Identification and quantification of extracellular polysaccharide biomarkers across soil organisms and plants

Soil extracellular polysaccharides (EPSac) are essential biopolymers in terrestrial ecosystems, playing key roles in soil aggregation, water retention, nutrient cycling, and carbon sequestration. These polysaccharides are produced by a wide range of organisms, including archaea, bacteria, fungi, plants, and soil fauna. However, their complex composition and low yet largely unknown abundance in environmental samples present significant challenges for their identification and quantification. In this study, we will culture and collect EPSac samples from various organisms (archaea, bacteria, fungi, algae, higher plants, and soil fauna) across different phyla. The extraction methods will be tailored to the specific sample types, such as cation exchange resin extraction for microbial cells and water extraction for plant roots and soil fauna. Dissolved extracellular polysaccharides will be separated from soil metabolites by ultrafiltration or precipitation before biochemical analysis. Plant samples will be cultivated hydroponically from seeds to minimize soil and microbial contamination of fine roots. To identify and quantify the monomeric composition of the polymers, we will employ an optimized acid hydrolysis method in combination with 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization, followed by analysis using ultra-high-performance liquid chromatography with high-resolution Orbitrap mass spectrometry (UPLC-Orbitrap MS). EPSac-specific monomers will be seeked for that do not occur in other known cell wall-based and/or storage polysaccharides of prokaryotes and eukaryotes. Multivariate analysis, such as non-metric multidimensional scaling (NMDS) and partial least squares discriminant analysis (PLS-DA), will be utilized to assess the variability of EPSac compounds across different taxonomic groups. Additionally, Indicator species analysis will be performed to evaluate the biomarker potential of these compounds. Finally, these biomarkers will be applied to various soil types (cropland, grassland, and forest soils) to assess their contributions to microbial metabolic and soil carbon cycling. This study aims to identify EPSac-specific and organism-specific biomarkers and precisely quantify these key EPSac monomers, advancing our understanding of soil biogeochemical processes and their role in ecosystem functioning and nutrient dynamics.