The discovery and engineering of metabolic pathways for the bioremediation and precipitation of heavy metals

To detoxify heavy metal pollutants, metals can be precipitated as metal sulfides and effectively removed from solution. While this is often accomplished industrially by the (abiotic) addition of hydrogen sulfide, sulfide-generating bacteria could be alternatively employed. Most research has focused on the use of sulfate-reducing bacteria, which convert sulfate to sulfide. However, sulfate-reducing bacteria cannot produce sulfide in the presence of oxygen and are thus limited to anaerobic applications. To thwart this limitation, this study investigated the capabilities and mechanisms of two aerobic microorganisms capable of precipitating metals as metal sulfides: a deep-sea isolate and a genetically engineered bacterium.; A new strain of Pseudomonas aeruginosa, designated strain CW961, was isolated from a deep-sea hydrothermal vent. This strain demonstrated the ability to precipitate cadmium as cadmium sulfide and remove 1 mM cadmium almost completely from solution. Electron microscopy and energy dispersive x-ray spectroscopy (EDXS) indicated that cadmium sulfide formed on the surface of the bacterium. Thiosulfate was found to be necessary for cell survival in the presence of cadmium and cadmium sulfide formation.; Unlike thiosulfate or other complex sulfur sources, sulfate is commonly found in the environment and would provide a cost-effective and accessible source of sulfur for sulfide production. To utilize sulfate as a sulfur source, a novel metabolic pathway was engineered and expressed in Escherichia coli. The assimilatory sulfate reduction pathway was redirected to over-produce cysteine and excess cysteine was converted to sulfide by cysteine desulfhydrase. Analysis of the individual components of the pathway revealed that cysteine generation was the rate-determining step. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve a balance between the production and degradation of cysteine. E. coli expressing the pathway were capable of precipitating 100 μM cadmium almost completely from solution. Electron microscopy and EDXS showed that cadmium sulfide formed on the surface of the bacterium. An analytical model demonstrated that this surface precipitation occurred because the reaction between cadmium and sulfide is extremely fast while the generation of sulfide is relatively slow.