Seasonal variations of biofilm C, N and S cycling genes in a pilot-scale chlorinated drinking water distribution system.

Biofilms in drinking water distribution systems (DWDS) host diverse microorganisms. However, the functional attributes of DWDS biofilms and their associations with seasonality remain unclear. This study aims to characterize variations in the microbial metabolic traits of DWDS biofilms collected during different seasons, using a pilot-scale DWDS in dark under plug-flow conditions during one-year operation period. Network analysis was used to predict the functional gene hosts. The overall functional attributes determined by shotgun metagenomics exhibited significant differences among seasons. Genes associated with aromatic metabolism, fatty acid biosynthesis and degradation, and capsular extracellular polymeric substance (EPS) were significantly upregulated in summer owing to the higher temperatures and chlorine in the influent of the DWDS. Moreover, the pathways associated with nitrogen, sulfur, glycolysis, and tricarboxylic acid (TCA) cycling, as well as carbon fixation were reconstructed and displayed according to the sampling season. Nitrogen reduction pathways [dissimilatory nitrate reduction to ammonium (DNRA) 73 %, assimilatory nitrate reduction to ammonium (ANRA) 21 %] were identified in DWDS biofilms, but nitrogen oxidation pathways were not. Sulfur cycling were involved in diverse pathways and genes. Glycolysis and TCA cycling offered electron donors and energy sources for nitrogen and sulfur reduction in biofilms. Carbon fixation was observed in DWDS biofilms, with the predominant pathway for fixing carbon dioxide being the reductive citrate cycle (38 %). Constructed functional gene networks composed of carbon, nitrogen, and sulfur cycling-related genes demonstrated synergistic effects (Positive proportion: 63.52-71.09 %). In addition, from spring to autumn, the network complexity decreased and network modularity increased. The assembly mechanism of carbon, nitrogen and sulfur cycling-related genes was driven by stochastic processes for all samples. These results highlight the diverse functional genes in DWDS biofilms, their synergetic interrelationships, and the seasonality effect on functional attributes.