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Nitrate amendment reduces biofilm biomass and shifts microbial communities in remote, oligotrophic ponds

Humans have increased the amount of reactive N available in the environment by over an order of magnitude since the industrial revolution. Most studies have been conducted in ecosystems with pervasive anthropogenic nutrient inputs, so little is understood about how naïve biofilm communities respond to elevated nutrients. Our nutrient-diffusing substrate (NDS) experiments, which were conducted in Alaskan freshwater ponds with very little anthropogenic nutrient inputs, suggest that P limits biofilm photoautotrophs. However, despite low water-column nutrient concentrations, overall biofilm biomass was not enhanced by the addition of N or P. Rather, we observed an ~60% biomass reduction with NO3– amendment in 15 oligotrophic ponds across 2 y. This widespread biomass reduction was accompanied by changes in microbial communities, but these trends were not observed with NH4+ or P amendment. Nonamended communities (i.e., no nutrient amendment other than lysogeny broth agar) were characterized by anaerobic heterotrophs and purple nonsulfur bacteria, whereas NO3–-amended communities were characterized by aerobic heterotrophs and facultatively aerobic heterotrophs (e.g., denitrifiers). These community patterns suggest that NO3– can strongly affect microbial interactions during biofilm formation by altering redox conditions. The effect of NO3– on microbial biomass may be caused by an NO3– toxicity effect or competitive shifts in taxa, both of which may shape biofilm formation and community assembly. Our results reveal possible consequences for low-NO3–, aquatic environments after novel exposure to anthropogenic NO3– inputs, suggesting that a legacy of anthropogenic NO3– inputs may have fundamentally changed microbial community assembly and biogeochemical cycling in aquatic ecosystems.