Cultures of a purple nonsulfur bacterium, Rhodobacter sphaeroides, amended with ϳ1 or ϳ100 ppm selenate or selenite, were grown phototrophically to stationary phase. Analyses of culture headspace, separated cells, and filtered culture supernatant were carried out using gas chromatography, X-ray absorption spectroscopy, and inductively coupled plasma spectroscopy-mass spectrometry, respectively. While selenium-amended cultures showed much higher amounts of SeO 3 2؊ bioconversion than did analogous selenate experiments (94% uptake for SeO 3 2؊ as compared to 9.6% for SeO 4 2؊ -amended cultures from 100-ppm solutions), the chemical forms of selenium in the microbial cells were not very different except at exposure to high concentrations of selenite. Volatilization accounted for only a very small portion of the accumulated selenium; most was present in organic forms and the red elemental form.The roles of selenium in the biosphere, both beneficial and deleterious, are gradually being determined (2,5,7,15), and it is becoming apparent that bacteria play a major role in the global selenium cycle. Selenate (SeO 4 2Ϫ ) and selenite (SeO 3 2Ϫ ) seem to be the most abundant forms of bioavailable selenium in the environment, and both can serve as electron acceptors for many microorganisms, including some phototrophic bacteria. These organisms are widespread and exhibit enormous metabolic and ecological diversity, and some are known to be able to use selenium oxyanions in their anaerobic metabolism (12,13,14,24). Rhodobacter sphaeroides is a purple nonsulfur bacterium that often serves as a model species for the group. It can tolerate high concentrations of selenite and selenate (13,14) and can reduce and/or methylate these compounds (24).Recent work with phototrophic bacteria has shown that some can grow in the presence of 0.1, 1.0, even 10 mM selenate or selenite. Though growth rates were somewhat decreased, five phototrophic species grown on Sistrom minimal medium were shown to exhibit overall stationary-phase biomass production similar to that of controls. Methylated organosulfur, organoselenium, and dimethyl selenenyl sulfide have been detected in anaerobic culture headspace, and elemental Se was produced in some cultures (13,21,23). This reduction to the elemental form may be an important process in the biosphere, since soils from an evaporation pond in the Kesterson Reservoir show that, nearest the surface, Se 0 is the most prominent selenium species (16).Although it is relatively clear that phototrophic bacteria can grow in the presence of selenium oxyanions and that small amounts are reduced and/or methylated, little is known of the extent and path of this process. Here we report the results of quantifying and speciating selenium in the culture medium, the bacteria, and the gaseous headspace above the culture after R. sphaeroides was grown phototrophically in the presence of different concentrations of selenite and selenate to stationary phase. Cell supernatants were analyzed for total selenium using inductively co...