Dynamics Of Denitrification And Related Functional Microorganisms In Estuarine Intertidal Wetland Sediments

Estuarine intertidal wetlands are the critical transitional zone between land and sea,and are a complex and multi-functional ecosystem.In recent decades anthropogenic reactive nitrogen has increased extensively due to the increasing use of fertilizer and the combustion of fossil fuels.However,considerable Nr is eventually transported into estuarine and coastal wetlands through river flow,groundwater discharge and atmospheric deposition,which has caused a series of environmental problems.Denitrification,the dissimilatory reduction of oxidized N compounds?NO₃^-and NO₂^-?to gaseous nitrogen?including NO,N₂O,and N₂?,has been considered an effective microbial nitrogen removal process.However,the microbial driving mechanisms and the links between the dynamics of denitrifiers and the physical,chemical and biological factors of estuarine ecosystem remain uncertain.In addition,the secondary environmental problems of greenhouse gas N₂O producted by denitrification process are the global hot topic.China harbored a long coastline and abundant estuaries,spanning multiple temperature zones from south to north.Particularly,the Yangtze estuary with extensive intertidal wetlands and situated at the mouth of the East China Sea,is a typical area to explore the regional response to the global change.In this study,we used ¹⁵N isotope-tracers and molecular techniques to explore the dynamics of denitrifying bacteria along the latitudinal gradient of China estuarine intertidal wetlands.Furthermore,the response of denitrifiers to the complex and changeable intertidal environmental factors?such as salinity and tidal dynamics?was identified in the Yangtze estuary.The main findings are as follows:?1?Clone library analysis revealed that the distribution and composition of nirS-harboring denitrifiers had a significant latitudinal differentiation along the coastal wetlands of China,probably due to the defference of climate and sediment structure.However,the community of denitrifiers did not display a significant seasonal shift,indicating that the denitrifiers distribution reflects an adaption to site-specific features.Diverse nirS sequences were obtained and almost all of them shared considerable phylogenetic similarity with sequences obtained from aquatic systems?marine/estuarine/coastal sediments and hypoxia sea water?,suggesting that all the microorganisms in this study derived from coastal rather than terrestrial environments.The PCoA and canonical correspondence analysis showed that temperature was a key factor affecting not only the nirS gene abundance but also the community structure of nirS-type denitrifiers.The nirS gene abundance ranged from 4.3×10⁵ to 3.7×10⁷copies g^-1 dry sediment,with a significant spatial heterogeneity.The denitrification rates ranged from 20.88 to 182.04?mol N kg^-1 d^-1,and positively correlated with nirS gene abundance.The denitrification rates were higher than anammox or DNRA rates,indicated the important role of denitrification in nitrogen reduction of China intertidal wetland systems.It was estimated that denitrification can remove about4.70×10⁵ t N yr^-1,which equaled to 19.5%of the total external terrigenous inorganic nitrogen annually transported into China intertidal wetland environment.?2?Due to the unique geographical location and estuarine processes,the Yangtze estuary harbors steep environmental gradients,especially salinity.And the diversity of denitrifying bacteria at the lower-salinity sites was significantly higher than the higher-salinity sites in the marsh sediments of the Yangtze estuary.Phylogenetic analysis revealed that the denitrifiers community also showed distinctive spatial heterogeneity along the Yangtze estuary.At the lower-salinity sites,the nirS-harboring bacterial community was co-dominated by Clusters?and?,while at the higher-salinity sites it was dominated by Cluster?.Quantitative PCR results showed that the nirS gene abundance of the lower-salinity sites(6.37×10⁶ to 9.00×10⁷ copies g^-1)was significantly higher than higher-salinity zones(1.01×10⁶ to 7.50×10⁶ copies g^-1),but without significant seasonal variation.In addition,the denitrifying bacterial nirS gene abundance was higher than anammox 16S rRNA gene in the marsh sediments of the Yangtze estuary.The denitrification rates showed significant seasonal shift in summer(41.82 to 67.13 nmol N g^-1 h^-1)and winter(12.04 to 20.03nmol N g^-1 h^-1).Denitrification process was estimated to remove approximate5.31×10³ t N yr^-1 from the Yangtze estuary.Therefore,the Yangtze estuary marsh sediment as an effective nitrogrn filter,is important for reducing the reactive nitrogen input into the East China Sea system.?3?The intertidal flat harbores three different habitats:high,middle,and low tidal flats,which may affect the community structure and distribution of denitrifiers.The high-throughput sequence method was used to further explore the response of denitrifying microorganisms to different habitats.The coverage of all libraries was more than 99%,which was higher than colne sequence method.The patterns of community composition of denitrifying bacteria differed across the estuary,especialy in the low tidal flat sites.Interestingly,there were lots of co-existed denitrifying bacterial species between middle tidal flat and low or high tidal flat environment,indicated that the denitrifying community showed differentiation gradually from land to sea.The potential denitrification rates and N₂O production rates ranged from 4.86to 9.22?mol N kg^-1 h^-1,and 49.87 to 236.01 pmol N g^-1 h^-1,respectively.However,there was no significant relationship between N₂O production rates and denitrification rates in this research.?4?The intertidal sediments were submerged periodically during tidal dry-wet cycles,which might shift the transformation and distribution of sediment nutrients.Hence,we simulated the different dry-wet cycles of high?H?,middle?M?,and low?L?tidal flat to explore the response of nitrogen transformation processes to tidal effects in estuarine intertidal systems.The results showed that the periodic submergence significantly affected the nitrogen transformation rates.After a period of exposure,the denitrification and N₂O production rates increased sharply when the overlying water added,which might due to the“Brich effect”.In summary,the rates of H group were lower than L and M groups,indicated that in the sediment-water interface,the nutrients was correspondingly diffused to the sediment phase through the overlying water,then promoting the nitrogen transformation process in the surface sediment.In addition,tidal dry-wet cycles enhanced the coupling of multiple nitrogen cycle.Moreover,both of the nirS gene abundance and denitrification rates in all groups were gradually lower than the previous results in natural environment.These results indicated that the land-sea interactions and the tidal dynamics could not only provide the substrate for denitrifying bacteria,but also promote the nutrients exchange within the sediment-water interface,further promoting the estuarine intertidal wetland ecosystems developing into a hot area of nitrogen loss.