Hydrophobic features of extracellular polymeric substances (EPS) extracted from biofilms: an investigation base on DAX-8 resin technique
Extracellular polymeric substances (EPS) are not only the “house” for the microbial cells, but they also facilitate several important cellular functions of the microorganisms. The hydrophobic properties of EPS exert a profound influence on the cell surface properties. However, many factors such as EPS extractions methods and substrate type influencing EPS characteristics as well as limited information regarding the hydrophobic features of EPS are reported. The main aim of this study is to develop a proper method to determine EPS hydrophobicity, and then investigate the hydrophobic features of the extracted EPS.
The hydrophobic fractionation of EPS 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 different EPS extraction methods on the hydrophobic features of EPS was also assessed. The results showed that the extraction methods and bulk solution pH dramatically influenced the EPS composition and the measured EPS hydrophobicity, respectively. 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, more complete aMW information of EPS 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 for the following analysis of EPS extracted from anaerobic granular sludge.
After identifying the proper conditions for DAX-8 resin fractionation, detailed qualitative analysis of the EPS hydrophobic features was investigated by size exclusion chromatography (SEC) and excitation and emission fluorescence matrix (EEM) techniques. The results showed that the humic-like substances (HS-like) were the major organic constituent of EPS extracted from the anaerobic granular sludge, and they were also the main molecular support of EPS hydrophobicity (> 50%). Those hydrophobic HS-like compounds were mainly small molecules ranging from 8 kDa to <1 kDa, and their hydrophobicity could be ascribed to their humic acid-like molecular structure. Proteins (PN) and polysaccharides (PS) contributed to the EPS hydrophobicity to a lesser extent (< 30%, respectively).
Although the hydrophobic features of HS-like compounds were demonstrated, 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 fungi are PN and PS. Ni2+ is the essential metal for the microorganisms and it is required as micronutrients at nano-molar concentrations, and it is also identified as the trace element for various biological systems such as anaerobic biochemical reactions. To explore the hydrophobic features of PN and PS, as well as to investigate the effect of metal addition (i.e. Ni2+) under sub-toxic concentration on the EPS hydrophobicity, fungus Phanerochaete chrysosporium was chosen as the model microorganism. The results showed that the contents of PN and PS in the extracted fungal EPS varied with the initial Ni2+ concentration added in the growth medium. With an increase in the initial Ni2+ concentration from 0 mg/L to 25 mg/L, the PN content was increased whereas the PS content eventually remained stable. The hydrophobicity of fungal EPS, determined by the DAX-8 resin treatment, was gradually decreased as the Ni2+ concentration increased. The zeta-potential of the fungus pellets was reduced by the presence of Ni2+. Besides, the peak intensity of the PN-like molecules ranging from 0.5 kDa to 14 kDa detected by SEC were intensified by the increased Ni2+ concentration, while the aMW distribution of the total organics in the EPS remained almost stable. These results indicated that, under sub-toxic concentration of Ni2+, the presence of Ni2+ promoted the fungus pellets becoming less hydrophobic, and the PN content was improved without changing its intrinsic fluorescence characteristics. Nevertheless, the molecular features of PS were still difficult to be elaborated and lack of specific spectroscopic properties that can be detected by EEM or SEC techniques.
Overall, the EPS extraction methods and bulk solution pH influenced both composition and hydrophobicity of the EPS. 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 fungal EPS played an active role in protecting the fungus under Ni2+ stress.