Study On The Change Of Nitrogen And Oxygen Isotope Ratio Of Nitrate During Denitrification In Vadose Zone Of Farmland Soil

As an important media space for biogeochemical circulation systems,unsaturated zone is of great significance for slowing down nitrate(NO₃^-)pollution in groundwater.Unsaturated zones where soil microbial communities exhibit wide distribution,high activity,and abundant diversity are hotspots for NO₃^-attenuation.Microbially-driven denitrification is one of the main pathways for the removal of NO₃^-from the inclusion zone.A constant ratio of the relative increase in?¹⁸O versus?¹⁵N in association with NO₃^-attenuation has been broadly used in the literature as a diagnostic tool to identify denitrification process.However,soils collected from unsaturated zones showed the ratios of??¹⁸O:??¹⁵N ranged from0.71 to 2.24,part of which were higher than the canonical ratios of 0.5 to 1 for denitrification reported in other terrestrial and freshwater systems.In this study,phylogenetic molecular ecological networks(p MENs)analysis was used to investigate the nitrogen microbial network interactions at different??¹⁸O/??¹⁵N ratios.The main results are as follows:(1)The physical and chemical properties of the six sampling sites showed obvious horizontal pattern,with the p H and salinity content of the soil at the southern sampling sites being the highest,followed by the central area sampling sites,and the northern sampling sites being the lowest.The trends of soil salinity in the profiles were different:the surface soil of the southern samples have a low salinity and salt accumulation in the deep layers,while the opposite is true in the north.With the NO₃^-attenuation in the profile,both?¹⁵N-and?¹⁸O-NO₃^-in the sampling sites showed an increasing trend,and they were significantly linearly correlated.According to the??¹⁸O/??¹⁵N ratios,the sampling sites could be divided into??¹⁸O/??¹⁵N<1 group and??¹⁸O/??¹⁵N>1 group.(2)The diversity and number of species of soil microbial communities differed among two??¹⁸O/??¹⁵N groups:the??¹⁸O/??¹⁵N>1 group had more diversity and number of species of soil microbial communities.At the taxonomic level of the phylum,the??¹⁸O/??¹⁵N>1 group of Thaumarchaeota and Chloroflexi had significantly higher relative abundance,while Proteobacteria and Bacteroidetes relatively lower.(3)The molecular ecological networks of the two??¹⁸O/??¹⁵N groups showed that both p MENs were modular and scale-free,and denitrifying bacterial were the main drivers.However,the topological properties of the p MENs and the dominant nitrogen transformation processes differed between the two groups.In the??¹⁸O/??¹⁵N<1-p MEN,denitrification and DNRA were the main nitrogen transformation processes,and under the influence of variable environmental factors in the unsaturated zone,denitrifying and DNRA bacteria showed complex cooperative and competitive relationships.In the??¹⁸O/??¹⁵N>1-p MEN,the cooperative relationship between denitrifying bacteria and NOB act as the main nitrogen transforming community,and the partnership between the two may occur as a result of the NOB possessing nitrite oxidoreductase(NXR),which undergoes nitrite oxidation simultaneously with denitrification under anoxic/anaerobic conditions.The"reverse"kinetic isotope effect of nitrite oxidation may be responsible for the appearance of??¹⁸O/??¹⁵N>1.