Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001, supplemented with carbon (glucose), nitrogen (NH4Cl), phosphorus (KH2PO4), or combined nutrients (CNP), with or without hexadecane, a model compound representing aliphatic hydrocarbons used to simulate a pollutant. In fall 1999 and fall 2001, comparable denitrification activities and catabolic potentials were observed in the biofilms, implying that denitrifying populations showed similar activity patterns and catabolic potentials during the fall from year to year in this river ecosystem, when environmental conditions were similar. Both nirS and nirK denitrification genes were detected by PCR amplification, suggesting that both denitrifying bacterial subpopulations can potentially contribute to total denitrification. Between 91.7 and 99.8% of the consumed N was emitted in the form of N2, suggesting that emission of N2O, a major potent greenhouse gas, by South Saskatchewan River biofilms is low. Denitrification was markedly stimulated by the addition of CNP, and nirS and nirK genes were predominant only in the presence of CNP. In contrast, individual nutrients had no impact on denitrification and on the occurrence of nirS and nirK genes detected by PCR amplification. Similarly, only CNP resulted in significant increases in algal and bacterial biomass relative to control biofilms. Biomass measurements indicated a linkage between autotrophic and heterotrophic populations in the fall 1999 biofilms. Correlation analyses demonstrated a significant relationship (P < or = 0.05) between the denitrification rate and the biomass of algae and heterotrophic bacteria but not cyanobacteria. At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification in both years, slightly more in the fall of 2001. This study suggested that the response of the anaerobic heterotrophic biofilm community may be cyclic and predictable from year to year and that there are interactive effects between nutrients and the contaminant hexadecane.