Investigation On The Mechanisms Of Efficient Biosynthesis Of Astaxanthin Induced By Two-stage Culture Combined With Butylated Hydroxytoluene In Haematococcus Pluvialis LUGU

Astaxanthin?3,3'-dihydroxy-?,?-carotene-4,4'-dione?is a xanthophyll carotenoid that has important applications in the nutraceutical,aquaculture and pharmaceutical industries and is produced by various microalgae under environmental stress conditions.Among these microalgae,Haematococcus pluvialis is believed to synthesize the highest level of natural astaxanthin.Although H.pluvialis is often used in the production of astaxanthin,it has high input and low output.Therefore,it is especially important to find effective cultivation methods to increase the biomass and astaxanthin accumulation of H.pluvialis.In this study,a high-yield astaxanthin of H.pluvialis LUGU was originally isolated from Lake Lugu in Yunnan province.The two-stage method was used.The effect of different media on the growth of H.pluvialis was studied in the first stage.The second stage,the most favorable medium for astaxanthin accumulation was selected in this study under high light and nitrogen deficiency.Based on it,the effect of adding exogenous antioxidant butylated hydroxytoluene?BHT?on the accumulation of astaxanthin in H.pluvialis was investigated.Then the response of intracellular reactive oxygen species?ROS?,enzymatic and non-enzymatic antioxidants,NO signal to BHT and the relationship with astaxanthin and lipid synthesis were discussed.This study preliminarily revealed the mechanism of high-efficiency synthesis of astaxanthin in H.pluvialis with BHT under high-light and nitrogen deficiency,with a view to bulid the BHT technology system for enhancing astaxanthin content of H.pluvialis.The specific results are as follows:?1?In the present study,the effects of four different culture media on the growth,astaxanthin production and morphology of H.pluvialis LUGU were studied.The interactions between astaxanthin synthesis and secondary messengers,ROS and mitogen-activated protein kinases?MAPK?were also investigated.In the frst green vegetative cell stage,maximal biomass(1.41 g L^-1)was obtained in BBM medium.In the induction stage,the highest astaxanthin content(21.5 mg g^-1)occurred in BG-11medium,which was higher than in any other media.The expressions of MAPK and astaxanthin biosynthetic genes in BG-11 were higher than in any other media,whereas the ROS content was lower.Biochemical and physiological analyses suggested that the ROS,MAPK and astaxanthin biosynthetic gene expression was involved in astaxanthin biosynthesis in H.pluvialis under high-light and nitrogen-deficiency conditions.?2?The aim of this study was to investigate the effects of BHT on the production of astaxanthin and lipids in H.pluvialis LUGU under high-light,nitrogen-deficiency and BG-11 conditions.Astaxanthin and lipid contents were increased by 86.89%and16.74%,respectively,in algal cells treated with 2 mg L^-1 BHT.The maximal contents of astaxanthin and lipids were 3.17%and 45.6%,respectively.In addition,BHT down-regulated intracellular carbohydrate content of treatment was 13.45%of the dry weight of the cells,which was 83.44%of the control group?16.12%?.BHT treatment may redistribute the primary metabolites?carbohydrates?in H.pluvialis.BHT up-regulated expression of some key genes involved in H.pluvialis LUGU astaxanthin and lipid biosynthesis.?3?The levels of ROS in the presence of BHT were lower than in the control,and this effect involved strong activation of several antioxidases.Additionally,BHT application upregulated endogenous NO production.These results showed that this approach is useful for stimulating production of astaxanthin in H.pluvialis and that exogenous BHT induces astaxanthin and lipid production,which is responsible for the signalling molecule responses against abiotic stress conditions in H.pluvialis.