Vermiculite-assisted enhancement and multimodal characterization of exopolysaccharide from Paenibacillus mucilaginosus CCASU-2024-68

Abstract

The exopolysaccharide (EPS) produced by Paenibacillus mucilaginosus CCASU-2024-68 and the enhancement of its production by vermiculite supplementation were investigated. The bacterium was cultivated in glycerol nitrate broth containing different vermiculite levels, and preliminary optimization indicated that 0.5 g L⁻¹ vermiculite at pH 7 and 30 °C for 4 days with agitation at 100 rpm yielded 6.0 g L⁻¹ EPS. To systematically identify the key variables controlling EPS production, a Plackett–Burman design was employed to screen medium components (glycerol, KNO₃, K₂HPO₄, MgSO₄, NaCl, CaCO₃, vermiculite) and culture conditions (initial pH, inoculum size, agitation speed, and fermentation time). Under the optimal Plackett–Burman condition (Run 2), EPS production increased to 8.44 g L⁻¹ at pH 7 with 30 g L⁻¹ glycerol, 2 g L⁻¹ KNO₃, 1 g L⁻¹ K₂HPO₄, 0.5 g L⁻¹ MgSO₄, 1 g L⁻¹ NaCl, 3 g L⁻¹ CaCO₃, 1 g L⁻¹ vermiculite, 20% inoculum, agitation at 100 rpm, and 4 days of fermentation. Transmission electron microscopy revealed pronounced morphological alterations in vermiculite-treated cells, including enlarged periplasmic regions, cell wall disintegration, cytoplasmic membrane rupture, and cytoplasmic leakage, indicating a stress-induced response. Scanning electron microscopy showed that control EPS formed a porous, fibrous network, whereas vermiculite-treated EPS exhibited compact, layered, sheet-like structures. Energy-dispersive X-ray analysis indicated substantial changes in elemental composition, with vermiculite-treated EPS enriched in O, Na, P, K, and Ca compared with the control. FTIR and ¹H-NMR spectroscopy confirmed typical polysaccharide functional groups and highlighted structural rearrangements upon vermiculite treatment, while HPLC revealed marked shifts in monosaccharide composition, including the appearance of mannose and glucose and the loss of fructose. Overall, these results demonstrate that vermiculite not only enhances EPS yield but also drives significant structural and compositional modifications in the exopolymer, suggesting a mineral-mediated strategy to engineer bacterial EPS for agricultural and industrial applications.