| With the development of aquaculture,the demand for active microalgae is increasing.It is a good way to supply fresh microalgae in aquaculture farm and reduce the cost of culture.Strengthening the research on the preservation technology of concentrated microalgae is of great significance and practical value for the application of active microalgae in aquaculture.Oocystis borgei is a common dominant population in high-level culture ponds of subtropical prawns,and is one of the beneficial microalgae that has been successfully developed and industrial production.In this paper,O.borgei was selected as the research object,and the concentrated O.borgei was stored at 5℃,15℃,25℃and 35℃for 100 days.The effects of temperature on the growth,biochemical components during the storage period and recovery of concentrated O.borgei were studied by measuring the biomass,chlorophyll content,fluorescence intensity of lipid,polysaccharide content,protein content,survival rate,biomass and specific growth rate after recovery.The transcriptome approach was used to analyze the deferentially expressed genes in the preservation process of O.borgei under different temperatures and different preservation time,in an attempt to reveal the effect of preservation temperature and time on the metabolic pathway of O.borgei.The main research results are as follow:1.The preservation temperature had a significant effect on cell density of O.borgei.The algal cell density of the 15℃and 25℃preservation group was significantly higher than that of the other groups after 100 days of preservation;35℃preservation group had the worst preservation effect,and the algal cell density decreased by 26.69%.The chlorophyll a,chlorophyll b and carotenoid contents of O.borgei decreased continuously during preservation at different temperatures,and the chlorophyll content of 35℃preservation group was significantly lower than that of other preservation groups.2.The preservation temperature had a significant effect on the lipid,polysaccharide and protein content of O.borgei.After 100 days of preservation,the polysaccharide content of 35℃preservation group increased by 17.96%compared with the initial value,while that of 5℃,15℃and 25℃preservation groups decreased by 7.90%,19.59%and18.96%,respectively.The lipid content of the 15℃group was the highest at 20 day while that of the remaining group was the highest at 50 day.During long-term preservation,the lipid content of the 15℃group was relatively low while that of the 5℃group was relatively high.The protein content of O.borgei decreased first and then increased under different preservation temperatures.After 100 days of preservation,the protein content of O.borgei under 35℃,5℃,and 15℃increased by 69.28%,19.45%,and6.94%compared with the initial value,while under 25℃the protein content reduced by4.40%.3.Both high temperature and low temperature preservation significantly reduced the survival rate of O.borgei.After 100 days of preservation,the survival rate of O.borgei cells of the 15℃and 25℃preservation groups was as high as 95%,significantly higher than that of the 5℃(84.77%)and 35℃(47.70%)preservation groups.The cell density and initial specific growth rate of resuscitated O.borgei decreased continuously with the increase of preservation time at different preservation temperatures.The specific growth rate of O.borgei with different preservation time tended to be stable after 4～5 days of resuscitation,the 35℃preservation group of O.borgei,although the survival rate was low,it could still revive and proliferate.4.The effect of preservation temperature on the biosynthesis of unsaturated fatty acids,photosynthesis and nitrogen metabolism of O.borgei was significant.When stored at 5℃,unsaturated fatty acid synthesis-related genes were significantly activated in O.borgei,which stimulated the accumulation of polyunsaturated fatty acids in O.borgei.However,low temperature inhibits the synthesis of PSⅡreactive central structural proteins,which reduces the photolysis efficiency of water and the release of electrons and hydrogen ions,further caused the strong upregulation of downstream electron transport chain and ATP synthesis related protein genes.At the same time,low temperature promotes nitrogen metabolism,and extracellular nitrate accelerates transport to the cell,while the conversion of formamide to ammonia and formic acid increases,indicating that low temperature promotes ammonia formation while promoting ammonia conversion to L-glutamate.Different from cryopreservation,the unsaturated fatty acid synthesis related genes were all down-regulated in O.borgei preserved under 35℃,and the synthesis of unsaturated fatty acids was blocked.Moreover,high temperature stimulates the synthesis of structural proteins of PSⅡreaction center and increases the release of electrons and hydrogen ions.However,the electron transfer between the cytochrome b6/f complex and the PSⅠand inside the reaction center may be inhibited,and the electron can not be transferred to the Fd-NADP reductase,reducing NADPH production.By contrast,the conversion of ammonia in 35℃of preserved O.borgei is inhibited,and the accumulation of intracellular ammonia at high temperature may be one of the possible reasons for cell death.5.The effect of preservation time on the photosynthesis and nitrogen utilization ability of O.borgei was significant,and the inhibition was increased with the preservation time increased.Compared with the initial preservation group,no matter after 50 or 90 days of preservation,the photosynthetic system and the electron transfer process were inhibited,the synthesis of NADPH and the synthesis of H~+and ATP were also inhibited.The absorption of inorganic nitrogen by O.borgei decreased at 50 days of preservation,the process of nitrite and formamide conversion to ammonia was inhibited,and the process of ammonia conversion to L-glutamate was inhibited.While the above pathway was still inhibited after 90 days of preservation compared with 50 days,indicating that the nitrogen utilization ability of O.borgei continued to be destroyed as the preservation time increased. |
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