Investigating biotic uptake of riverine organic nitrogen using a compound-specific stable-isotope probing approach.

The flux of nutrients into rivers is rising due largely to inputs from the expansion and intensification of agriculture along with inputs from treatment of human waste. This trend is set to continue due to changing climate and increasing population while we attempt to balance food security and environmental impact. While water quality legislation focuses on inorganic nutrients due to their bioavailability, the proportion of the total nitrogen (N) flux, which is organic in its molecular composition is important in many riverine systems. Despite this, the impact of organic N on ecosystem function is currently poorly understood. Here we address part of this knowledge gap using compound-specific stable isotope probing to investigate the extent to which dissolved organic matter substrates are bioavailable to stream biota and if they can be directly assimilated.Stable isotope probing was used to identify and quantify the routes of biotic uptake of organic N and carbon (C) into stream biota. Here, we added 15N labelled (nitrate, ammonium, glucosamine, sheep urine) and doubly labelled (15N/13C) substrates (glutamic acid, urea, glycine) to in-stream mesocosms containing water and epilithon, and bryophyte communities from the River Conwy North Wales, UK. Samples of epilithon and bryophyte were removed from the incubations after 2, 6, 12, 24 and 48 h and rates of assimilation of the labelled substrate were determined using bulk 15N/13C, followed by compound-specific 15N/13C analysis of extracted amino acids. This method allowed us to demonstrate the assimilation of labelled organic substrates into newly biosynthesised proteinaceous amino acids and to determine if they were utilised as intact organic molecules.The findings showed that the majority of the organic N substrates tested were directly bioavailable for utilisation as intact molecules by the stream biota, except for urea where transformation occurred before uptake. The data also showed that there were differences in the rates of assimilation both between the organic substrates added and between the epilithon and bryophyte communities. This work illustrates the analytical power of using doubly labelled 13C, 15N compounds in a stable isotope probing experiment, as the ability to trace the utilisation of both the N and C simultaneously had provided significant new insights in the biotic assimilation of organic-N substrates. Our findings confirm the importance of organic nutrients in ecosystem function and the need for changes to water quality legislation to reflect this.