The purpose of this research was to compare the relative toxicity of selenite and selenate with a proposed biomethylation intermediate, dimethyl selenone, and analyze bacterial media to determine the distribution of selenium species in bacterial cultures.Methods
Pseudomonas fluorescens K27 was used and a minimal medium (DM-N medium) was applied for anaerobic growth of this bacterium. Bacterial cultures were amended with selenate, selenite and freshly synthesized dimethyl selenone; growth inhibition and doubling time methods were used to determine the toxicity.Findings
The distribution of selenium species in amended cultures was measured in a series of time course experiments. Volatile methylated selenium species produced by the bacteria were detected by gas chromatography coupled with a fluorine-induced chemiluminescence detection. Elemental selenium precipitates and total selenium oxyanions in the supernatant were determined by atomic absorption spectroscopy. The selenite anions in the solution were determined by a colorimetric test via UV/VIS spectrophotometry over time. Furthermore, the change of nitrate concentration over time in bacterial cultures was quantified by using UV/VIS spectroscopy.
The relative toxicity of the three selenium species examined using both growth inhibition and doubling time methods with Pseudomonas fluorescens K27 increased in the order of selenite < selenate < dimethyl selenone. However, the maximum concentration at which this bacterium was observed to survive is in an increasing order of dimethyl selenone (0.7 mM) < selenite (35 mM) < selenate (200 mM).
The growth rates of K27 were dramatically slowed when cultures were amended with selenate in the range of 1 mM to 5 mM; they were even two times slower than 100 mM selenate amended cultures. The higher the concentration of selenate amended, the longer the lag phase observed for this bacterium. However, in selenite amended cultures, growth rates were smoothly decreased with the increasing of the concentration of selenite, and the higher the concentration of selenite amended, the more elemental selenium was produced. Most interesting, more than one exponential growth phase was observed in this case.
Nitrate is the limiting reagent for the anaerobic growth of K27 in our DM-N medium according to these experiments. Nitrate reduction does not inhibit selenite reduction while selenite reduction does inhibit nitrate reduction: selenite was reduced simultaneously with nitrate reduction and only about 3/4 of added nitrate was consumed in the 10 mM selenite amended culture even 120 hours after stationary phase was achieved. For the selenate amended cultures, on the other hand, nitrate inhibits selenate reduction but selenate does not inhibit nitrate reduction: only when nitrate was almost consumed, (less than 1 mM nitrate in the solution in 10 mM selenate amended culture), could K27 start to reduce selenate.
The reduction of selenate was accompanied by the production of volatile selenium and sulfur compounds with little elemental selenium being produced. However, the reduction of selenite mainly involved the production of elemental selenium and volatile selenium compounds; much less organosulfur compounds were observed in selenite amended cultures than selenate amended cultures. No dimethyl selenenyl sulfide was observed in selenite amended culture; while this is one of the major volatile organosulfur compound present in the headspace of selenate amended cultures of K27.
Thomas G. Chasteen