BACKGROUNDThis study aimed to clarify how a variation in the carbon to nitrogen (C/N) ratio in influent wastewater affects the production and composition of extracellular polymeric substances (EPS) in activated sludge. In the literature such a comparative analysis is rarely presented, most studies on EPS focus on simultaneous organic carbon removal and nitrification whereas little attention is paid to the case of low organic load or separate-stage nitrification. RESULTSAmong the very loosely bound, loosely bound and tightly bound EPS fractions, tightly bound EPS prevailed in semi-continuously fed batch activated sludge reactors that were fed at a C/N ratio of 10, 5 and 0, respectively. The C/N ratio had no clear effect on total EPS production, however it changed the proportion of proteins and carbohydrates. In each sludge proteins constituted the dominant part of EPS, but the EPS of the nitrifying sludge (C/N=0), had a definitely higher protein content. As evidenced by HPSEC fingerprints, in all EPS fractions most proteins had MWs 16 kDa. Depending on the C/N ratio in a reactor, high MW proteins in EPS shifted from tightly bound fractions to others. CONCLUSIONThe study is among the few that highlight how the protein and carbohydrate content in EPS changes at different C/N ratios. Since wastewater composition in a treatment system affects the chemical composition of EPS, each sludge might have different responses to uptake of xenobiotics such as metals. Further, this difference in composition might affect sludge settling, dewatering and membrane fouling. (c) 2014 Society of Chemical Industry