Microbial Mechanism Of Coupled Nitrification-denitrification Mediated By Suspended Particulate Matter In Hangzhou Bay Estuary

Human activities lead to coastal nitrogen overload and eutrophication, and then result in a suit of environmental problems. Under the background of global change, to investigate the mechanism of nitrogen transport and circulation in estuarine zone, and further explore the mechanism and contribution of microbial-driven nitrogen cycling processes, are important for evaluation and prediction of changes in coastal ecosystem services and management decision. However, in estuarine ecosystem, the majority of previous studies have considered coupled nitrogen-cycling processes to be exclusively sediment based, with little focus on the water column. Based on this, taking a typical macrotidal estuary-Hangzhou Bay estuary as the research object, a variety of molecular biology techniques were used to investigate the distribution and diversity of nitrogen-cycling microbes, the spatial distribution of nitrogen-cycling microbes on the suspended particulate matter (SPM), the evolution rule of microbial community structure and its environmental regulation information in Hangzhou Bay estuary. The results of this study provide a theoretical basis for us to understand the nitrogen cycle of estuarine ecosystem and enrich biogeochemical processes in estuaries. The main conclusions are as follows:(1) Based on the study of Hangzhou Bay estuary, the distribution characteristics and changing regularity of environmental factors in Hangzhou Bay estuary (water column and sediment) were systematically analyzed. The results showed that the water bodies in Hangzhou Bay estuary were high oxygen (4.90-7.36 mg/L), high nitrogen(61.75-182.76 ?M) and high SPM (0.02-8.07 g/L). Ranging from 61.47-182.13 ?M,nitrate was found to be the main source of dissolved inorganic nitrogen (DIN) in the water column of Hangzhou Bay estuary, accounted for 95.85-99.80% of the DIN. And ammonium was the main form of DIN in sediments, and its concentration ranged from 3.84 to 86.52 mg/kg. The chlorophyll a concentration in the water column was ranged from 0.26 to 1.75?g/L, indicative of poor phytoplankton growth in the water column of Hangzhou Bay estuary. The results of correlation analysis showed that SPM was found to be negatively correlated with nitrate and positively correlated with nitrite.(2) The abundance and diversity of main functional genes (AOA amoA, AOB amoA, nirK, nirS) of nitrogen cycle microorganisms in Hangzhou Bay estuary (water column and sediment) were systematically analyzed by fluorescence quantitative PCR technique. In the water column, the abundances of AOB awmoA and AOA amoA genes were 105-108 copies/L and 105-107 copies/L, respectively. The abundance of ammonia-oxidizing bacteria was larger than that of ammonia-oxidized bacteria in most of the water samples; the abundances of nirS and nirK genes were 106-108 copies/L and 105-107 copies/L, which showed the absolute superiority of nirS type denitrifiers in the water column. The results of correlation analysis showed that there were significant positive correlations between the concentration of SPM and the abundances of nitrifying, denitrifying and anaerobic ammonium oxizing genes. At the same time, the abundances of AOB amoA and nirK genes were negatively correlated with the concentration of nitrate, while the abundance of AOA amoA and nirS genes showed no significant correlation with nitrate concentration. In the sediment, the abundances of AOB amoA and AOA amoA genes were both 105-107 copies/g dry weight; the abundances of nirS and nirK genes were 106-107 copies/g dry weight and 104-106 copies/g dry weight, which showed the absolute superiority of nirS type denitrifiers in the sediment. For the abundance of anammox bacteria, it ranged from 103-106 copies/g dry weight. In general, the abundance of major functional genes in nitrogen cycle was in the order of nirS>AOB amoA> AOA amoA>AMB 16S>nirK. The correlation analysis showed that nitrite nitrogen and pH were the main environmental factors affecting the abundance distribution of nitrifying and denitrifying bacteria. At the same time, the abundance of AOB amoA and nirS genes were significantly positively correlated with nitrite concentration, while the abundance of AOA amoA gene showed no significant correlation with nitrite concentration.(3) The spatial distribution characteristics of main functional microbes in nitrogen cycle of Hangzhou Bay estuary (water column and sediment) were systematically investigated by fluorescence in situ hybridization. In the water column,the presence of nitrifiers and denitrifiers in the bacterial consortia of the SPM was examined by fluorescence in situ hybridization (FISH) analysis. In situ hybridization clearly indicated that ammonia oxidizing bacteria and denitrifiers were not uniformly distributed within the SPM, and ammonia oxidizing bacteria were in greater abundance than acetate-denitrifying cells, while nearly no methanol-denitrifying cells were detected. Specifically, the majority of NSO190 probe-stained AOB and DEN1 24 probe-stained acetate-denitrifying cells formed irregularly shaped (2-10 ?m), dense aggregates, with ammonia-oxidizing microcolonies found mainly in the outer part of the aggregate and acetate-denitrifying cells in the middle. In the sediment, the presence of nitrifiers and denitrifiers in the bacterial consortia of the sediment was examined by fluorescence in situ hybridization (FISH) analysis. In situ hybridization clearly indicated that ammonia oxidizing bacteria were in greater abundance than acetate-denitrifying cells, while nearly no methanol-denitrifying cells were detected.Specifically, the majority of NSO190 probe-stained AOB and DEN124 probe-stained acetate-denitrifying cells formed irregularly shaped (20 ?m), dense aggregates.Ammonia-oxidizing bacteria and acetate-denitrifying cells were evenly distributed in aggregates.(4) Based on the second generation high-throughput sequencing technique, the succession regularity of the total bacterial community structure and the environmental regulation information in the Hangzhou Bay estuary (water body and sediments) were systematically analyzed. In the water column, Proteobacteria, Planctomycetes and Bacteroidetes were the most abundant phylum/class. The results of redundancy analysis (RDA) suggested that depth, temperature, SPM and particulate organic carbon played important roles in shaping the bacterial communities in the water column. The Betaproteobacterial Nitrosomonas and Nitrosospira, and the Gammaproteobacterial Nitrosococcus were observed in this study. The Nitrosomonas group was the dominant genus of AOB. In addition, the relative abundance of Nitrosomonas showed strongly negative correlations with SPM, NO2- and AOB amoA gene abundance, and a weakly positive correlation with NO3-. The denitrifying genera, such as Bradyrhizobium Comamonas, Thauera, Stenotrophomonas, Acinetobacter, Anaeromyxobacter,Sulfurimonas, Paenibacillus and Sphingobacterium, which may play important roles in the water column denitrification process. In the sediment, Proteobacteria,Planctomycetes and Bacteroidetes were the most abundant phylum/class. The results of the RDA suggested that pH and nitrite played important roles in shaping the bacterial communities. The Nitrosococcus group was the dominant genus of AOB. The denitrifying genera, such as Pseudomonas, Halomonas, Anaeromyxobacter,Paenibacillus, Flavobacterium, Flexibacter, Sphingobacterium and Chryseobacterium, which may play important roles in the sediment denitrification process.