Measuring the soil-microbial interface: Extraction of extracellular polymeric substances (EPS) from soil biofilms

Abstract

Many soil microbes exist in biofilms. These biofilms are typified by variable quantities of extracellular polymeric substances (EPS: predominantly polysaccharides, glycoconjugates, and proteins) and the embedded microbial cells. A method to measure soil-EPS (the biofilm exclusive of microbial cells) has not yet been described. The present work investigates the potential of five extraction methods to estimate changes in soil-EPS content. A rationale for selection of appropriate EPS extraction and methodology is discussed, including the crucial consideration of both intracellular and extracellular contamination.EPS was developed in situ by provision of labile C (glycerol) to the microbial biomass of a moist soil and then applying desiccation stress. Only two out of the five extraction methods showed statistically significant increases in polysaccharide production responding to substrate addition. Humified organic matter, estimated by its humic acid equivalent (HAE) was used to indicate the degree of extracellular contamination, and/or creation of humic artefacts – both of which affect detection of changes in EPS. The HAE concentration was very high when applying original and modified methods designed to extract glomalin related soil protein (GRSP). Extraction methods involving heating with dilute sulphuric acid appeared to overestimate EPS-polysaccharide. Using microbial ATP as an indicator of cell-lysis, confidence could only be ascribed to EPS extraction with cation exchange resin. Using this method, the expected increases in EPS-polysaccharide were clearly apparent. The HAE/protein ratios of EPS extracts were also lowest with cation exchange – indicating this method did not cause excessive contamination from humified soil organic matter or create related artefacts.