Insight into correlation of advanced nitrogen removal with extracellular polymeric substances characterization in a step-feed three-stage integrated anoxic/oxic biofilter system

As a core component of the biomass, the important role of extracellular polymeric substances (EPS) on treatment performance has been recognized. However, the comprehensive understanding of its correlation with nitrogen removal remains limited in biofilm-based reactors. In this study, the relevance between EPS and advanced nitrogen removal in a novel step-feed three-stage integrated anoxic/oxic biofilter (SFTIAOB) was specifically investigated. The operation showed as high as 81% TN removal was achieved under optimal conditions. Among the whole reactor, 2nd anoxic (A2) zone was the largest contributor for nitrogen removal, followed by the 3rd anoxic (A3) and 2nd oxic (O2) zones. EPS composition analysis found that high content of polysaccharides in tightly bound-EPS (A2 and A3) and protein in loosely bound-EPS and tightly bound-EPS (O2). Fourier transform infrared spectroscopy, three-dimensional fluorescence spectrum further verified stratified EPS subfractions containing different secondary protein structures, while 3-turn helix and tryptophan-like protein was the main reason for nitrogen removal. High-throughput sequencing revealed the co-existence of nitrogen removal-associated genera accomplished nitrification/denitrification combined with aerobic denitrification and anammox. Moreover, the correlation of EPS and microbial composition with nitrogen removal was clarified by redundancy analysis (RDA). Finally, potential mechanism for nitrogen removal was illuminated. This research gives more insight into EPS characteristics in enhancing nitrogen removal during the operation and optimization of a step-feed multi-stage A/O biofilm process.