Insight into the role of extracellular polymeric substances in denitrifying biofilms under nitrobenzene exposure

Abstract

Denitrifying biofilm promises to be very useful for remediation of nitro-aromatic compounds (NACs) and nitrates in wastewater. Little is known about the role of extracellular polymeric substances (EPS) in nitrobenzene (NB, a typical NAC) remediation, despite the indispensability of EPS for biofilm formation. Herein, the significance of the mechanistic role of EPS in the response of denitrifying biofilms to various levels of NB was investigated. The removal of NB was predominantly controlled via absorption, with little biodegradation during the short-term exposure. Specifically, NB was adsorbed by EPS, as shown by a total adsorption of 40.06% at the initial step, which declined to around 10.52% in the equilibrium stage, while sorption via cells gradually increased from 59.93% to 89.47% over the same period. The results suggested that EPS might act as an important reservoir for NB, which endows inner cells with increased adsorption ability. The presence of EPS might also alleviate the negative impacts of NB toxicity on inner cells, thus protecting microorganisms. This was indicated by the difference in denitrification performance and cell integrity between intact and EPS-free biofilms. High-throughput sequencing data demonstrated that EPS could maintain the stability of microbial communities under NB stress. The fluorescence quenching analysis further indicated that EPS formed stable complexes with NB mainly through hydrophobic interactions with protein-like fractions (tryptophan and tyrosine). Moreover, Fourier transform infrared spectroscopy identified that the hydroxyl, amino, carboxyl, and phosphate groups of EPS were the candidate functional groups binding with NB. Protein secondary structures were also significantly affected, resulting in a loose structure and enhanced hydrophobic performance for EPS. These results provide insights into the role of EPS in alleviating NB-caused cellular stress and the underlying binding mechanisms between NB and EPS.