Abstract
Microbial exopolysaccharides (EPS) are structurally diverse biopolymers with ecological and biotechnological relevance due to their physicochemical and functional properties, including emulsification, metal chelation, and contributions to biofilm formation. In this study, an EPS-enriched extracellular fraction (EPS-EF) produced by Vreelandella zhaodongensis BS253, isolated from the Brazilian Pantanal, was characterized using an integrated approach combining microscopic, physicochemical, molecular, genomic, and transcriptomic analyses. Morphological and structural profiling by microscopy revealed a highly porous, reticulated web-like supramolecular network. The EPS-EF exhibited cation-dependent colloidal stability, with turbidity decreasing by approximately 95% after divalent-cation chelation. The emulsification index after 24 h (E24) against kerosene reached approximately 67%, and the surface tension of water was reduced to approximately 45 mN/m, with an apparent surface-tension inflection (STI) near 10.7 mg/mL. X-ray diffraction (XRD) revealed a predominantly amorphous profile with discrete crystalline reflections. Thermogravimetric analysis (TGA) demonstrated thermal stability with a multi-step degradation profile and a maximum decomposition rate centered at 280 °C. CHNS/O and FTIR were consistent with a carbohydrate-associated, nitrogen-poor molecular profile containing carbonyl/carboxylate and glycosidic functionalities. UHPLC–ESI–QTOF–MS/MS resolved a reproducible set of oligomeric features across four chromatographic zones, without establishing monosaccharide composition, together with minor hydrophobic features tentatively associated with lipid-related compounds. One-dimensional 1H NMR provided complementary molecular-fingerprint information, but the primary structure remains unresolved. EPS-EF material analysis may also include contributions from co-extracted components because the analyzed material is an enriched extracellular fraction rather than a chemically homogeneous polysaccharide. Genome mining and targeted RNA-seq analysis of 47 EPS-associated genes identified candidate pathways for precursor metabolism, glycan assembly, and export. Collectively, these findings indicate that V. zhaodongensis BS253 produces an ionic-sensitive EPS-EF with emulsifying activity and support a working hypothesis for EPS-EF biosynthesis and export.