| Due to the strongest antioxidant known in nature,natural astaxanthin has been widely used in food supplements,health products,feed,and cosmetic ingredients.Haematococcus pluvialis is regarded as the best biological source of natural astaxanthin.However,the two-stage culture method based on the photoautotrophy of H.pluvialis for the currently commercial production of astaxanthin has low efficiency and high cost.A novel cultivation technology named“Sequential Heterotrophy-Dilution-Photoinduction"(SHDP)was developed by our research team.The technology replaced the photoautotrophic culture in the first stage for cell growth by heterotrophic culture,and thus greatly improved the efficiency of biomass accumulation,but has not yet been industrialized.During heterotrophic culture,the cell morphology of H.pluvialis changed significantly,affecting not only the growth ability but also the ability of astaxanthin accumulation.Meanwhile,the complex cell morphology in the heterotrophic culture also brought difficulties to the collection and utilization of heterotrophic cells,and thus restricted the industrialization of heterotrophic technology.In addition,the SHDP technology still relies on light in the second stage,and still has the defect of photoautotrophy.In view of the above problems,this thesis took the heterotrophic cell characteristics of H.pluvialis as the starting point,studied the transformation law of cell morphology and its impact on cell growth and astaxanthin accumulation,guided the optimization and amplification of heterotrophic culture,and finally realized the industrialization of heterotrophic culture of H.pluvialis.In addition,on the basis of in-depth study of cell morphological transformation,a novel cultivation mode named "Sequential heterotrophy induction in situ" was successfully established in this thesis to realize the production of astaxanthin under dark conditions.The main contents and conclusions of this thesis are as follows:(1)The transformation law of cell morphology during heterotrophic culture of H.pluvialis was clarified.And the relationship between cell morphology and proliferation was analyzed.During the heterotrophic culture,cells were transformed from motile cells to non-motile cells and then to akinetes.The order of cell morphology according to the ability of division and proliferation:motile cell>non-motile cell>akinete.Cells of different morphologies had distinct differences in microstructure and biochemical composition.Furthermore,the differential expression of cytoskeleton-related genes caused synergistic changes in cell morphology and the ability of proliferation.(2)Heterotrophic culture of H.pluvialis was optimized based on shear stress.The critical superficial gas velocity and average shear stress affecting the heterotrophic growth of H.pluvialis were determined to be 3.2×10-3 m·s-1 and 4.84×10-3 Pa,respectively.0.05%of Pluronic F-68 was selected as a shear protectant suitable for heterotrophic culture of H.pluvialis,and could increase the cell density by 30.76%in the 500 L Fermenter;Based on the shear stress,the most suitable impeller for heterotrophic culture was selected as hydrofoil impeller.(3)A model of cell morphology and specific growth rate was established.Based on this model,the seed age of 50 L and 1 m3 seed fermenter were optimized to realize the step-by-step amplification of heterotrophic culture from 50 L seed fermenter to 20 m3 culture fermenter.It was determined that the content of chlorophyll and protein was the key index of astaxanthin accumulation ability of heterotrophic cells.Based on the dry cell weight and astaxanthin accumulation ability of heterotrophic cells,the standard for the end of culture in the culture fermenter that could obtain the maximum astaxanthin yield was determined.(4)The strategy of harvesting and comprehensive utilization of heterotrophic cells of H.pluvialis was established.The recovery rate of natural sedimentation in the fermenter reached 92.14%.And the volume concentration factor reached 9.85.The cells harvested by natural sedimentation could be directly photoinduced to accumulate astaxanthin,which was similar to that of cells from the original culture.Aiming at the remaining supernatant cells after sedimentation harvest,the culture strategy of first indoor photoautotrophy and then photoinduction was established,and significantly increased the astaxanthin production of supernatant cells.The membrane filtration device suitable for the collection of supernatant cells was selected.And the recovery rate and the volume concentration factor could reach 95.10%and 6.63,respectively.(5)A novel cultivation mode named "Sequential heterotrophy induction in situ" was developed,and astaxanthin accumulation of H.pluvialis under dark conditions was realized in the 50 L tank.The cell morphology suitable for light-independent induction was determined as non-mobile cell.The medium for light-independent induction was optimized,and the astaxanthin production of H.pluvialis under dark conditions was increased by adding 1%ethanol as an exogenous stress substance.Finally,heterotrophic culture and light-independent induction was sequentially carried out to accumulate biomass and astaxanthin based on the above optimization in the 50 L fermenter,the volume content and productivity of astaxanthin reached 71.23 mg·L-1 and 6.77 mg·L-1·d-1,respectively.The results in this thesis clarified the transformation law of cell morphology during the heterotrophic culture of H.pluvialis,and analyzed the relationship between cell morphology and proliferation,laying a theoretical basis for the optimization and scale-up of the heterotrophic culture of H.pluvialis.The industrialization of the heterotrophic culture of H.pluvialis provided an important support for the industrialization of SHDP technology;In addition,a novel cultivation mode named "Sequential heterotrophy induction in situ" was developed successfully,and would be expected to get rid of light dependence from the astaxanthin production by H.pluvialis completely. |
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