N2-fixing bacteria convert atmospheric N2 gas to biologically available forms, yet N limits primary production in many ecosystems. Despite an abundant source of N, other elements may limit N input via N2-fixation in benthic environments. We examined how N, P, and the micronutrients Fe and Mo (cofactors of nitrogenase) might affect N2-fixation. We used short-term nutrient additions to assess macronutrient effects on N2-fixation of intact epilithic biofilm in Ditch Creek, Wyoming (USA). Additions of NH4 , NO3−, and PO43− at varying concentrations did not alter N2-fixation over 4 h. We assessed macro- and micronutrient effects on N2-fixation, biofilm biomass, and composition of the biofilm algal assemblage at Ditch, Spread, and Kimball Creeks, Wyoming, via 6-wk nutrient amendments with nutrient-diffusing substrata. Fe and Mo additions did not affect N2-fixation or biofilm biomass, but NO3− and PO43− altered the biofilm biomass, algal assemblage structure, and N2-fixation. N and P colimited biofilm biomass in Ditch Creek, and P limited biofilm biomass in Spread Creek. N2-fixation was inhibited by NO3− and stimulated by PO43−. Inhibition by NO3− was stronger than stimulation by PO43−. Trends in accumulation of N2-fixers, mostly Epithemia sorex with N2-fixing endosymbionts, corresponded to effects of nutrient additions on N2-fixation. The algal assemblage shifted from N2-fixer dominance in control and P treatments to fewer N2-fixers and increased biovolume of nonN2-fixing diatoms in the N and N P treatments. N2-fixation/unit E. sorex biovolume was inhibited by NO3− and stimulated slightly by PO43−. A combination of demographic and mutualistic responses to nutrients altered N input via N2-fixation.
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