Nitrogen (N) is a key element sustaining life and is one of the most abundant elements on earth . In 2050, 60% of global production of N is expected to come from anthropogenic sources  such as industrial fertilizer production, biological fixation of N in agricultural systems, and combustion. The increases in N production have resulted in large increases in the fluxes of N in nature . Unintentional N enrichment has a variety of negative environmental impacts, including soil acidification , reduction of global terrestrial biodiversity , increased nutrient runoff to aquatic ecosystems, and eutrophication worldwide , and such changes can ultimately impact ecosystem services and human well-being .
In aquatic ecosystems, bacteria-mediated denitrification is potentially an important pathway for N removal by converting nitrate/nitrite into gaseous products (N2, NO, N2O) . Mitigation of anthropogenic N2O release is not least now in focus as N2O is an important greenhouse gas and a major cause of ozone layer depletion . As the only known N2O sink, enzymatic reduction to N2, controlled by denitrifiers harboring the nitrous oxide reductase gene (nosZ, consisting of type I and type II) , has received increasing attention [11-13]. For example, N fertilizer has been shown to reduce N2O emission from field crops , and N addition increased the denitrification potential in the Broadbalk wheat experiment . It has also been shown that high N2O emissions could be attributable to the legacy effect from previous N addition to cropland or to an interactive effect of N addition and climate change . However, the change of N2O-producing bacteria at different nitrogen loadings is unclear.
N addition increases the microbial biomass , decreases fungal diversity and alter the fungal community composition  in the soil. In lake sediments, N input enhanced the relative abundances of the genera Flavobacterium, Pseudomonas, Arenimonas, Novosphingobium, Massilia, Aquabacterium, and Bacillus but inhibited those of Sporacetigenium, Gaiella, Desulfatiglans, Nitrospira, and Haliangium . As ecological functions tie up closely with microbial communities, the changes of microbial communities inevitably impact functions . Soil studies have revealed that N addition decreased the population of microbial nitrogen fixers , weakened the biological nitrogen fixation capacity , and led to loss by denitrification [22, 23]. However, Kramer et al.  found that the application of N in orchards significantly enhanced the activity and efficiency of soil denitrifiers and reduced nitrate leaching. The ultimate effect of N addition on denitrifiers, however, remains unclear and needs to be elucidated at contrasting N loadings in different ecosystems.
In this study, the clade I group of nosZ gene was selected to quantify the abundance and communities of denitrifiers, because most microbes with nosZ clade I are complete denitrifiers and play an important role in the N2O reduction in aquatic systems , while the nosZ clade II is more relevant in soils . In order to investigate the direct influence of N on the abundance and composition of nosZ I denitrifying bacteria, we collected sediments (0-20 cm) from the long-term (4 years) experimental ponds of five nitrogen fertilization treatment (TN10, 10 kg NH4Cl per month; TN20, 20 kg NH4Cl per month; TN30, 30 kg NH4Cl per month; TN40, 40 kg NH4Cl per month; TN50, 50 kg NH4Cl per month) in the north-eastern part of Bao’an lake in Wuhan, China. We hypothesize that (a) high N concentration will reduce the abundance of denitrifying bacteria, (b) denitrifying bacteria communities will cluster according to the N gradient, and (c) the differences of nosZ I communities will reflect adaptive shifts by the microbial communities to the N concentration that they face.