| There are many kinds and wide distribution of microalgae.It is of scientific research and application value to find more algae strains with excellent properties from nature.In this study,high-throughput sequencing was used to analyze the microalgae biodiversity of greenhouse soil samples from several geographic locations,and the abundance,taxonomy and dominant species of microalgae detected in different samples were compared.At the same time,the culturable microalgae were isolated and purified from one of the soil samples and a fresh water sample collected from a river that flows through downtown Baoding by plate streaking and classified by the internally transcribed spacer of ribosomal RNA genes(ITS)sequencing and phylogenetic analysis combined with microscopic observation in order to provide the basis for the collection,preservation and subsequent research of microalgae resources.Chlamydomonas reinhardtii was cryopreserved in liquid nitrogen by two-step freezing method in order to provide a long-term preservation method for the above purified species.The main results are as follows:1.High-throughput sequencing analysis showed that at the genus level,the relative abundance of genera and dominant algal groups in the samples from various greenhouse farming sites were different.The top five relatively abundant eukaryotic microalgae in sample1A-1 were Tetracystis,chlorococcum,Ochromonas,Chlorella and Chlamydomonas;the top five relatively abundant eukaryotic microalgae in sample 2B-1 were Chlorococcum,Bumilleriopsis,Klebsormidium,Chlamydomonas and Chlorella;the top five ones in sample2B-2 were: Bumilleriopsis,Chlorococcum,vaucheria,Klebsormidium and Chlorella;the top five ones in sample 3C-1 were: Chlorococcum,Chlamydomonas,Graesiella,Bumilleriopsis and Chlorella;and the top five ones in sample 4D-1 were: Chlorococcum,Graesiella,Bumilleriopsis,Chlamydomonas and Klebsormidium respecctively.The results indicated that Chlorococcum showed the highest abundancy in the five soil samples and Bumilleriopsis,Chlorella and Chlamydomonas were less abundant.2.16 microalgae were successfully isolated from the Langfang greenhouse soil sample.The ITS sequence analysis showed that: 8 of the 16 microalgal isolates belonged to genus Chlamydomonas,4 of them belonged to genus Protosiphon,3 belonged to genus Scenedesmus and 1 isolate was classified as the member of genus Bracteacoccus.3.46 microalgal isolates were purified from the fresh water sample.Among them,25 were identified as members of genus Chlorella,10 of them belonged to Micraninium pusillum,5 isolates were members of genus Coelastrella,both of the genus Desmodesmus and Kirchneriella got 3 members,and the genus Coelastrum got 2 members;4 isolates were classified to each of genus Monoraphidium,Nephrochlamys yushanlensis,Selenastraceae sp.and Scenedesmus.4.The model microalga,Chlamydomonas reinhardtii cc503 was preserved by cryopreservation,and the cells were stained by dual fluorescence staining of fluorescein diacetate(FDA)and propidium iodide(PI)after 15 days,40 days,17 months and 21 months of liquid nitrogen cryopreservation.The cell viability was detected by flow cytometry,and the survival rates were 76.10%,67.60%,49.87% and 39.47%,respectively.In addition,the maximum photosynthetic efficiency Yield(Y)of algal cells at different storage times was detected by PHYTO-PAM phytoplankton analyzer in which the maximum photosynthetic efficiency Yield(Y)after 21 months of storage decreased by 83% compared with the control.The growth curve was plotted by measuring the concentration of chlorophyll.The above analysis of cell survival rate,maximum photosynthetic efficiency and chlorophyll concentration showed that the growth potential and physiological state of Chlamydomonas reinhardtii after21 months of storage were basically the same as those of the control,which proved that liquid nitrogen cryopreservation can effectively maintain the vitality of Chlamydomonas reinhardtii cells,and also provided a reference for the long-term preservation of other microalgae. |
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