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Bacillus sp. Abq, belonging to Bacillus cereus sensu lato, was isolated from an aquifer in New Mexico, USA and phylogenetically classified. The isolate possesses the unusual property of precipitating Pb(II) by using cysteine, which is degraded intracellularly to hydrogen sulfide (H2S). H2S is then exported to the extracellular environment to react with Pb(II), yielding PbS (galena). Biochemical and growth tests showed that other sulfur sources tested (sulfate, thiosulfate, and methionine) were not reduced to hydrogen sulfide. Using equimolar concentration of cysteine, 1 mM of soluble Pb(II) was removed from Lysogeny Broth (LB) medium within 120 h of aerobic incubation forming black, solid PbS, with a removal rate of 2.03 µg L-1 h-1 (∼8.7 µM L-1 h-1). The mineralogy of biogenic PbS was characterized and confirmed by XRD, HRTEM, and EDX. Electron microscopy and electron diffraction identified crystalline PbS nanoparticles with a diameter <10 nm, localized in the extracellular matrix and on the surface of the cells. This is the first study demonstrating the use of cysteine in Pb(II) precipitation as insoluble PbS and it may pave the way to PbS recovery from secondary resources, such as Pb-laden industrial effluents.
Lead (Pb) ranks as a major anthropogenic pollutant because it is used extensively by industry and it has no known biological function. The toxic effects of Pb(II) include inhibition of protein synthesis, alteration of the osmotic balance, enzyme inhibition, nucleic acid damage, disruption of membrane functions and oxidative phosphorylation, and is neurotoxic for young children and fetuses. Lead enters bacterial cells using uptake pathways for essential metals such as Mn(II) and Zn(II) . An interesting bioremediation approach connects the biological production of sulfide with metal removal through the formation of sparingly soluble metal sulfides. We have found that cultures of Bacillus cereus and Escherichia coli would remove 70% to 90% of Pb(II), respectively, from solution when grown in media containing 1 mM lead acetate. The removal of Pb(II) with the formation of a black precipitate by E. coli required the addition of cysteine to complex medium while lead precipitation by B. cereus did not require the addition of cysteine to the growth medium. Extracellular matrix material consisting of capsule and other extracellular polymeric materials isolated from cells of both B. cereus and E. coli were found to bind Pb(II) by mechanisms that could be described by Freundlich isotherms. Analysis of the extracellular matrix of B. cereus revealed the presence of aspartic acid and glutamic acid which effectively bind Pb(II). A considerable amount of PbS was produced outside of the cells and using electron diffraction X-ray analysis coupled to electron microscopy to assess element composition, the particles were determined to be PbS and were 280-400 nm in diameter. Additionally, B. cereus and E. coli produce PbS nanocrystals (~4 – 10 nm) that are associated with the cell when the bacteria are grown in complex organic media containing Pb(II). In complex media containing yeast extract and tryptone, B. cereus does not require addition of cysteine for sulfide production because the cells have sufficient proteolytic activity to release appreciable levels of hydrogen sulfide from sulfur-containing amino acids. However, E. coli requires cysteine for hydrogen sulfide production. While it is unknown how many enzymes are used by B. cereus for hydrogen sulfide production, E. coli has at least 4 distinct enzymes that can use cysteine as the substrate for release of hydrogen sulfide.