Investigation On The Related Metabolic Mechanisms Of Lipid Accumulation By Combined Fulvic Acid And Salt Stress In Monoraphidium Sp. QLY-1

Nowadays,the conventional energy reserve based on fossi fuel are dwindling,the serious environmental pollution is caused by the massive burning of fossil fuels.Therefore,the developing of clean and renewable energy becomes the research hot topic in recent years.Biodiesel is the best alternative for fossil fuels because of its sustainable development.Microalgae are an advantageous raw material for biodiesel production,which can effectively use light energy to fix CO₂ in the air and convert it into biomass energy.Among the microalgae,the Monoraphidium species is a novel oleaginous microalgae,which can growth and promote the lipid under the heterotrophic and autotrophic culture.In this study,Monoraphidium sp.QLY-1 is as the research object,fulvic acid(FA)is the basic inducer and salt stress(Na Cl)is the abiotic stress for inducing the lipid accumulation during the autotrophic condition.Based on the physiological and biochemical characteristics,such as biomass production,lipid content,chlorophyll,carbohydrates,proteins,and endogenous signal molecules,including the reactive oxygen species(ROS),peroxidase(POD),superoxide dismutase(SOD),catalase(CAT),ascorbic acid peroxidase(APX),nitric oxide(NO)and Mitogen-activated protein kinase(MAPK),as well as the research and analysis of genes related lipid synthesis.Moverover,the metabolic regulation mechanism of GABA and Ca²⁺in the process of lipid synthesis by combined fulvic acid and salinity stress was investigated by chemical genetic method.The specific results are as follows:(1)A culture system for Monoraphidium sp.QLY-1 high efficiency synthetic lipid was established.25 mg L^-1 FA was used as the main concentration parameter,and different concentrations of Na Cl(0-30 g L^-1)were combined to cultivate QLY-1.Through the analysis of biomass,lipid content and other indicators,the 20 g L^-1 Na Cl with the highest lipid content(59.53%)was finally selected as the optimal parameter at day 6,which increased by 46%compared with the control group.(2)Dynamic analysis biomass,lipid content,chlorophyll,carbohydrates and protein contents under the combination of FA and Na Cl with optimal parameters,the different results was acquired.Compared with the control,the biomass had lightly reduced,lipid content was significantly increased,the chlorophyll production was remarkable reduced,carbohydrate contents was decreased,and protein contents had no significant effect.(3)Under the combined treatment of FA and Na Cl,the antioxidant capacity and related endogenous response mechanism of Monoraphidium sp.QLY-1 were changed,the ROS rapid response stimulated the increase of antioxidant enzyme,the increase of NO level and the cascade effect of MAPK were affected(4)GABA was used to explore the relevant metabolic regulation of the lipid synthesis pathway in microalgae under FA combined with Na Cl treatment,and the optimal concentration of GABA was optimized.The biomass increased slightly under0.1 m M GABA treatment,and the lipid content was 57.89%at 24 h,which was 1.26times higher than that of the control group(45.94%)(5)Monoraphidium sp.QLY-1 was treated with GABA in FA and Na Cl culture,and it was found that during the whole 60-hour culture period,the biomass of microalgae increased slightly,the lipid content increased significantly in the early stage of culture,ROS and GSH changed to different degrees,and Ca²⁺level and Ca M concentration were also affected to some extent(6)In addition,the chemical genetic method was used to study the GABA shunt metabolic-related signals involved in the synthesis pathway of lipid in microalgae.It was found that when exploring the signal transduction of GABA,the lipid content was significantly affected by 3-MP,and the Ca²⁺level was also affected.When exploring the signal transduction of Ca²⁺,the lipid content was also significantly affected by EGTA,while the GABA content was also affected.Besides,studies on antioxidant systems have found that when microalgae are subjected to abiotic stress,regulation by plant hormones can affect the antioxidant capacity of microalgae by participating in GABA shunt metabolism through Ca²⁺and thereby promoting lipid accumulation.In conclusion,the mechanism of promoting the lipid accumulation Monoraphidium sp.QLY-1 was discussed from the aspects of physiological and biochemical epigenetic characteristics and protein and molecular levels under FA and salinity stress this study,and the highest lipid content(59.53%)was obtained.On the other hands,under the action of FA combined with Na Cl,the mechanism of the action of GABA and Ca²⁺on the accumulation of lipid in QLY-1 was analyzed.This study provides a theoretical and experimental basis for using genetic engineering strategies to improve the rapid accumulation of lipid in microalgae and to clarify the ability of microalgae to resist oxidative stress under stress conditions.