NO3- concentrations have been rising in Loch Vale Watershed (LVWS) located along the eastern slope of Rocky Mountain National Park from a mean annual concentration of 0.23 +/- 0.02 mg L-1 between 1991 and 1999 to a contemporary concentration of 0.34 +/- 0.06 between 2000 and 2006. While total nitrogen in the precipitation has been increasing over this period the signal has become increasingly dominated by NH4+ rather than NO3-. We collected precipitation from two national atmospheric deposition sites (NADP CO89 and CO98) within the watershed, a snow pit dug in May below tree line, and an alpine snowfield located above the highest lake in the watershed in September. In addition we collected surface water samples at four times during the ice free season, from two headwater lakes, a connecting stream, and the lowest lake in the watershed during 2011. At the outlet to the watershed we collected surface water samples weekly, year-round. We used dual (15N,18O) isotope analysis of NO3- to evaluate whether NO3- in the watershed was arriving in precipitation and accumulating (inert), or whether NH4+ was being converted to NO3- by Bacteria and Archaea (reactive).
While the δ15N-NO3- of all precipitation samples ranged from ~ -5 to 5‰ the δ15N-NO3- of the all surface water samples was constrained within that range between -1 to 2‰. However, the δ18O-NO3- for precipitation was enriched in O18 (60 to 80‰) relative to the surface water (0 to 50‰) and thus the two pools of NO3- did not overlap in oxygen isotope concentration suggesting that the nitrate in the surface water had been nitrified by microorganisms and was distinct in origin from NO3- in precipitation. In addition, the δO18-NO3- of the surface water became increasingly depleted in O18 suggesting an increasing contribution of nitrification to the dissolved NO3- pool as the season progressed. We used bacteria community analysis of the active (16S rRNA) and total community (16S rDNA) coupled with analysis of relative nitrifier abundance (qPCR of amoA gene) to identify hotspots for nitrification in time and space within LVWS. Understanding the microbial mechanism for this watershed scale biogeochemical signal has important implications for how alpine ecosystems are being altered in the face of global change.