Hydrophobic features of extracellular polymeric substances (EPS) extracted from biofilms : an investigation based on DAX-8 resin technique

The hydrophobic properties of extracellular polymeric substances (EPS) exert a profound influence on the cell surface properties. However, many factors such as EPS extractions methods, substrate type influence EPS characteristics, and limited information regarding to the hydrophobic features of EPS can be found. The main aim of this study is to develop a proper method to study EPS hydrophobicity, and then investigate the hydrophobic features of EPS.The hydrophobic fractionation by Supelite™ DAX-8 resin was first applied on the EPS extracted from anaerobic granular sludge, two elution pH conditions i.e. pH 2 and 5 were tested. The impact of seven EPS extraction methods on the hydrophobic features of EPS was assessed. The results showed that the extraction methods and bulk solution pH dramatically influenced the EPS composition and their hydrophobicity. Besides, the EPS extracting reagents namely formaldehyde, ethanol, sodium dodecyl sulfate (SDS) and Tween 20 not only introduced extra carbon content during total organic carbon (TOC) measurement, but also interacted with the DAX-8 resin. By comparing the apparent molecular weight (aMW) distribution of the untreated and pH-adjusted EPS samples detected by size exclusion chromatography, more complete EPS aMW information was preserved at pH 5. Thus, the hydrophobic fractionation by DAX-8 resin at pH 5 and physical EPS extraction methods were preferred in this study.After identifying the proper conditions for DAX-8 resin fractionation, detailed qualitative analysis of the EPS hydrophobic features was further investigated. The results showed that the humic-like substances (HS-like) were the major organic constituent of the EPS extracted from the anaerobic granular sludge, and they were also the main molecular support of the EPS hydrophobicity. Those hydrophobic HS-like compounds were mainly small molecules ranging from 8 kDa to <1 kDa. Proteins (PN) and polysaccharides (PS) contributed to the EPS hydrophobicity to a lesser extent.The role of PN and PS in the EPS hydrophobicity was difficult to be shown. It is known that the major organic constituents of the EPS extracted from bacteria, algae and fungi are PN and PS. Therefore, to explore the hydrophobic features of PN and PS, as well as to investigate the impact of Ni(II) addition, on the EPS hydrophobicity, the fungus Phanerochaete chrysosporium was chosen. The results showed that the contents of PN and PS in the extracted fungal EPS varied with the Ni(II) concentration. With an increase in the Ni concentration from 0 mg/L to 25 mg/L, the PN content was decreased whereas the PS content was increased. The fungal EPS hydrophobicity, determined by the DAX-8 resin treatment, was decreased as the Ni concentration increased.Besides, the peak intensity on the size exclusion chromatography (SEC) corresponding to the PN-like molecules (Ex/Em = 225/345 nm) ranging from 1.9×103 to 10 kDa were intensified by the Ni addition, while the aMW distribution of the total organics (UV/210) in the EPS remained almost stable. These results indicated that those PN-like compounds may determine the hydrophobicity of fungal EPS under stress conditions.For the more heterogeneous EPS extracted from anaerobic granular sludge, HS-like compounds were identified as the major organic component, as well as the main molecular support of the EPS hydrophobicity. By studying the hydrophobic features of the EPS extracted from the fungus Phanerochaete chrysosporium, it showed that the PN and PS in the EPS played an active role in protecting the fungus under Ni stress. The increased Ni concentration decreased the hydrophobicity of fungal EPS, but it increased the cell surface hydrophobicity of the fungus. It seems that the presence of Ni promoted the fungus becoming more hydrophobic