Temporal And Spatial Variations Of Dark Carbon Fixation Process In The Yangtze River Estuary And Associated Influencing Factors

Dark carbon fixation(DCF)driven by nitrifying microorganisms has an important influence on carbon and nitrogen cycling in estuary waters,but the contribution of ammonia-oxidizing microorganisms to the DCF process in estuary waters remains unclear.In this study,the Yangtze River Estuary was taken as a typical research area.Based on 14C and 15N isotope tracer technology and molecular biological methods,the study investigated the variation characteristics of DCF rates and nitrification rates under the tidal cycle and different salinity gradients in the Yangtze River Estuary,further quantified the abundance of related functional genes,revealed the dynamics of DCF functional genes of nitrifying microorganisms,and clarified the functional gene composition,the community structure change and the contribution to carbon and nitrogen conversion of the chemoautotrophic microorganisms.This study can provide scientific reference for the assessment of estuary ecosystem function in the context of global change.The main research results obtained in this study are as follows:(1)DCF rates and nitrification rates in the overlying water of tidal flat wetlands had obvious temporal and spatial variations under different salinity gradients in the Yangtze River Estuary.DCF rates were in the range of 700.34-4048.85 nmol C L-1 d-1 in summer and 422.00-731.10 nmol C L-1d-1 in winter,while nitrification rates ranged from 32.05-499.63nmol N L-1 h-1 and 4.78-42.24 nmol N L-1 h-1 in summer and winter,respectively.The rates were higher in summer,which might be due to the combination of precipitation,water temperature,salinity,and other environmental conditions.In addition,DCF rates had significant spatial variation,with a higher rate at the sites with higher concentration of inorganic nitrogen.Pearson correlation analysis also showed that NH4+and NO2-were the main environmental factors affecting the spatial variation of DCF rates in the overlying water of tidal flat wetlands in the Yangtze River Estuary.(2)The abundance of accA,cbbL and cbbM and flora in the overlying water of each sampling site were measured.The results showed that the average abundance of cbbL gene was significantly higher than that of accA and cbbM,and there was a significant positive correlation between the abundance of cbbL gene and the DCF rates,revealing that microorganisms containing cbbL gene might play a dominant role in the variation of DCF rates in the overlying water of estuary tidal flat wetlands.Phylogenetic analysis showed that microorganisms of different clusters,including cbbL,cbbM and accA genes,had different spatial and temporal distribution patterns,and temperature was an important environmental factor regulating the composition of chemolithotrophic microorganism communities.(3)During the tidal period of the Yangtze River Estuary,DCF rates and nitrification were ranged from 170.72-1007.35 nmol C L-1 d-1 and 1.45-70.75 nmol N L-1 h-1,respectively.These rates were relatively high in spring tide and relatively low in neap tide.The rates of bottom water were significantly higher than those of surface water.The concentration of ammonium and soluble inorganic carbon in water was the key factor affecting the DCF rates and nitrification rates.Quantitative PCR results showed that the abundance of cbbL copies was 0.40× 108-3.40×108 copies L-1 in spring tides and 0.49×108-2.27×108 copies L-1 in neap tides,and both were higher than that of cbbM copies(spring tides:0.67×106-9.84×106 copies L-1,neap tides:0.75×106-5.73×106 copies L-1).The abundance of accA genes in neap tides(0.16×108-2.65×108 copies L-1)was higher than that in spring tides(0.20×108-3.92×108 copies L-1),and the bottom layer was higher than the surface.In the whole tidal cycle,the abundance of autotrophic carbon sequestration genes increased at high tide and decreased at low tide.Ammonia-oxidizing archaea(AOA)and Ammonia oxidizing bacteria(AOB)were the major contributors to the DCF process.The abundance of AOA amoA and AOB amoA was significantly different between spring and neap tide.In spring tide,the abundance of AOA amoA,with a range of 0.22×107-3.59×107 copies L-1,was significantly higher than that of AOB amoA,with a range of 0.26×107-1.61 ×107 copies L-1,while the abundance of AOB amoA was dominant with a range of 0.92×106-1.32×106 copies L-1 in neap tide,indicating that tidal action could affect DCF by changing the composition of chemolithotrophic microorganism communities in waters.Overall,this study reveals the importance of the DCF process driven by nitrifying microorganisms in estuary waters,deepens the understanding of carbon-nitrogen coupling mechanism in estuary coastal wetlands,and has important significance for assessing the carbon sink function of estuary ecosystems.