Fluorescent quenching for biofilm extracellular polymeric substances (EPS) bound with Cu(II)

The Cu(II) binding properties of loosely bound extracellular polymeric substances (LBEPSs) and tightly bound EPSs (TBEPSs) extracted from biofilm samples at two apparent molecular weight (AMW) ranges, >14 kDa and 1–14 kDa, were investigated using three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy. The protein-like and aromatic protein fluorescence peaks were identified in EEM fluorescence spectra as peaks A and B, respectively. The intensities of peaks A and B were generally quenched when Cu(II) was bound with LBEPSs or TBEPSs with AMWs > 14 kDa at various pH levels. Conversely, for 1–14 kDa EPSs, fluorescence intensities of peaks A and B were not quenched when Cu(II) was bound with LBEPSs at pH 4 or with both LBEPSs and TBEPSs at pH 8. The Stern–Volmer constant (log Ksv) for the Cu(II)–LBEPSs and Cu(II)–TBEPSs binding processes were 2.38–4.37. The capability of EPSs to bind with Cu(II) increased as pH increased. At pH > 4, the protein-like substances and aromatic proteins in TBEPSs had greater Cu(II) binding capability than LBEPSs. Additionally, the EPSs with AMWs > 14 kDa had stronger binding capability with Cu(II) than EPSs with AMWs of 1–14 kDa. The difference in Cu binding behavior of LBEPS and TBEPS significantly affect the mobility, bioavailability, and toxicity of Cu in aquatic environments.

► We investigated the Cu(II) binding properties of extracellular polymeric substances (EPS) from biofilm samples.
► The EPS–Cu complex was probed using three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy.
► The Cu(II) is bound with EPS at different molecular weights with binding coefficients evaluated.
► EPS bind most Cu(II) is identified.