The role of cell surface sulfhydryl and amine binding sites in the removal of Cr(VI) from solution by Bacillus subtilis bacterial cells

Abstract

A range of bacterial species are known to be capable of reducing soluble Cr(VI) to Cr(III), causing the precipitation of Cr(III) phases from solution, but the mechanism responsible for the initial interaction between anionic aqueous Cr(VI) and the negatively charged cell surface has not been identified. Our study examines Cr(VI) removal from solution by the Gram-positive soil bacterial species Bacillus subtilis. We measured the kinetics of Cr(VI) removal as a function of pH in B. subtilis suspensions with different Cr(VI):cell ratios, and we measured the effect of pH on the extent of Cr(VI) removal at a fixed time in experiments with B. subtilis biomass (cells plus exudates) and in experiments involving B. subtilis exudates alone. The roles of sulfhydryl and amine binding sites in Cr(VI) removal were constrained using site–specific blocking molecules, and the impact of the site blocking on Cr(VI) removal was studied as a function of pH. Our results indicate that Cr(VI) removal by B. subtilis cells under the experimental conditions is at least partially non-reversible and is dependent on binding site concentration, pH, and metal loading. Our results are consistent with a two step removal process: Cr(VI) first adsorbs reversibly onto a cell wall binding site, followed by Cr(VI) reduction to Cr(III) likely via electron transfer from cell wall electron transport chain molecules. B. subtilis exudates are capable of removing a relatively small fraction of Cr(VI) from solution, and hence our results indicate that the dominant mechanism of Cr(VI) removal requires interaction with bacterial biomass. The presence of either the sulfhydryl-specific blocking molecule or the amine-specific blocking molecule or both in the experimental systems dramatically reduces the extent of Cr(VI) reduction, especially under circumneutral pH conditions, strongly suggesting that both sulfhydryl and amine binding site types participate in the initial attachment of Cr(VI) onto the cell surface. The experiments with either sulfhydryl or amine sites blocked both exhibited a similar reduction in Cr(VI) removal to the experiments with both types of sites blocked, strongly suggesting that both types of sites are involved simultaneously in binding Cr(VI) species to the cell surface. For example, these sites could both be involved in forming a bidentate bond with Cr(VI) species, or the positive charge of an amine site in close proximity to a sulfhydryl site could be necessary in order to enable the Cr(VI) species to approach the cell wall and to bind to a sulfhydryl site. The results of this study are the first to propose a viable mechanism that can explain the binding of anionic Cr(VI) onto an overall negatively charged cell surface as a first step in the reduction to Cr(III) and subsequent removal of Cr from solution.