Heavy metals polluting the 100-year-old waste heap in Bolesław (Poland) are acting as a natural selection factor and may contribute to adaptations of organisms living in this area, including Trifolium repens and its root nodule microsymbionts-rhizobia. Exopolysaccharides (EPS), exuded extracellularly and associated with bacterial cell walls, possess variable structures depending on environmental conditions; they can bind metals and are involved in biofilm formation. In order to examine the effects of long-term exposure to metal pollution on EPS structure and biofilm formation of rhizobia, Rhizobium leguminosarum bv. trifolii strains originating from the waste heap area and a non-polluted reference site were investigated for the characteristics of the sugar fraction of their EPS using gas chromatography mass-spectrometry and also for biofilm formation and structural characteristics using confocal laser scanning microscopy under control conditions as well as when exposed to toxic concentrations of zinc, lead, and cadmium. Significant differences in EPS structure, biofilm thickness, and ratio of living/dead bacteria in the biofilm were found between strains originating from the waste heap and from the reference site, both without exposure to metals and under metal exposure. Received results indicate that studied rhizobia can be assumed as potentially useful in remediation processes.
Keywords: adaptation to toxic metals; biofilm; confocal laser scanning microscopy (CLSM); exopolysaccharide (EPS); gas chromatography-mass spectrometry (GC-MS); heavy metals; nucleic acid staining dyes; retention index; rhizobia.