Regulation Mechanism Of High Effective Astaxanthin Accumulation In Chromochloris Zofingiensis By Induction

Astaxanthin is a high-value keto-carotenoid with a wide range of application in food,feed and pharmaceutical industries because of its super powerful anti-oxidative activity.The green microalga,Chromochloris zofingiensis,has the capability of accumulating relatively high content of astaxanthin esters under stress conditions;therefore,it is regarded as a potential producer for natural astaxanthin.This study mainly focused on stress conditions enhancing astaxanthin accumulation effectively in C.zofingiensis and these underlying regulation mechanisms.First of all,the flow cytometry-based method for rapid estimation of astaxanthin content in C.zofingiensis was established for screening favorable stress conditions for astaxanthin accumulation.Furthermore,metabolome profile analysis associated with multivariate data analysis was applied to explore underlying regulation mechanisms of stress conditions enhancing astaxanthin accumulation.The main results were listed as follows:?1?Based on the correlation of autofluorescent characteristics of C.zofingiensis cells with its cellular pigments,the rapid method for astaxanthin determination was established by linar regression analysis.Moreover,the flow cytometry-based rapid method for Car/Chl ratio was established by the similar procedure.Method validation suggested that the FCM method for rapid,non-destructive and online estimation of astaxanthin content could fulfill the requirements of the analytical methodology with the acceptable accuracy and precision.?2?Mixotrophic C.zofingiensis was cultivated in shaking flasks and then in microplate-based culture system in order to explore the effects of different light sources,different ligh intensities and different nitrogen concentraions on astaxanthin accumulation.The results suggested that astaxanthin accumulation would be effectively promoted when C.zofingiensis was first cultivated in shaking flasks under low light irradiation and then subjected to continuous high light irradiation.In the culture using microplate-based culture system,when the light intensity was 150?mol m^-2 s^-1,blue light was more favorable for astaxanthin accumulation compared to white light.When light intensity was increased to 300?mol m^-2 s^-1,the cell growth and astaxanthin biosynthesis of C.zofingiensis were both inhibited by blue light.However,white light with the same intensity significantly enhanced astaxanthin accumulation,and the highest astaxanthin content and yield were obtained at 7.1mg/g and 30.6 mg/L,respectively.What's more,the maximum astaxanthin yield of 38.9 mg/L was achieved when 0.1 g/L nitrate was added in the culture,which was increased by 27.1%.?3?Based on the rapid method for astaxanthin determination and the developed microplate-based culture system,certain stress conditions significantly favoring astaxanthin accumulation were compared and selected.The results demonstrated that among various stress conditions,indole-3-propionic acid?IPA?and abscisic acid?ABA?were the most favorable inducers for astaxanthin accumulation.The highest astaxanthin content in C.zofingiensis was obtained above 13 mg/g when induced by IPA;however,the cell growth of C.zofingiensis was significantly inhibited.Conversely,indole-3-acetic acid?IAA?and pyruvic acid?Py?could enhance astaxanthin accumulation without inhibiting cell growth,and then greatly promoted the astaxanthin production by C.zofingiensis.The highest astaxanthin yield of 87.0 mg/L was obtained after the cultivation which significantly promoted the capability of astaxanthin production by C.zofingiensis.?4?Physiological and biochemical changes of C.zofingiensis under IPA-induced stress conditions were investigated and compared in this study.It was found that after the cultivation,chloroplasts and thylakoids in C.zofingiensis cells almost disappeared along with the formation of large amounts of oil droplets.Compared to the control,IPA inhibited the glucose consumption of C.zofingiensis as well as lipid accumulation,but significantly stimulated astaxanthin biosynthesis along with the inhibition of chlorophyll degradation.Finally,the highest astaxanthin content and yield were achieved at 13.9 mg/g and 49.6 mg/L,respectively.?5?GC-TOF-MS-based metabolome profile analysis combined with multivariate data analysis was applied to identify metabolic biomarkers in C.zofingiensis induced by IPA and then was used to depict central metabolic pathways.The results suggested that carohydrates,amino acids and fatty acids were the main metabolic biomarkers in C.zofingiensis,which mainly involved in galactose metabolism,alanine,aspartate and glutamate metabolism,biosnhthesis of unsaturated fatty acids,starch and sucrose metabolism and glutathione metabolism.It was shown in central metabolic pathways that metabolic changes induced by IPA were significantly different from that in the control.Under the IPA-induced stress condition,glycolysis and amino acid metabolism were significantly inhibited which led to the reduction of intracellular nitrogen supply from the degradation of amino acids and chlorophylls.Furthermore,the carbon chain extension and desaturation of fatty acids were also inhibited due to nitrogen limitation,but organic acids involved in TCA cycle accumulated significantly.Finally,astaxanthin accumulation was significantly promoted by the combined effects of these biochemical changes of C.zofingiensis cells induced by IPA.These findings in the study are meaningful for investigating regulation mechanisms of microalgae in response to environmental stress.