Regulation Of Starch Synthesis In Chlorella Sorokiniana By Sulfur Limitation Culture And Its Application

Microalgae culture can not only fix CO2,but also can accumulate energy storage substances such as starch used for biofuel production.Therefore,the development of microalgae biomass resources is of great significance for the realization of the targets in carbon neutralization and carbon peak in China.Nutrient limitation culture is an effective method to improve the starch accumulation efficiency of microalgae.However,the growth of microalgae is inhibited under nutrient stress,which affects the total starch yield.To solve this problem fundamentally,it is very important to understand the mechanism of efficient starch accumulation in microalgae and explore the key genes regulating the starch accumulation.It is beneficial to improve the accumulation of microalgae starch by means of genetic engineering and lay a foundation for the further use of microalgae biomass resources to produce biofuel.In our previous study,the efficiency of carbohydrate and starch accumulation in Chlorella sorokiniana UTEX1230 under different sulfur limitation methods was investigated.The key differential expressed genes of C.sorokiniana UTEX1230 under sulfur limitation were identified by RNA-seq analysis.Also,a FOS culture system which is suitable for large-scale culture of C.sorokiniana was established.Based on these,this study conducted the sulfur limitation culture under the FOS system,trying to improve the growth and starch yield of C.sorokiniana,and also,carried out the test of bio-fuel conversion with wet algae,exploring the feasibility of microalgae fuel.Meanwhile,the key differential genes obtained by RNA-seq as candidates were used to prepare antibodies.The expression of key genes at the protein level were identified and the key proteins regulated the starch synthesis of C.sorokiniana were screened.The results are as follows:Sulfur Limitation(SL)culture for 2 d,the content of carbohydrate in C.sorokiniana UTEX1230 was 50.6%of the dry weight,and the starch content was 49.5%of the dry weight.The starch content in low concentration sulfur(50%)limitation culture reached to 18.5%after 6 d,which was significantly higher than that of the control.Protein content decreased significantly under SL,while lipids content increased slightly.After 6 d of SL culture,the OD750 value of C.sorokiniana reached to 4.3,the cell dry weight was 1 g/L,and the cell number was 72×106 cells/mL.Under 50%S condition,the dry weight was 1.9g/L and the cell number was 281 ×106 cells/mL,which were all lower than those of the control.Under SL condition,the Fv/Fm of C.sorokiniana decreased.C.sorokiniana UTEX1230 was cultured in SL,and then inoculated to Sulfur Replishment(SR)culture,the starch content decreased rapidly,the protein content increased,the lipids content did not change much.The OD value,dry weight and cell number,and the Fv/Fm value all increased,and returned to control level.Western blot analysis showed that the protein related to sulfur metabolism,such as sulfate transporter SULTR,was expressed after 2 days in SL condition,and continued to be expressed in SR stage.The cysteine synthetase OASTL was expressed after 1 day in SL,and the expression gradually decreased in SR.The expression of photosynthesis related protein such as Rubisco was down-regulated in SL and up-regulated in SR,while the expression of GAPDH was up-regulated in SL and down-regulated in SR.AGPase A.a gene related to starch metabolism,was expressed after 2 days in SL,and then downregulated in SR;AGPase B was stably expressed in SL and SR;SBE was expressed after 1 day in SL,and then down-regulated in SR;GBSS was down-regulated in SL,and then upregulated in SR;GBE was down-regulated in SL,and then up-regulated in SR.Starch-rich wet cells of C.sorokiniana were treated with dilute sulfuric acid,which could release 88.5%glucose into the hydrolysate,while direct enzymatic hydrolysis yielded 78.3%glucose.The addition of cellulase had little effect on glucose yields.Direct enzymatic hydrolysate of wet biomass was used for Acetone-Butnaol-Ethanol(ABE)fermentation,7.2 g/L ABE was obtained,and the yields of butanol and ABE were 0.22 and 0.35 g/g glucose,respectively.These results indicated that SL culture could significantly increase the starch accumulation in C.sorokiniana UTEX1230,but affected the growth of C.sorokiniana cells.SR culture verified the rapid response of cell composition and growth of C.sorokiniana to the presence and absence of sulfur element.The active expression of SULTR and OASTL under SL condition promoted the transport and metabolism of sulfur,and the increase of starch content might be related to the high expression of enzymes related to the regulation of starch synthesis such as AGPase A and SBE.The down-regulation of Rubisco might directly reduce the photosynthetic rate of C.sorokiniana,leading to the slow growth.Compared with flask culture,FOS system can increase the accumulation of Chlorella biomass,and the potential of using wet algae to produce biobutanol is demonstrated by direct enzymatic hydrolysis without cell wall disruption.