Cell Transformation Mechanism And "Three-stage" Culture Technique Of Haematococcus Pluvialis Of The Astaxanthin Production

In this study,the cell morphology,life cycle,cell transformation,and cell reproduction of Haematococcus pluvialis were studied by microscopy and electron microscopy.Then,differential expression gene analysis was carried out on the four types of H.pluvialis cells.The gene expression patterns related to encystment of H.pluvialis cells were determined by GO and KEGG enrichment analysis.An efficient preparation technology of nonmotile cells was developed by regulating the environmental factors.After that,the differences in the accumulation of astaxanthin between the motile-and nonmotile cells of H.pluvialis under different light intensities were compared.At last,we proposed the three-stage cultivation strategy to produce astaxanthin.The main contents and conclusions as follow(1)Morphological transformation and cell reproduction of H.pluvialis.The green motile cells of H.pluvialis can be transformed into red motile cells or green nonmotile cells.The green nonmotile cells can be transformed into red cysts under stress conditions.H.pluvialis has asexual reproduction and sexual reproduction.The sexual reproduction is carried out in isogamy.(2)Transcriptomic analysis of encystment of H.pluvialis,19 genes associated with the cell wall and 156 genes associated with endocytosis were significantly up-regulated in the encystment group(green nonmotile cell and red cyst),while the expression level of these genes in non-encystment group(green motile cell and red motile cell)was very low,indicating that the encystment of H.pluvialis was related to cell wall and endocytosis.(3)Effects of phosphorus starvation and NaCl on the formation of green nonmotile cells of H.pluvialis.The motile cells of H.pluvialis could be induced to form encvstment by adding NaCl or phosphorus starvation.Adding a high concentration of NaCl was able to promote cell encystment,but excessive NaCl concentrations were able to cause large numbers of cells deaths.In addition,the effect of encystment of H.pluvialis cells was significant under the synergistic effect of phosphorus starvation and NaCl addition.After motile cells stressed in the phosphorus starvation+0.1%NaCl medium for 72 h,the percentage of nonmotile cells reached 82%.(4)Differences between motile-and nonmotile cells of H.pluvialis in the production of astaxanthin at different light intensities.The concentration of astaxanthin and biomass in the nonmotile cell cultures and the size of red cysts formed by nonmotile cells were significantly increased compared to the motile cells.Astaxanthin content was closely related to cell size,and the nonmotile cells were more likely to form astaxanthin-rich large cysts than motile cells.The cyst enlargement and astaxanthin accumulation of H.pluvialis were both affected by light intensity,and a general trend was that the higher the light intensity,the larger the cysts formed,and the larger the quantity of astaxanthin accumulated.In addition,the relatively low cell mortality rate indicated that the nonmotile cells have a stronger tolerance to photooxidative stress.(5)Study on the three-stage cultivation strategy of H.pluvialis for astaxanthin production.The three-stage cultivation strategy has a great advantage in biomass and astaxanthin production compared to the traditional two-stage cultivation strategy.The biomass,astaxanthin concentration and astaxanthin content in three-stage cultivation strategy were up to 1.84,2.44 and 1.29 times as much as in the two-stage cultivation strategy.In summary based on the biological characteristics and cell transformation rules of H.pluvialis,our work proposed a three-stage cultivation strategy,which solved the problem of large cell death occurred during the red stage and achieved efficient production of astaxanthin.Our technology provided an effective technical support for the development of industrialization of H.pluvialis.