High-efficient Cultivation And Induction For Astaxanthin Accumulation In Chromochloris Zofingiensis

Astaxanthin,a kind of ketocarotenoid with powerful anti-oxidative activity,has anti-cancer,anti-diabetic,anti-inflammation and other physiological functions.Chromochloris zofingiensis can grow autotrophically,mixotrophically and heterotrophically and accumulate high content of astaxanthin under stress conditions.Thus,it is considered as a potential microalga for natural astaxanthin production.This study mainly investigated the nutritional growth characteristics and astaxanthin accumulation of C.zofingiensis in mixotrophic culture.First of all,the cell growth conditions of C.zofingiensis in mixotrophic culture were optimized,the aims were to obtain the highest biomass along with a relatively high content of astaxanthin in a short period of time.Subsequently,The effects of different stress conditions on enhancing astaxanthin accumulation of C.zofingiensis were studied in shaking flasks and further in 300 L photobioreactor for validation.Then,the conditions of enhancing the astaxanthin accumulation of C.zofingiensis were studied in culture with the complex carbon source,after that,the former optimal conditions in culture with constant high light,low-high light intensity and low-high light intensity with supplemental hydrogen peroxide were studied in 5 L fermenter.The main experimental results were as follows:1.Different carbon to nitrogen ratios,glucose concentrations,nitrogen sources,initial cell density had significant effects on the growth and astaxanthin accumulation of C.zofingiensis in mixotrophic culture?p<0.05?.The result showed that in the mixtrophic culture with 30 g/L glucose concentration,sodium nitrate as nitrogen source,carbon to nitrogen ratio of 34 and initial cell density of 2.30 g/L,the biomass,astaxanthin content and yield of C.zofingiensis achieved the maximum values of 11.65 g/L,1.52 mg/g and 17.74 mg/L,respectively,which was increased by 19.49%,12.59%,34.29%compared with before optimization.2.The cells cultivated at the optimal conditions for cell growth described above were served as the seed for induction study.Carbon source and nitogen source concentrations,feeding mode,pH,ethanol and magnesium chloride with the sole carbon source of sodium acetate in the medium had significant effects on astaxanthin accumulation in C.zofingiensis?p<0.05?.The result showed that the optimal conditions of 2.50 g/L sodium acetate,nitrogen free,feeding sodium acetate?2.50 g/L?every 6 days,maintaining pH at 6.5⁸.3 and 2%etanol addition was more suitable to enhance astaxanthin accumulation of C.zofingiensis in the culture,the highest astaxanthin content of astaxanthin was 3.71 mg/g,which was increased by 31.71%compared with pre-optimization,and the astaxanthin yield was 5.37mg/L.The highest astaxanthin content of 3.44 mg/g and astaxanthin yield of 1.10 mg/L were obtained in the tubular photobioreactor.3.The cells cultivated at the optimal conditions for cell growth were served as the seed for astaxanthin induction study.The mixed carbon sources and concentration,initial inoculation density had significant effect on astaxanthin accumulation of C.zofingiensis under complex caobon source culture?p<0.05?.The result demonstrated that mixed carbon source of20 g/L glucose and 2.50 g/L sodium acetate,the initial cell density of 1.22×10⁸ cfu/mL were applied for the culture,the maximum biomass and yield of astaxanthin was 16.96 g/L and60.28 mg/L,which was higher 188%and 502%than before optimization?0.24×10⁸cfu/mL?.4.Based on the mixed carbon source,the light intensity had significant effect on astaxanthin accumulation of C.zofingiensis?p<0.05?.when the light intensity was 240?mol m^-2 s^-1,the highest biomass of 11.28 g/L and astaxanthin yield of 32.20 mg/L were achieved.When the light intensity was increased to 320?mol m^-2 s^-1,the biomass and astaxanthin content were inhibited.5.Based on the former optimal conditions with complex caobon source,the stress conditions?hydrogen peroxide and ethanol?had significant effects on astaxanthin accumulation of C.zofingiensis?p<0.05?.The result showed that range of H₂O₂ concentration?107.5¹37.5 mg/L?was the optimal condition for cell growth and astaxanthin accumulation,resulting in the hightest yield and productivity of astaxanthin of 73.45 mg/L,4.59 mg/L/d,respectively.The addition of ethanol enhaned the axtaxnthin content,but decreased biomass concentreation When 2%ethanol was added.the maximun content and yield of astaxanthin was 3.27 mg/g and 42.32 mg/L,respectly,6.The stress conditions for enhancing astaxanthin above in mixotrophic culture with complex caobon source were validated by 5 L fermenter.Constant high light intensity,low-high intensity and low-high light intensity with supplemental H₂O₂ had significant effects on astaxanthin accumulation of C.zofingiensis?p<0.05?.The result showed that the growth of C.zofingiensis was inhibited under constant high light intensity,the highest yield and productivity of astaxanthin was 21.46 mg/L and 2.76 mg/L/d,respectively.Biomass concentration and astaxanthin accumulation of C.zofingiensis were enhanced when the cell was first cultivated under low light intensity and then high light intensity,resulting in high yield and productivity of astaxanthin of 38.38 mg/L,6.25 mg/L/d,respectively,which were increased by 78.84%,126%compared with constant high light intensity.When the cell were first cultivated under low light intensity and then at high light intensity with supplemental H₂O₂,the yield and productivity of astaxanthin were achieved at the maximum value of 41.41mg/L,6.77 mg/L/d,respectively,which were increased by 92.96%,163%than that in culture with constant high light intensity.