Nitrogen Removal Process And Microbial Characteristics Of Artificial Ponds In Small Watersheds In Mountainous Areas

The construction of artificial ponds dates back nearly 3000 years in China.As an integral part of agricultural landscape,ponds can effectively regulate the hydrological processes in a watershed,such as storing water for irrigation and reducing the rate of runoff,and play a key value for agricultural development.With the development of agriculture and the influence of human activities,nitrogen pollution has become one of the most crucial environmental issues in the 21st century.Ponds,the special type of wetland systems,are considered as biological filters that provide security for water resources such as estuaries,groundwater,lakes and streams.Ponds retain agricultural runoff through interception and remove it through internal transformation,while the role of microbes is often overlooked in this process.As a hotspot for nitrogen removal,the form of nitrogen in ponds is still unclear,the microbial nitrogen removal processes and dominant pathways are rarely discussed.Besides,studies on the microbial mechanisms and environmental influences of the nitrogen removal process in ponds are also insufficient.In this thesis,typical artificial ponds in small mountainous watershed are selected and monitored for four seasons a year.By analyzing the physicochemical parameters of water and sediments in different periods,to clarify the spatial and temporal variation patterns of nitrogen pollution in ponds.The combination of isotope tracing techniques and molecular biology methods is conducted to investigate the spatial and temporal variations of nitrogen removal in the ponds,and then to reveal the microbial mechanisms of nitrogen removal in artificial ponds.Finally,the main influencing factors of microbial nitrogen removal in pond sediments are studied through the regulation of environmental factors.The main conclusions are as follows:(1)This study seasonally analyzes the spatial and temporal characteristics of the overlying water and sediment physicochemical parameters in the pond samples.Overall,the p H of the overlying water is generally neutral.Dissolved inorganic nitrogen(DIN)is mainly in the form of NO₃^-.There are no significant seasonal differences in DIN.TN and TP show lowest contents in May in overlying water.The pond sediments are abundant in nutrients,especially TN(2.3±1.1 g kg^-1)and TOM(63.7±8.4 g kg^-1),and the TOM content decreased with increasing depths.DIN in the sediments is dominated by NH₄⁺,whose concentration is highest in May and August and lowest in December and February.the content of TN and TP is lowest in May.Besides,the p H of Pond 1(neutral)is significantly lower than the other four ponds(alkaline).(2)Based on isotope tracing and slurry incubation experiments,the spatial and temporal distribution of nitrogen removal from pond sediments is investigated.Denitrification is found to dominate microbial nitrogen removal process in ponds,with variations ranging from 0.3±0.1 to 13.6±1.0 nmol N g^-1 h^-1.Denitrification rates in the pond sediments are mainly regulated by p H,NH₄⁺,TN and TOM.Anammox rates are relatively low,ranging from 0 to 1.3 nmol N g^-1 h^-1,while the rates in May are significantly higher than the other three months.NH₄⁺,TN and TP are the main environmental factors determining the activity of anammox.(3)The abundance and diversity of denitrifiers and anammox bacteria are investigated in artificial pond sediments with the molecular biology method.Microbial mechanisms of denitrification and anammox processes are preliminarily analyzed in conjunction with the potential nitrogen removal rates in pond sediments.The abundance of nir S gene is significantly higher than that of nir K gene,in which nir S gene is highest in May and decreases with increasing depths.The spatial and temporal patterns of nir K gene are similar to those of nir S.Besides,the abundance of nos Z?gene is highest in May and August.16S rRNA and hzs B gene abundance of anammox bacteria are both lower(10⁴-10⁵ copies g^-1).Correlation analysis between environmental factors and abundance of denitrifiers shows that nir S gene abundance had a significant positive correlation with TOM,while unlike nir S gene,nir K showed a significant positive correlation with NH₄⁺and TP.As for the potential denitrification rates,there are significant positive correlations with nir S gene abundance and nir/nos Z ratio.In addition,anammox rates,which are well coupled with anammox 16S rRNA gene.The nir S-type denitrifiers are all affiliated to Proteobacteria at phylum level,which are divided into?,?and?Proteobacteria at the class level.As for the genus level,nir S-type denitrifiers are dominated by Azoarcus,Pseudomonas,Steroidobacter,Dechloromonas,Thermomonas,Sulfuritortus,Thauera and Aromatoleum,which show significant spatial and temporal divergence.Anammox bacteria are affiliated with Ca.Kuenenia,Ca.Brocadia and unclassified genus,with differences in composition mainly between ponds.(4)The effects of NO₃^-,NH₄⁺,DO,and glucose concentrations on the nitrogen removal processes in pond sediments are also studied by regulation experiments.NO₃^-addition stimulates the potential denitrification rates within a certain range,while the responses to NO₃^-addition are varied for overlying water with different NO₃^-concentrations in situ.The rates of anammox are little influenced with increasing NO₃^-concentrations.The addition of NO₃^-also promotes N₂O production rates,but the denitrification shows more complete(N₂O/(N₂O+N₂)ratio is reduced).Potential nitrogen removal processes in pond sediments are unaffected by NH₄⁺addition overall.Increasing DO concentrations show an inhibitory effect on denitrification process in pond sediments.Besides,denitrification process is more complete as DO concentrations increases when DO concentrations are below 2 mg L^-1.Glucose could promote the denitrification of pond sediments,while no effect is observed in the anammox process.