Investigation On Abiotic Stress-coupled Chemical Induction To Accumulation Of Astaxanthin And GABA And Mechanism In Haematococcus Pluvialis

Astaxanthin(3,3'-dihydroxy-?,?-carotene-4,4'-dione)has 13 conjugated double bonds.This special molecular structure makes it a super natural antioxidant,which is widely used in health products,cosmetics,food and breeding industries.Increasing the production and reducing production costs of astaxanthin in Haematococcus pluvialis have attracted significant attention since Haematococcus pluvialis is considered as the best natural source of astaxanthin.Abiotic stress and chemical induction are effective methods to promote the accumulation of astaxanthin by Haematococcus pluvialis.Melatonin(MT)and Ca²⁺,as plant hormones and common second messengers,can participate in the regulation of microalgae growth and metabolism,but its in-depth mechanism for inducing Haematococcus pluvialis efficient accumulation of astaxanthin remains to be studied.In addition,mining high-value by-products can also effectively reduce production costs.?-aminobutyric acid(GABA),a four-carbon non-protein amino acid,widely exists in microorganisms,plants and animals,has a variety of physiological functions,and can also participate in signal transduction and plant stress resistance.This study combined with two-stage cultivation,studied the effect of salt stress on the accumulation of GABA and astaxanthin in Haematococcus pluvialis.Meanwhile,the effects of salt stress on the related physiological indicators and the expression of GABA synthesis related genes in Haematococcus pluvialis were analyzed.On the other hand,the cultivation system of Haematococcus pluvialis with MT-coupled Ca²⁺to accumulate astaxanthin and lipid efficiently was established;in addition,the relationship between signals of Ca²⁺,GABA,ROS and the synthesis of astaxanthin and lipid under the induction of MT was explored.The regulation mechanism of MT-coupled Ca²⁺on Haematococcus pluvialis efficient accumulation of astaxanthin and lipid under abiotic stress was preliminary analyzed.This article provides new insights for reducing the production cost and increasing the production of astaxanthin accumulation in Haematococcus pluvialis,thereby promoting the rapid development of the commercialization of Haematococcus pluvialis astaxanthin production,which has certain theory and practice significance.The specific research results are as follows:(1)A two-stage cultivation method is adopted.In the first stage,under suitable temperature and light conditions,2 g/L sodium acetate(Na Ac)is added as an additional carbon source for the mixotrophic cultivation of Haematococcus pluvialis,In addition,the addition of 5 mg/L phytohormonal fulvic acid(FA)further promoted the growth of Haematococcus pluvialis.The biomass reached 1.84 g/L on the 7th day of cultivation,which was 2.27 times higher than that of autotrophic conditions alone.The growth cycle is shortened by half.(2)In the second stage,the effects of high light and salt stress on the growth and GABA accumulation of Haematococcus pluvialis were investigated.The results show that 0.4 g/L Na Cl can significantly increase the GABA content up to 38.57mg/g,which is 1.25 times that of the control group;at the same time,it reduces the carbohydrate,protein and chlorophyll levels in the algae cells and upregulates the the expression level of gad and cam related to GABA synthesis.The results showed that microalgae can be used as raw materials for the production of GABA.(3)This study established a culture system of MT-coupled Ca²⁺to promote the accumulation of astaxanthin and lipid efficiently by Haematococcus pluvialis under high-light and nitrogen deficiency stress.The study found that the astaxanthin and lipid content of Haematococcus pluvialis increased by 65.89%and 27.38%,respectively with induced of MT compared with the control group;Under the condition of coupling MT and Ca²⁺,the astaxanthin and lipid content in algal cells have further increased up to 38.2 mg/g and 49.53%,which were 1.13 and 1.21 times higher than those induced with induce of MT alone;meanwhile,the intracellular GABA content increased(51.83mg/g).In addition,the coupling of MT and Ca²⁺also increased the expression level of astaxanthin and lipid synthesis related genes and GSH content,while reducing the ROS level to improving the resistance of microalgae.When the endogenous Ca²⁺decreased,the content of astaxanthin,lipid and GABA in algal cells decreased simultaneously.The above results indicate that MT-coupled Ca²⁺can increase the accumulation of astaxanthin and lipid by regulating the expression levels of astaxanthin and lipid synthesis related genes and ROS,GABA signals.