Characterising soil extracellular polymeric substances (EPS) by application of spectral-chemometrics and deconstruction of the extraction process

Extracellular polymeric substances (EPS) exuded by microbes are found in diverse habitats, including soil. The quantification and characterization of EPS are essential prerequisites for understanding the ecological functions of extracellular products in soil. However, few studies have conducted in-depth profiling of soil EPS extraction steps and chemical characteristics. Here we examine the C and N contents, biopolymer contents, and optical properties of EPS involving three soil pre-treatment methods (moist soil, moist soil pre-incubated, and air-dried soil pre-incubated) and simultaneously track the compositional changes during the two-step extraction process (extract with CaCl2 in Step 1 and phosphate buffer solution (PBS) with cation exchange resin (CER) in Step 2). The optical properties of soil EPS are investigated using ultraviolet-visible adsorption (UV-Vis), excitation-emission-matrix fluorescence (EEM), and fourier transform infrared (FTIR) spectroscopy coupled with multi-variate analytical methods such as parallel factor analysis (PARAFAC), principal component analysis (PCA), and correlation analysis. Results show that Step 2 releases significantly more dissolved organic carbon and bio-polymers (polysaccharides, proteins, and uronic acids) from soils. UV-Vis and EEM spectroscopy suggest that the fractions extracted in Step 2 exhibit higher mean aromaticity, molecular weight, and humification degree than those extracted in Step 1. When using the standard method to extract EPS from moist aggregates (Step 2), EPS has a distinct fluorescence profile, rich in tryptophan-and tyrosine-like signals. Reassuringly, the fluorescence-profiles found in pre-extracts (Step 1) are very different -except where i) aggregates have an experimental drying step added, and ii) pure water is used as a pre-extractant. Drying of aggregates is thus problematic, and the use of pure water as an alternative pre-extractant is not advisable. Soil treatments have significant effects on the qualities of the various extracts which are detected by both the FTIR-PCA and EEM-PCA analyses. EEM fluorescence profiles of EPS suggest that both tryptophan-like and tyrosine-like substances are key components of the EPS extracted from all soils regardless of pretreatment. Our findings indicate that chemical characterization of soil EPS combined with spectral-chemometric approaches can be applied to identify and characterise the extracellular responses of soil microbes and thus help reveal the ecological functions of extracellular products in soils.