the algal cell survival rate and relative growth rate constant K value of various cryoprotectants,algal cell densities,and cooling procedures.The results showed 15%glucose was the best cryoprotectant for C.vulgaris,the concentration of algal cells was 1.0×10⁷ind./m L,and the cooling procedure was 4°C（60 h）,-20°C（5 h）,-40°C（2 h）,-80°C（2 h）,liquid nitrogen（24 h）,with a survival rate of 94.511%～97.291%,and a relative growth rate of 0.235 d^-1～0.243 d^-1.The survival rate of algal cells after 3months of storage can reach 91.153%～96.185%,and the relative growth rate of 0.231d^-1～0.237 d^-1.The best cryoprotectant for O.borgei is 5%ethylene glycol,the algal cells concentration was 7.5×10⁷ind./m L,and the cooling procedure was 4℃（36 h）,-20℃（3h）,-20℃（3 h）,-40°C（3 h）,-80°C（1 h）,liquid nitrogen（24 h）,the algal cell survival rate was 92.688%～97.128%,while the relative growth rate of 0.143 d^-1～0.153 d^-1.,Algal cells can survive up to 3 months in storage,with a survival rate of 92.125%～96.623%,and a relative growth rate of 0.141 d^-1～0.147 d^-1.