Chemical forms of selenium in the metal-resistant bacterium Ralstonia metallidurans CH34 exposed to selenite and selenate

Abstract

Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se(0)). We have studied the kinetics of selenite (Se(IV)) and selenate (Se(VI)) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se(0) and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se(0). Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se(0) was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se(IV) was detected as a transient species in the first 12 h after selenate introduction, Se(0) also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.

FIG. 1.

XANES spectra for some reference compounds (in solid state unless otherwise specified). From top to bottom: sodium selenate, sodium selenite, selenourea, S-methyl seleno l-cysteine, seleno-dl-cystine, selenodiglutathione (in solution), red and gray elemental selenium. The position of the maximum of the white line is indicated in parentheses.

FIG. 2.

Concentration of Se species in R. metallidurans CH34 exposed to selenite as determined by XANES linear combination fitting and ICP-MS analyses and time course of growth for the bacteria exposed to selenite (open circles). Error bars correspond to ±5% of total Se.

FIG. 3.

Selected Se K-edge XANES spectra for R. metallidurans CH34 at various incubation times after introduction of selenite into the culture medium at A₆₀₀s (optical densities [O.D.]) of 0.3, 1, and 3 and the distribution of Se species determined by linear combination fitting.

FIG. 4.

Concentration of Se species in R. metallidurans CH34 exposed to selenate as determined by XANES linear combination fitting and ICP-MS analyses and time course of growth for the bacteria exposed to selenate (open circles) and for the control culture in the absence of added selenium oxyanion (filled circles). Error bars correspond to ±5% of total Se.

FIG. 5.

Selected Se K-edge XANES spectra for R. metallidurans CH34 at various incubation times after the introduction of selenate into the culture medium at A₆₀₀s (optical densities [O.D.]) of 0.3 and 1 and the distribution of Se species determined by linear combination fitting.