Mitchell J. Herbel

Structural and Spectral Features of Selenium Nanospheres Produced by Se-Respiring Bacteria

ABSTRACT Certain anaerobic bacteria respire toxic selenium oxyanions and in doing so produce extracellular accumulations of elemental selenium [Se(0)]. We examined three physiologically and phylogenetically diverse species of selenate- and selenite-respiring bacteria, Sulfurospirillum barnesii, Bacillus selenitireducens, and Selenihalanaerobacter shriftii, for the occurrence of this phenomenon. When grown with selenium oxyanions as the electron acceptor, all of these organisms formed extracellular granules consisting of stable, uniform nanospheres (diameter, ∼300 nm) of Se(0) having monoclinic crystalline structures. Intracellular packets of Se(0) were also noted. The number of intracellular Se(0) packets could be reduced by first growing cells with nitrate as the electron acceptor and then adding selenite ions to washed suspensions of the nitrate-grown cells. This resulted in the formation of primarily extracellular Se nanospheres. After harvesting and cleansing of cellular debris, we observed large differences in the optical properties (UV-visible absorption and Raman spectra) of purified extracellular nanospheres produced in this manner by the three different bacterial species. The spectral properties in turn differed substantially from those of amorphous Se(0) formed by chemical oxidation of H2Se and of black, vitreous Se(0) formed chemically by reduction of selenite with ascorbate. The microbial synthesis of Se(0) nanospheres results in unique, complex, compacted nanostructural arrangements of Se atoms. These arrangements probably reflect a diversity of enzymes involved in the dissimilatory reduction that are subtly different in different microbes. Remarkably, these conditions cannot be achieved by current methods of chemical synthesis.

Fractionation of selenium isotopes during bacterial respiratory reduction of selenium oxyanions

Reduction of selenium oxyanions by microorganisms is an important process in the biogeochemical cycling of selenium. Numerous bacteria can reduce Se oxyanions, which are used as electron acceptors during the oxidation of organic matter in anoxic environments. In this study, we used a double spike (82Se and 74Se) thermal ionization mass spectrometry technique to quantify the isotopic fractionation achieved by three different species of anaerobic bacteria capable of accomplishing growth by respiratory reduction of selenate [SeO42− or Se(VI)] or selenite [SeO32− or Se(IV)] to Se(IV) or elemental selenium [Se(0)] coupled with the oxidation of lactate. Isotopic discrimination in these closed system experiments was evaluated by Rayleigh fractionation equations and numerical models. Growing cultures of Bacillus selenitireducens, a haloalkaliphile capable of growth using Se(IV) as an electron acceptor, induced a 80Se/76Se fractionation of −8.0 ± 0.4‰ (instantaneous ϵ value) during reduction of Se(IV) to Se(0). With Bacillus arsenicoselenatis, a haloalkaliphile capable of growth using Se(VI) as an electron acceptor, fractionations of −5.0 ± 0.5‰ and −6.0 ± 1.0‰ were observed for reduction of Se(VI) to Se(IV) and reduction of Se(IV) to Se(0), respectively. In growing cultures of Sulfurospirillum barnesii, a freshwater species capable of growth using Se(VI), fractionation was small initially, but near the end of the log growth phase, it increased to −4.0 ± 1.0‰ and −8.4 ± 0.4‰ for reduction of Se(VI) to Se(IV) and reduction of Se(IV) to Se(0), respectively. Washed cell suspensions of S. barnesii induced fractionations of −1.1 ± 0.4‰ during Se(VI) reduction, and −9.1 ± 0.5% for Se(IV) reduction, with some evidence for smaller values (e.g., −1.7‰) in the earliest-formed Se(0) results. These results demonstrate that dissimilatory reduction of selenate or selenite induces significant isotopic fractionation, and suggest that significant Se isotope ratio variation will be found in nature.

Reduction of Elemental Selenium to Selenide: Experiments with Anoxic Sediments and Bacteria that Respire Se-Oxyanions

A selenite-respiring bacterium, Bacillus selenitireducens, produced significant levels of Se(-II) (as aqueous HSe−) when supplied with Se(0). B. selenitireducens was also able to reduce selenite [Se(IV)] through Se(0) to Se(-II). Reduction of Se(0) by B. selenitireducens was more rapid in cells grown on colloidal sulfur [S(0)] or Se(IV) as their electron acceptor than for cell lines grown on fumarate. In contrast, three cultures of selenate-respiring bacteria, Sulfurospirillum barnesii, B. arsenicoselenatis, and Selenihalanaerobacter shriftii either were unable to reduce Se(0) to Se(-II) or had only a very limited capacity to achieve this reduction. Biological reduction of Se(0) to Se(-II) was observed during incubation of estuarine sediment slurries, while no such activity was noted in formalin-killed controls. The majority of the Se(-II) produced was found in the sediments as a solid precipitate of FeSe, rather than in solution as HSe−. These results demonstrate that certain anaerobic bacteria have the capacity to reduce Se(0) to Se(-II), providing a possible biological explanation for the occurrence of the selenide species in some sedimentary rocks.

Stable isotope fractionation of selenium by natural microbial consortia

The mobility and bioavailability of Se depend on its redox state, and reduction of Se oxyanions to less mobile, reduced species controls transport of this potentially toxic element in the environment. Stable isotope fractionation of Se is currently being developed as an indicator of Se immobilization through reduction. In this study, Se isotope fractionation resulting from reduction of Se(VI) and Se(IV) oxyanions by natural microbial consortia was measured in sediment slurry experiments under nearly natural conditions, with no substrate added. Experiments were conducted with a wide range of initial Se concentrations and with sediment and water from three locations with contrasting environmental settings. The products of Se(VI) and Se(IV) reduction were enriched in the lighter isotopes relative to the reactants. Shifts of −2.6‰ to −3.1‰ and −5.5‰ to −5.7‰, respectively, were observed in the 80Se/76Se ratio. These isotopic fractionations did not depend significantly on initial Se concentrations, which were varied from 22 μg/l to 8 mg/l, or on geochemical differences among the sediments. These results provide estimates of Se isotope fractionation in organic-rich wetland environments but may not be appropriate for substrate-poor aquifers and marine sediments.

Dissimilatory arsenate reductase activity and arsenate-respiring bacteria in bovine rumen fluid, hamster feces, and the termite hindgut

Abstract Bovine rumen fluid and slurried hamster feces completely reduced millimolar levels of arsenate to arsenite upon incubation under anoxic conditions. This activity was strongly inhibited by autoclaving or aerobic conditions, and partially inhibited by tungstate or chloramphenicol. The rate of arsenate reduction was faster in feces from a population of arsenate-watered (100 ppm) hamsters compared to a control group watered without arsenate. Using radioisotope methods, arsenate reductase activity in hamster feces was also detected at very low concentrations of added arsenate ( approximately 10 muM). Bacterial cultures were isolated from these materials, as well as from the termite hindgut, that grew using H(2) as their electron donor, acetate as their carbon source, and arsenate as their respiratory electron acceptor. The three cultures aligned phylogenetically either with well-established enteric bacteria, or with an organism associated with feedlot fecal wastes. Because arsenite is transported across the gut epithelium more readily than arsenate, microbial dissimilatory reduction of arsenate in the gut may promote the bodys absorption of arsenic and hence potentiate its toxicity.