The Effect of the Growth Medium on the Composition and Metal Binding Behaviour of the Extracellular Polymeric Material of a Metal-Accumulating Citrobacter sp.

Abstract

Cells of a heavy metal-accumulating Citrobacter sp. appeared electron-opaque under the electron microscope when pre-grown in medium containing lactose and inorganic phosphate as the respective carbon and phosphorus sources, compared to cells supplied with glycerol and glycerol 2-phosphate. This was attributable to a greater proportion of bound metal cations, as analysed using proton induced X-ray emission analysis of extracted extracellular polymeric material (EPM). This was associated with a greater content of EPM-associated phosphorus and also an enhanced activity of isolated EPM to accumulate uranyl ion, to 23% of the polymer dry weight. EPM from glycerol 2-phosphate-grown cells, analysed using 31P nuclear magnetic resonance spectroscopy, gave several major phosphorus resonances between −1 and 2 ppm, in accordance with published spectra for enterobacterial lipopolysaccharide. Addition of Cd2+ promoted a shift downfield of the P resonances to a new, common, resonance at 3 ppm which was similar to that observed in native, Cd-unchallenged EPM obtained from inorganic phosphate-pregrown cells. The two types of polymer were morphologically distinct, with a shift to a more defined structure on addition of Cd2+ to the EPM from glycerol phosphate-grown cells. The two types of 31P spectra of the EPM were influenced by the pregrowth phosphorus source, and not by the carbon source. In contrast, analysis of the monosaccharide content of the EPM by gas chromatography and high performance liquid chromatography showed that the main components, rhamnose, glucose and ribose, were unaffected by the pre-growth phosphate source but cells pre-grown in glycerol medium had a higher proportion of glucose residues (ratios of glucose:rhamnose of 4:1 and 2.5:1 in glycerol and lactose-pregrown cells, respectively), and a higher content of EPM monosaccharides, overall, in the lactose-grown cultures. Restriction of carbon or nitrogen source by growth in continuous culture and analysis of the EPM gave major 31P resonances in the region of −1 to 3 ppm, while growth under phosphate-limiting continuous culture showed only one major species, at 1 ppm. The data illustrate that the composition of the cell surface polymers, and the ability of these to sorb heavy metal, are functions of the environment in which the cells are grown. This has implications in the application of biosorption techniques to the removal of heavy metals from wastes.