Sodium Bicarbonate Strengthen C4 Carbon Fixation Pathway And Its Effect On Lipid Accumulation In Phaeodactylum Tricornutum

Microalgae can efficiently fix CO₂ through photosynthesis and accumulate energy precursors,among which lipid is the ideal raw material for renewable energy refining.The C4 pathway of microalgae was reported to have more potential for efficient CO₂fixation and lipid accumulation than the C3 pathway.Therefore,strengthening microalgae C4 fixation pathway and revealing the mechanism of carbon fixation have positive significance for CO₂ pollution prevention and energy pressure relief.In this paper,Phaeodactylum tricornutum was used as the research object,and the C4 carbon fixation pathway of P.tricornutum was enhanced by Na HCO₃ regulation.The effects of C4 carbon fixation pathway on the growth and intracellular components of microalgae were analyzed.Furthermore,the mechanism of C4 carbon fixation pathway regulating lipid synthesis and accumulation was further revealed.The main results are as follows:1)Low concentration Na HCO₃（50 mg/L）,medium concentration Na HCO₃（196mg/L）and high concentration Na HCO₃（400 mg/L）were used to treat P.tricornutum.Under low concentration Na HCO₃,Biomass,chlorophyll,protein,lipid and starch were1.13 g/L,13.81 mg/g,272.22 mg/g,36.3%and 186.19 mg/g.Under medium concentration of Na HCO₃,the biomass,chlorophyll,protein,lipid and starch were 1.14g/L,11.55 mg/g,282.92 mg/g,45%and 172.42 mg/g.Moreover,under high concentration of Na HCO₃,biomass,chlorophyll,protein,lipid and starch were 1.15 g/L,10.12 mg/g,308.48 mg/g,47.3%and 153.46 mg/g.These results indicate that low concentration of Na HCO₃ has no inhibitory effect on the growth of P.tricornutum,and is beneficial to the accumulation of chlorophyll and starch inside the cell.Therefore,low concentration of Na HCO₃ makes P.tricornutum more inclined to photosynthetic carbon fixation.However,the effects of medium and high concentrations of Na HCO₃increased the contents of intracellular lipid and protein,making it easier to enrich lipid and synthesize protein.2)Based on 1)the effect of low concentration（50 mg/L）and medium concentration（196 mg/L）of Na HCO₃ on P.tricornutum.Low concentration Na HCO₃induced algal cells as the control group,medium concentration Na HCO₃ induced algal cells as the experimental group,Analysis of intracellular C4 pathway for carbon fixation related enzyme activity.The results showed that the enzyme activities of CA,Ru Bis CO,PEPC,PPDK and NAD-MDH in algae cells were significantly increased at medium concentration Na HCO₃,suggesting that C4 carbon fixation pathway was strengthened in P.tricornutum.Further,by analyzing the correlation between CA,PEPCK,NADP-ME,NAD-ME and Ru Bis CO enzyme activity,it was concluded that there may be NAD-ME type C4 carbon fixation pathway in P.tricornutum at medium concentration Na HCO₃,which is regulated by the negative feedback of Ru Bis CO enzyme activity.Subsequently,through ¹³C non-target metabolic flow,it was found that the pathway of pyruvate formation from malic acid under the action of NAD-ME enzyme was credible,it was confirmed that the effect of medium concentration Na HCO₃ could strengthen the NAD-ME C4 carbon fixation pathway of P.tricornutum.3)Further analysis of ¹³C marker flow direction showed that the effects of medium concentration of Na HCO₃ could degrade chlorophyll-chlorophyll,provide precursors for lipid synthesis and accumulation,and promote starch conversion into lipid.Therefore,the enhancement of NAD-ME C4 carbon fixation pathway was conducive to lipid synthesis and accumulation.Moreover,the correlation between lipid content and C4 pathway carbon fixation enzyme activity also confirmed the positive regulation of C4 pathway on lipid synthesis and accumulation.In addition,the fatty acid composition of Na HCO₃ treated algae showed that the content of saturated fatty acid and monounsaturated fatty acid accounted for 96.8%of the total fatty acid content,in which C16:1,C24:0,C18:1 and C16:0 accounted for 85.6%.The correlation analysis between the fatty acid composition and the C4 pathway showed that the enhancement of C4 pathway was more conducive to the synthesis and accumulation of monounsaturated fatty acids and hexadecanoic acid,these fatty acid components are ideal raw materials for refining high quality biodiesel.