Spatial And Temporal Variation And Microbial Mechanisms Of N₂O Emissions From Artificial Pond Systems In Small Mountain Watersheds

As the most commonly used water conservancy facility in southern farmland,artificial ponds with a long history are widely distributed throughout China.With the development of agriculture and human activities,the conflict between irrigation water demand and the spatial and temporal heterogeneity of precipitation has become more and more pronounced.In addition,water sources are being contaminated by nitrogenous nutrients as a result of the unreasonable extraction and usage of resources.Although the surface area of artificial ponds is smaller than that of other large lakes or rivers,they have a higher potential for N₂O emissions.In order to evaluate the impact of small water bodies in greenhouse gas（GHG）emissions,it is crucial to define the geographical and temporal distribution of N₂O emissions from artificial reservoirs.However,the environmental characteristics and spatial and temporal variation of artificial reservoirs are still unclear.The environmental conditions and mechanisms of microbiological activities involved in the nitrogen cycle that concentrate on generating N₂O emissions are all still in development.In this study,small hilly watershed with typical manmade ponds were chosen as the study region and were observed during four seasons of the year.To understand the variations in the temporal and geographical distribution of N₂O emissions from artificial water pond systems in small mountainous watersheds,the physicochemical characteristics and in situ monitoring of N₂O dissolution and emission were examined in each season.At the same time,the microbial mechanisms of N₂O emission in artificial water pond systems were investigated by combining isotope tracing and molecular biology techniques.In order to clarify the various characteristics of N₂O emissions from freshwater ecosystems under various temperatures,we further investigated the impact of temperature,the primary environmental factor,on N₂O emissions from artificial water pond systems in the context of indoor regulation experiments.The main conclusions are as follows:（1）In this study,the characteristics of physical and chemical properties of sediments from artificial water ponds were analyzed from the perspective of different sampling seasons.As a whole,the pH of the overlying water of the pond was neutral or weakly alkaline;NH₄⁺,the main form of inorganic nitrogen（DIN）in the sediment,was significantly higher in summer than that in autumn and winter;the sediment possessed a higher average TN concentration（2.85g/kg）;and the TP content of the sediment was significantly higher in winter than that in other three seasons.（2）A combination of in situ monitoring and the model for calculating N₂O emission fluxes at the water-air interface was used.The N₂O concentrations in the overlying water of the reservoir were measured seasonally and continuously for 24h.The concentration of N₂O dissolved in the overlying water was found to be higher in summer and fluctuated throughout the day which was inconsistent at the start and end times.Corresponding to the pattern of N₂O dissolved in the overlying water,N₂O emission fluxes at the water-gas interface of the reservoir were the highest in the summer of 2021 and fluctuated throughout the day.（3）Seasonal distribution of nitrogenous nutrient transformation processes and N₂O production in artificial pond sediments were based on the isotope tracing and slurry incubation experiments.Denitrification rate was relatively high,which varied from 7.79μmol/kg·h to27.15μmol/kg·h.The denitrification rate of sediments was mainly influenced by pH,NH₄⁺,NO₃^-,NO₂^-and TP.The rate of anaerobic ammonia oxidation in sediments was relatively low,ranging from 0.17μmol/kg·h to 1.34μmol/kg·h,which was significantly higher in winter than that in other sampling seasons.The main environmental variables affecting the rate of anaerobic ammonia oxidation were NO₂^-and TP.The high potential for N₂O emissions was measured in February and July in 2022.The surface sediments of the ponds,with the exception of Pond 3,exhibited the characteristics of a"source"of N₂O emissions among the sediments of different depths.（4）The abundance,diversity and community composition of denitrifying bacteria in artificial pond sediments were investigated.It was also combined with the characteristics of N₂O emission processes in pond sediments to preliminarily analyze the microbial mechanisms of N₂O emission processes by denitrifying microorganisms.In the pond,nir K gene abundance was noticeably higher than nir S gene abundance,with the highest abundance of nir K genes in winter.nir S gene abundance showed no significant patterns in different sampling months.The results of Pearson correlation analysis showed that nir K gene abundance was significantly positive with pH,NO₃^-,NO₂^-and denitrification rate,but was negative with NH₄⁺.Unlike nir K genes,nos ZⅠand nos ZⅡgenes showed significant positive correlations with NH₄⁺.Besides,nir S-type denitrifying bacteria in pond sediments at the class level（except for the unannotable）consisted mainly ofα,βandγamoebae,with significant spatial and temporal divergence between sampling seasons as well as between ponds.（5）Temperature,which was found to significantly affect the denitrification process,was used in the study to further investigate the response pattern of N₂O emissions in freshwater sediment to temperature through regulation experiments.The results showed that freshwater sediments had the highest N₂O emission fluxes at high temperatures.But the highest emission potential appeared at room temperature.And the sediment denitrification rate gradually increased and NO₃^-content gradually decreased with the increasing temperature.Combined with molecular biology,it was concluded that nir K-type denitrification functional genes were dominant in denitrification process and N₂O production.