Using a stable isotope tracing technique to elucidate the effect of substrate C/N ratio on the formation of different constituents of extracellular polymeric substances in an aerobic-anoxic sequencing batch reactor

Abstract

Carbon to nitrogen (C/N) ratios in wastewater vary with time and sources, which greatly affect the production and composition of extracellular polymeric substances (EPS) in biological treatment processes. In this study, the EPS yield and their compositional changes were examined using stable isotopes (13C-glucose and 15NH4Cl) during the operation of aerobic-anoxic sequencing batch reactors (SBR) with different C/N ratios (C/N = 5, 10, and 15). Spectroscopic characterization revealed that humic-like aromatic compounds tended to be more enriched in loosely bound EPS (LB-EPS) at a lower C/N ratio, whereas protein-like fluorophores prevailed in tightly bound EPS (TB-EPS) (>90 %) regardless of the feed C/N ratio. Variations in isotopic enrichment revealed that the rates of substrate assimilation into TB-EPS were different between organic carbon and nitrogen, with an earlier replenishment of nitrogen regardless of C/N ratios. Yield estimation based on individual carbon and nitrogen isotopes indicated that maximum 17.8 % of organic carbon was converted into TB-EPS at a lower C/N ratio, whereas nitrogenous EPS exhibited higher yield coefficients with a wider range from 26.0 % (C/N = 5) to 44.0 % (C/N = 15). The results suggest that varying C/N ratios might exert more pronounced effects on the production of nitrogenous EPS constituents than their carbon counterparts. This suggested isotope labeling approach can be further applied to determine the mass balances among the substrate, biomass, and bound/soluble EPS within activated sludge systems.