Our study objectives were to investigate both the nutrient limitation status of these ponds using nutrient diffusing substrate experiments and to examine how pond biofilm communities were affected by nutrient amendments using targeted high-throughput gene amplicon sequencing.

Aquatic ecosystems are often limited by nitrogen and/or phosphorus. Our results from nutrient-diffusing substrate experiments suggest that phosphorus may limit pond photoautotrophs. However, despite low water column nutrient concentrations, biofilm biomass was not enhanced by the addition of nitrogen and phosphorus; instead, we observed a ~60% biomass reduction with nitrate addition. We observed widespread reduction in biomass in 15 ponds (Copper River Delta, Alaska) across two years, even when log-lower concentrations of nitrate were added. Biofilm nutrient responses? also translated into differences among microbial communities. Control communities were characterized by anaerobic heterotrophs and purple nonsulfur bacteria, which are phototrophs that use either organic carbon or H2 as electron sources. In contrast, nitrate-amended communities were characterized by aerobic heterotrophs and facultatively aerobic heterotrophs, which are capable of fermentation or of using both oxygen and nitrate as electron acceptors (i.e., denitrifiers). These community patterns suggest that nitrate can drastically affect microbial interactions during biofilm formation by altering redox conditions. The reduced biomass observed on nitrate-amended substrates could be due to a nitrate toxicity effect or potential antimicrobial compound release by denitrifiers, both of which could shape biofilm formation and community assembly. Our experiment may be indicative of what happens in low-nitrate, aquatic environments after novel exposure to anthropogenic nitrate inputs, which could result in temporary or long-term shifts in microbial communities and ecosystem function depending on resiliency. Therefore, these results have important implications for ecosystem restoration and successional processes.