The Mechanism Of Microcystis Carbohydrate Accumulation Regulated By Nitrogen Availability And Its Resource Utilization

The cyanobacterial blooms caused by eutrophication is one of the major threats to the water quality of global lakes,and their occurrence and disappearance have become a hot issue of concern for experts and scholars at home and abroad.The mainstream view holds that the nitrogen content in the water body is one of the key factors affecting the proliferation of cyanobacteria.Studies have shown that nitrogen availability affects the cyanobacterial biomass and intracellular carbohydrate content of water bodies,and further changes its sedimentation performance by affecting the specific gravity of cells,which promotes the disappearance of blooms.However,the mechanism by which nitrogen availability regulates cell carbohydrate accumulation is still not clear.In this study,the changes of carbohydrate content of Microcystis in Taihu Lake were investigated during the outbreak of blooms,the changes of cell composition of Microcystis under different nitrogen availability was determined,and the proteomics results as well as carbohydrate metabolites were analyzed comprehensively to preliminary explain the effect mechanism of nitrogen depletion on the accumulation of carbohydrates in Microcystis.Based on that,the feasibility of producing bioethanol from bloom-forming cyanobacteria was explored,which provided a technical reference for the resource utilization of cyanobacteria in Taihu Lake.The main conclusions are as follows:(1)During the outbreak of cyanobacteria,the carbohydrate content in Microcystis cells is affected by the availability of nitrogen in the water.Microcystis could grow when nitrogen is sufficient,and its growth could be promoted by the input of CO₂ and NaHCO₃.Microcystis grew under stress when nitrogen was deprived,but the input of CO₂ and NaHCO₃ can alleviate this stress.On the day 7,the dry weight was significantly increased after supplemented with CO₂ and NaHCO₃ and getting up to 4.4 times,the carbohydrate increased from 13.4% to 56.6%,the specific gravity increased relative to the control group,but the pigment content as well as the photosynthetic and respiration rate didn't increase.(2)The proteomics results showed that under nitrogen depletion for 48 h,955 proteins changed,among which 504 proteins were up-regulated and 451 proteins were downregulated.KEGG results showed that NADPH produced by photosynthetic system I and CO₂ entering the nitrogen metabolism pathway were both reduced,while the carbon fixation pathway and glycolysis pathway were both down-regulated.The results indicated that the accumulation of carbohydrates is mainly due to the inhibition of its decomposition process rather than the synthesis under nitrogen depletion.In addition,the proteomics verification results showed that the trend of key protein changes was consistent with the proteome results,proving that the results are true and reliable.The carbohydrate metabolites results showed the content of disaccharides in Microcystis cells was much higher than that of monosaccharides.Under nitrogen-depleted condition,the content of carbohydrate metabolites in Microcystis increased.Glucose increased most obviously,which was 8-16.3 times higher than the initial value;followed by fructose,which was 4.4-8.3 times higher than the initial value.Besides,the increase in NaHCO₃ resulted in a decrease or a less increase of carbohydrate metabolites per cell.(3)Under nitrogen depletion,the optimal light-dark cycle of Microcystis growth was 24L:0D,and the addition of NaHCO₃ significantly promoted the dry weight and carbohydrates accumulations in Microcystis.Under nitrogen depletion for 4 days,the dry weight reached 1018.5 mg/L and the intracellular carbohydrate content reached 63.2%.In the ethanol production stage,the optimal concentration of the sulfuric acid during hydrolysis was 0.5%(v/v)with consideration of the hydrolysis rate and cost,and the hydrolysis rate reached 93.4%;the optimal fermentation time was 24 h with consideration of the ethanol concentration and the cost,at which time the fermentation efficiency reached 90.4%.The cyanobacteria in Taihu Lake were cultured under nitrogen depletion using the above conditions,and the intracellular carbohydrate content reached 51.8%.Compared with uncultured cyanobacteria,the ethanol yield increased from 0.02±0.00 to 0.22±0.02,and fermentation efficiency also increased from 34.1±1.2% to 82.4±2.3%.The results fully verify the feasibility of producing bioethanol from Microcystis and provide a technical reference for the resource utilization of bloom-forming cyanobacteria.