Integrated Investigation on the Production and Fate of Organo-Cr(III) Complexes from Microbial Reduction of Chromate

Our objective is to investigate the complexity of chromium biogeocycling. Our results clearly support more complexity. In short, the chromium cycle is not as simple as the conversion between Cr(III) and Cr(VI) in inorganic forms. We have obtained more evidence to prove the formation of soluble organo-Cr(III) complexes from microbial reduction of Cr(VI). The complexes are relatively stable due to the slow ligand exchange of Cr(III). However, some microorganisms can consume the organic ligands and release Cr(III), which then precipitates. Efforts are being made to characterize the organo-Cr(III) complexes and investigate their behavior in soil. Progress and efforts are summarized for each task. Task 1. Production of soluble organo-Cr(III) complexes by selected microorganisms A total of eight organisms were screened for production of soluble organo-Cr(III) complexes by culturing in both growth and non growth media containing 4 mg/L of Cr(VI); three were Gram positive and five were Gram negative. The Gram-positive bacteria were Cellulomonas sp. ES 6, Rhodococcus sp., and Leafsonia sp., while Shewanella oneidensis MR 1, Desulfovibrio desulfuricans G20, D. vulgaris Hildenborough, Pseudomonas putida MK 1 and Ps. aeruginosa PAO 1 were Gram negative. Purifications of the soluble organo-Cr(III) complexes produced by Cellulomonas sp. ES 6, Shewanella. oneidensis MR 1, Rhodococcus sp., and D. vulgaris Hildenborough were carried out. The culture supernatants were lyophilized and extracted first with methanol followed by water. The extracts were then analyzed for soluble Cr. The majority of the Cr(III) was present in the water-soluble fraction for all of the bacteria tested (data not shown), revealing a general phenomenon of soluble Cr(III) production. Cellulomonas sp. ES6 produced the highest amount of soluble Cr(III) (364 ppm) and D. vulgaris Hildenborough produced the least (143 ppm). Seventy eight percent of the soluble Cr(III) produced by Shewanella. oneidensis MR 1 was water soluble, while 45% was water soluble for the Cellulomonas sp. ES6. The water-soluble fractions were further purified by anion exchange chromatography. All soluble Cr(III) was bound to the anion exchange column. The bound organo-Cr(III) was eluted by gradient elution, (0.25M-2M) using ammonium acetate. Preliminary characterization confirmed the nature of organo-Cr(III) complexes. Further characterization of these species by electrospray ionization mass spectrometry (ESI-MS) is in progress. Task 2. Demonstrate that chromate reduction produces organo-Cr(III) complexes with microbial cellular components. In the past year, further research on the formation of organo-Cr(III) complexes has been completed. Formation of soluble complexes with cell free extracts as the organic portion has resulted in the formation of organo-Cr(III) complexes, approximately 27% Cr(III) remained soluble after 14 days. In addition, complexes formed between individual organic components and Cr(III) have been tested for changes in solubility due to changes in pH