Study On Key Problems In The Industrial Production Of Astaxanthin By The Photoinduction Of Heterotrophic Haematococcus Pluvialis

Astaxanthin has been widely used in food supplements,cosmetic ingredients,and aquaculture feed.Haematococcus pluvialis is regarded as the best biological source of natural astaxanthin.At present,the two-stage cultivation of H.pluvialis results in a high cost of astaxanthin derived from H.pluvialis due to the low yield and vulnerability to biological contaminants and weather.The "Sequential Heterotrophy-Dilution-Photoinduction"(SHDP)strategy developed by the author’s team can be used to obtain green cells stably and efficiently.The problems of low yield and vulnerable to contaminants in the green stage have been solved by heterotrophy instead of photoautotrophy.Our research group had conducted a preliminary study on the photoinduction process of the heterotrophic cells for astaxanthin production,but the technology had not yet been industrialized.Based on our previous work,five key problems(including the lack of a reliable method for evaluating the astaxanthin accumulation ability of heterotrophic green cells at the photoinduction stage,excessive light in the early stage and insufficient light in the later stage when photoinduced,the contradiction between heterotrophy and photoinduction site selection,biological contaminants,the lack of a method to dry algal powder on a production scale)in the industrial production of astaxanthin by the photoinduction of heterotrophic H.pluvialis were studied in this thesis to realize the industrialization of this technology.The main contents and conclusions of this paper are shown as follows:(1)Astaxanthin production was improved by using multivariate statistical analysis to evaluate green cells of H.pluvialis.The results showed a significant correlation among the green cell parameters that influenced astaxanthin accumulation.Multivariate statistical analysis could be used to evaluate the astaxanthin accumulation ability of green cells.The combination of principal component analysis with cluster analysis showed that chlorophyll content,protein content,dry weight of single cell,and akinetes proportion were positively correlated with astaxanthin accumulation.The larger these parameters,the greater the comprehensive evaluation value,and the stronger the astaxanthin accumulation ability.(2)The stepwise dilution strategy of the horizontal tube photobioreactor(HTPBR)was developed for producing astaxanthin and verified in outdoor large-scale HTPBR.The average light intensity was maintained as much as possible by adjusting the biomass density,which strengthened the light stress in the late induction period,ensured the maximum increase of astaxanthin content,and promoted the efficient accumulation of astaxanthin.In a 16.5 m3 HTPBR with 5-row parallel tubing,the best dilution strategy was two times dilution,with an initial biomass density of 1 g·L-1 in 3 rows.By the stepwise dilution strategy,the astaxanthin content in algal cells and the yield of astaxanthin per heterotrophic cell increased from 2.56%to 3.40%and from 41.0 mg·g-1 to 55.0 mg·g-1,respectively.(3)A new paradigm for producing astaxanthin by the photo induction of heterotrophic H.pluvialis in a separate location was established to solve the contradiction between heterotrophy and photoinduction site selection.The effects of refrigeration time on the cell viability of heterotrophic cells were investigated.The cell viability was still more than 90%after 4 days of refrigeration at 4℃.The volume content of astaxanthin with 0.60 mM nitrate addition increased by 25.7%as compared with that of without addition.Finally,by using the refrigerated and transported heterotrophic cells in a separate location,the volume content of astaxanthin reached 50.57 and 29.14 mg L-1 respectively,meanwhile the astaxanthin content reached 3.63%and 2.75%,after the photoinduction in Shidian and Shangri-La.Yunnan Province.(4)The control strategy for biological contaminants on outdoor large-scale cultivation was established.A degradation model of sodium hypochlorite was established to quantify the concentration of residual sodium hypochlorite in HTPBR.By comparing the multiple disinfection effects on outdoor HTPBRs,a low level of the total bacterial count was observed in HTPBR with double disinfection.By optimizing the disinfection strategy,the biological contaminants were reduced,and the volume content of astaxanthin was 2.37 times that of the unoptimized disinfection strategy.Increasing the salinity to 6‰ during light induction could inhibit the breeding of biological contaminants and reduce the losses caused by biological contaminants.(5)Optimization and scale-up spray drying for H.pluvialis.The key factors affecting the quality of algae powder were the drying time for droplets and the peclet number for heat,both of which can be adjusted by the outlet temperature.According to the key factors,the scale-up of the spray drying process of H.pluvialis powder was finally achieved by optimizing the outlet air temperature,with the production capacity of 37.04 kg·h-1,the yield of 98.57%,and the annual astaxanthin degradation rate of less than 10%.In this thesis,five key problems in the industrialization process of producing astaxanthin by the photoinduction of heterotrophic H.pluvialis were studied.The industrialization of astaxanthin production by heterotrophic cells of H.pluvialis was realized,and laid the foundation for the large-scale popularization of SHDP technology.Meanwhile,this study also provides a valuable reference for other oleaginous microalgae cultivation.