Biofilms mediate crucial biochemical processes in aquatic ecosystems.It was hypothesized that eutrophication may promote the growth of biofilms,resulting in larger numbers of functional genes.However,the metabolic activity and the roles of biofilms in N cycling will be affected by ambient inorganic nitrogen availability,not by the abundance of functional genes.Biofilms were cultured either with replete inorganic nitrogen (N-rep) or without exogenous inorganic nitrogen supply (N-det) in a flow incubator,and the N-cycling gene abundances (nifH-,N2 fixation;amoA,ammonia oxidation,archaea and bacteria;nirS and nirK,denitrification) and enzyme activities (nitrogenase and nitrate reductase) were analyzed.The results showed that,comparing the N-def and N-rep biofilms,the former contained lower nifHgene abundance,but higher nitrogenase activity (NA),while the latter contained higher nifH gene abundance,but lower NA.Different pattems of NA diel variations corresponded to the dynamic microbial community composition and different stages of biofilm colonization.Ammonia oxidizing bacteria (AOB),detected only in N-def biofilms,were responsible for nitrification in biofilms.N-rep biofilms contained high nirS and nirK gene abundance and high denitrification enzyme activity,but N-def biofilms contained significantly lower denitrification gene abundance and activity.In general,the strong N2 fixation in N-def biofilms and strong denitrification in N-rep biofilms assured the balance of aquatic ecosystems.The results suggested that evaluation of the functional processes of N cycling should not only focus on genetic potential,but also on the physiological activity of biofilms.