Flocculation and Transparent Exopolymer Particles Formation of Microbial Mat Extracellular Polymeric Substances from a Hypersaline Lake

This study investigated the mechanism of flocculation and transparent exopolymer particles (TEP) formation of extracellular polymeric substances (EPS) from mixed algal-bacterial biofilm mat from hypersaline lakes. Various spectrophotometric techniques were employed along with parallel factor (PARAFAC) modelling on the sample excitation-emission matrix (EEM) spectra. EPS with exposure to sunlight was found to form larger TEP and floc particles of variable sizes, confirmed from turbidity test, ζ−potential (−mV) and PSD (d.nm) assay in the first few hours of sunlight exposure. It further suggested that floc/TEP formation from EPS could occur by charge neutralization and bridging mechanism. Correspondingly, EEM-PARAFAC identified humic-like and protein-like or tryptophan-like components in both raw and treated EPS samples. After the fifth day of solar irradiation, humic-like substances were entirely decomposed whilst the regenerated protein-like substances from EPS were the key components remained in irradiated samples. Degradation of EPS, particularly protein molecules of EPS was observed from the result of HPLC−SEC, which was confirmed from degradation of DOC, total protein and total polysaccharide analysis. FTIR of treated EPS and filtered EPS had more –COOH, C−H, and –C–O groups indicating acidic polysaccharide typical of TEP. With increasing pH, EPS flocculated with increasing PSD (d.nm) while a decrease in PSD (d.nm) was observed with decreasing pH. Microscopic images with AB dye assay showed a significant increase in floc/TEP at pH 10 followed by pH 8.Incubation over 24 hours with different concentration of NaCl (0 mM to 60 mM) had noteworthy effect on floc/TEP formation. But AB dye assay image with NaCl treatment showed no significant differences in floc/TEP size by changing the concentration of NaCl. ATR-FTIR spectroscopy analysis confirmed C=O and C–N groups. The carboxylic and phenolic (–COO− /–O − ) containing protein molecules in EPS might be responsible for EPS-metal (Mn+)–protein or EPS- metal (Mn+)-polysaccharide complexation by higher pH and Na+/Cl−leading to TEP/flocformation.The TEP/floc formation mechanism of EPS in response to different divalent cations (Fe2+, Ca2+ and Mg2+) with different concentrations (2 mM, 0.5 mM and 10µM) were observed. In the course of time a significant charge drop was observed with increasing concentration of Fe2+ followed by a larger floc/TEP formation in the first six hours. However, the larger floc/TEP broken down into daughter particles in the next 24 hours (approximately ~1652 d.nm). Microscopic investigation of AB dye confirmed fibrillar amorphous floc/TEP formation after 24 hours of incubation EPS adjusted with FeCl2 , CaCl2 and MgCl2. PSD assay confirmed highest floc/TEP formation in FeCl2 treatment compared to CaCl2 and MgCl2 treated EPS. ζ−potential, PSD and turbidity assay confirmed that particle bridging and particle breakage were dominant. Excitation-emission (EEM) spectroscopy along with parallel factor (PARAFAC) analysis confirmed protein-like, tryptophan-like and humic-like substances of EPS formed complexes through donation of their electrons to Fe2+, Ca2+,Mg2+ and that subsequently formed TEP/floc. It is, therefore, evidence from the above experiments that EPS can transform itself into the floc/TEP in a natural environment by the influence of sunlight, pH, salinity and different types of divalent cations that alter the geochemical interaction in natural waters