Denitrifying Anaerobic Methane Oxidation And Its Influencing Mechanism In Estuarine Intertidal Wetlands

In recent decades,estuarine and intertidal wetland ecosystems receive a large amount of nitrogen（N）from industrial,agricultural and domestic sources.At the same time,they are also one of the significant natural sources of atmospheric methane（CH₄）,which plays an important role in regulating global atmospheric CH₄ concentration.Therefore,it is of great significance to explore the coupling between N cycle and CH₄metabolism,which further help understand the ecological service functions of estuarine and intertidal wetland ecosystems.Denitrifying anaerobic methane oxidation（DAMO）is a crucial link between carbon（C）and N cycles in estuarine and intertidal wetland ecosystems.This process can achieve the removal of inorganic N with anaerobic oxidation of methane,which has the dual potential to reduce the greenhouse effect and alleviate aquatic eutrophication.However,the dynamics of DAMO microbes and associated driving mechanisms in estuarine and intertidal wetland ecosystems remain unclear.Thus,this study took the Yangtze Estuary as a typical study area to explore the dynamic characteristics of composition,diversity,abundance and activity of DAMO microbes and associated driving mechanisms via ¹³C stable isotope technology and molecular biological methods under the effects of salinity gradient,environmental deviation,salt plant and tidal wave,which can provides the scientific reference for the CH₄ emission mitigation in estuarine wetland ecosystem.The main findings are as follows:（1）Along the Yangtze Estuary salinity gradient,there was a relatively higher abundance of the DAMO bacteria than the DAMO archaea.The abundance and activity of DAMO microbes showed significant spatiotemporal variations.The DAMO microbial abundance and activity tended to be higher in the warm season and in the upstream freshwater and low-salinity estuarine habitats.The abundance of the DAMO bacteria was closely associated with sediment pH and ammonium,and the co-regulation mechanism of multiple electron receptors might lead to the differentiation of abundance in DAMO archaea.The DAMO potential was mainly controlled by sediment pH,total organic carbon,Fe（II）,and Fe（III）contents.The methane oxidation capacity of DAMO bacteria was greater than that of DAMO archaea in estuarine and intertidal wetlands of the Yangtze Estuary.In addition,the contribution of nitrite-DAMO process to nitrogen removal was similar to that of anammox process,but lower than that of denitrification process.（2）DAMO bacteria and DAMO archaea coexisted in the sediment profile of the intertidal marshes,and the community composition and diversity showed a profile distribution pattern with the depth of sediment.The diversity of DAMO bacteria at the sediment-tidal water interface layer is higher than that at the middle and sediment-groundwater interface layers,and the community composition of DAMO bacteria at the sediment-tidal water interface layer is significantly different from that at the sediment-groundwater interface layer.DAMO archaea showed high diversity at the sediment-tidal water interface and sediment-groundwater interface layers,and their community structure was different along the vertical environmental changes of the intertidal marshes.The diversity of DAMO archaea was higher than that of DAMO bacteria.The abundance of DAMO bacteria is similar to that of DAMO archaea.In the sediment profile of the intertidal marshes,the abundance and activity of DAMO microbes showed a significant vertical distribution.The DAMO bacteria abundance was higher at the sediment-tidal water interface and sediment-groundwater interface layers,while the DAMO archaea abundance was higher than that of DAMO bacteria at the middle layer.DAMO process rates were higher active at the sediment-tidal water interface layer than those at the middle and sediment-groundwater interface layers.The variation of Eh and NO₂^-/NO₃^-availability in sediments induced by tidal action might be an important potential factor for the vertical distribution pattern of the community composition,diversity,and abundance of DAMO microbes in intertidal marshes.（3）DAMO bacteria and archaea co-existed in estuarine saltmarsh ecosystem,and the abundance of DAMO bacteria was higher than that of DAMO archaea.Compared with bare mudflat stands,the DAMO bacteria abundance in saltmarsh stands was relatively high,while the DAMO archaea abundance was relatively low.Estuarine saltmarsh ecosystems had a high potential for nitrite-DAMO and nitrate-DAMO.Mean total DAMO rates ranked as Spartina alterniata stands>Scirpus mariqueter stands>Bare mudflat stands>Phragmites australis stands.Nitrification,DNRA,Fe-ammox and anammox processes might be important processes controlling DAMO bioprocesses in estuarine saltmarsh ecosystems.In addition,the variations in sediment moisture content,pH,TOC,C and N matrix and CH₄ availability under the influence of different salt plant types might be important potential factors for the DAMO bioprocesses.Saltmarsh plants significantly altered the rhizosphere sediment methane anaerobic oxidation pattern,which tightly depended on saltmarsh plant types.Among all the methane anaerobic oxidation pathways,DAMO bioprocesses were dominant in bare mudflat,Scirpus mariqueter and Spartina alterniflora stands,which accounted for22.4-27.5%and was second only to SAOM.The estimated contribution of the nitrite-DAMO process to N removal followed the order of Scirpus mariqueter stands>Spartina alterniflora stands>Bare mudflat stands>Phragmites australis stands.In addition,compared with tidal flat wetland of the Yangtze estuary,methane reduction capacity by nitrite-DAMO and nitrate-DAMO processes increased by about 25%and200%,respectively,while nitrogen removal capacity by nitrite-DAMO process increased by about 40%.Its contribution to nitrogen removal was greater than that of anammox process but was less than the denitrification process.（4）The coexistence of DAMO bacteria and archaea in the tide-fluctuating environments was detected,and there was generally higher biodiversity under reflooded conditions than consecutive inundation or emersion.The DAMO microbial abundance and associated activity varied significantly during alternative exposure and inundation,with higher abundance and activity under the waterlogged than desiccated conditions.In the reflooding conditions,the abundance and activity of DAMO bacteria and archaea decreased after desiccated treatment,and then increased with sediment waterlogged treatment.Tidal-fluctuating environments significantly affected the DAMO bioprocesses.Sediment moisture content,Fe（III）,NO₃^-and NH₄⁺were important factors affecting DAMO bioprocesses under inundation,while sediment moisture content,salinity,pH and enzyme activity were important factors affecting DAMO bioprocesses under the desiccation.Reflooding of intertidal wetlands might intensify DAMO activities,indicating the resilience of DAMO microbial metabolisms to the wetting-drying events.In addition,the methane reduction and nitrogen removal capacities of DAMO processes under tidal conditions were relatively weak compared with the tidal flat wetland and salt marsh wetland of the Yangtze Estuary.（5）Based on the measured DAMO rates,it was estimated that the annual CH₄content removed by nitrite-and nitrate-DAMO processes in estuarine and coastal wetlands accounted for about 17.9%and 17.1%of the total annual methane emissions in estuarine and coastal wetlands.Meanwhile,potential N elimination attributed to nitrite-DAMO was roughly evaluated at 1.3×10⁵ tons N.It accounted for 9.8%of the total nitrogen reduction,which was second only to denitrification process（82.4%）and larger than anammox process（7.8%）.Therefore,DAMO bioprocess in estuarine and coastal wetlands is an important pathway of CH₄ reduction and N removal which has been previously neglected,and thus it has a potential contribution to elimination of CH₄greenhouse emissions and aquatic eutrophication in estuarine wetland ecosystems.