A Mechanism Study On Co-producing Lipid And Extracellular Polysaccharide From Auxenochlorella Protothecoides By Fed Batch Culture With NaCl Stress

Microalgae is a sustainable biological resource that has attracted much attention,and its lipids and exopolysaccharides have great potential for applications in many fields,such as food,medicine,and biofuels.However,the development and utilization cost of microalgal metabolites is still high,resulting in poor market competitiveness and hindered large-scale application.Co-production of various high-value products is an important strategy to solve the economic problem of microalgae.In this study,Auxenochlorella protothecoides was taken as the research object,the effects of NaCl concentrations and application time on lipids and exopolysaccharides accumulation of Auxenochlorella protothecoides were first explored,a co-producting system for the accumulation of lipids and exopolysaccharides in Auxenochlorella protothecoides under NaCl stress in fed-batch culture is constructed,and then the effects of fed NaCl on the growth,structure and metabolite accumulation of Auxenochlorella protothecoides cells in the co-producting system are investigated.The main findings are as follows:1)NaCl concentrations,stress application time and their interaction have significant effects on the accumulation of lipids and exopolysaccharides in Auxenochlorella protothecoides.When the same concentration of NaCl is applied at different growth stages,the effects on production of lipids and exopolysaccharides production in Auxenochlorella protothecoides with the delay of the stress application time point.However,under the stress of different concentrations of NaCl in the same growth period,the production of lipids and exopolysaccharides in Auxenochlorella protothecoides showed different changing laws:the production of exopolysaccharides first increased and then decreased with the increase of NaCl concentrations,and achieve 0.49 g·L^-1,0.46 g·L^-1 and 0.46 g·L^-1 under the stress conditions of 0 h feeding with 0.2 mol·L^-1 NaCl,48 h feeding with 0.2 mol·L^-1 and 48 h feeding with 0.4 mol·L^-1 NaCl.The lipids production of Auxenochlorella protothecoides cells decreased with the increase of NaCl concentrations in the early growth stage,while NaCl stress had almost no effect on lipid accumulation in the later stage of growth.Considering the production of Auxenochlorella protothecoides lipids and exopolysaccharides comprehensively,feeding 0.2 mol·L^-1 NaCl after 48 h of culture is selected as the culture condition for stressing Auxenochlorella protothecoides to produce lipids and exopolysaccharides,and the yields reached 0.46 g·L^-1 and 5.28 g·L^-1,which are1.14 times and 2.88 times of Basal culture group,respectively.2)Under the co-producting culture conditions,the changes of Auxenochlorella protothecoides biomass,exopolysaccharides,lipids,starchs and proteins accumulation with time were measured,the results showed that the exponential growth period of Auxenochlorella protothecoides in the co-producting system was extended from 144 h to168 h,the secretion of exopolysaccharides showed a continuous increase trend,and the maximum accumulation rate was 16.89 mg·g^-1·d^-1 at 216 h;cell lipids accumulated rapidly in exponential phase and deceleration phase,reaching a maximum accumulation rate of93.34 mg·g^-1·d^-1 at 144 h.However,starch content and protein content began to decrease at 144 h and 96 h respectively,indicating that starch and protein synthesis were restricted,and intracellular carbon flow was redistributed to provide carbon precursors for the accumulation of lipids and exopolysaccharides.3)Taking the chlorella cultured in Basal as the control group and one in the co-production system as the experimental group,the combined transcriptome-metabolome analysis was carried out.The results showed that at 48 h fed 0.2 mol·L^-1 NaCl cultured Auxenochlorella protothecoides cells leading to down-regulate the gene expression of citrate synthase,glucose-1-phosphate adenylatetransferase,and glutamate synthase,4-α-glucanotransferase gene expression was up-regulated,protein,starch and sucrose synthesis was inhibited,and carbon flux was directed toward lipids and exopolysaccharides synthesis.The up-regulation of genes encoding phosphoenolpyruvate carboxykinase,pyruvate kinase and pyruvate dehydrogenase promoted the increased synthesis of pyruvate and acetyl-CoA,which provided precursor substances for lipid synthesis.At the same time,genes of encoding long-chain acyl-CoA synthase,phosphatidate phosphatase and diacylglycerol diphosphate phosphatase/phosphatidate phosphate,acetyl-CoA acyltransferase 1significantly up-regulate,indicating that the fatty acid synthesis pathway was enhanced and the fatty acid degradation pathway was inhibited,which promoted the accumulation of lipids in cells.In addition,significant upregulation of genes encoding UDP-glucose 6-dehydrogenase,3,5-epimerase/4-reductase and phosphoacetylglucosamine mutase promotes the up-regulation of amino sugar and nucleotide sugar metabolic pathways,and the contents of UDP-glucose,D-galacturonic acid and UDP-glucuronate increased,which provided sufficient precursors for the synthesis of exopolysaccharides.