Reduction of Selenite to Red Elemental Selenium by Rhodopseudomonas palustris Strain N

The trace metal selenium is in demand for health supplements to human and animal nutrition. We studied the reduction of selenite (SeO₃ ⁻²) to red elemental selenium by Rhodopseudomonas palustris strain N. This strain was cultured in a medium containing SeO₃ ⁻² and the particles obtained from cultures were analyzed using transmission electron microscopy (TEM), energy dispersive microanalysis (EDX) and X ray diffraction analysis (XRD). Our results showed the strain N could reduce SeO₃ ⁻² to red elemental selenium. The diameters of particles were 80–200 nm. The bacteria exhibited significant tolerance to SeO₃ ⁻² up to 8.0 m mol/L concentration with an EC₅₀ value of 2.4 m mol/L. After 9 d of cultivation, the presence of SeO₃ ²⁻ up to 1.0 m mol/L resulted in 99.9% reduction of selenite, whereas 82.0% (p<0.05), 31.7% (p<0.05) and 2.4% (p<0.05) reduction of SeO₃ ⁻² was observed at 2.0, 4.0 and 8.0 m mol/L SeO₃ ²⁻ concentrations, respectively. This study indicated that red elemental selenium was synthesized by green technology using Rhodopseudomonas palustris strain N. This strain also indicated a high tolerance to SeO₃ ⁻². The finding of this work will contribute to the application of selenium to human health.     

Selenate Reduction to Elemental Selenium by Anaerobic Bacteria in Sediments and Culture: Biogeochemical Significance of a Novel, Sulfate-Independent Respiration

Interstitial water profiles of SeO₄²⁻, SeO₃²⁻, SO₄²⁻, and Cl⁻ in anoxic sediments indicated removal of the seleno-oxyanions by a near-surface process unrelated to sulfate reduction. In sediment slurry experiments, a complete reductive removal of SeO₄²⁻ occurred under anaerobic conditions, was more rapid with H₂ or acetate, and was inhibited by O₂, NO₃⁻, MnO₂, or autoclaving but not by SO₄²⁻ or FeOOH. Oxidation of acetate in sediments could be coupled to selenate but not to molybdate. Reduction of selenate to elemental selenium was determined to be the mechanism for loss from solution. Selenate reduction was inhibited by tungstate and chromate but not by molybdate. A small quantity of the elemental selenium precipitated into sediments from solution could be resolublized by oxidation with either nitrate or FeOOH, but not with MnO₂. A bacterium isolated from estuarine sediments demonstrated selenate-dependent growth on acetate, forming elemental selenium and carbon dioxide as respiratory end products. These results indicate that dissimilatory selenate reduction to elemental selenium is the major sink for selenium oxyanions in anoxic sediments. In addition, they suggest application as a treatment process for removing selenium oxyanions from wastewaters and also offer an explanation for the presence of selenite in oxic waters.     

Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ~600 kDa. The individual subunit sizes are ~100 kDa (α), ~55 kDa (β), and ~36 kDa (γ), with a predicted overall subunit composition of α₃β₃γ₃. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent K_m for selenate was determined to be ~2 mM, with an observed V_max of 500 nmol SeO₄²⁻ min⁻¹ mg⁻¹ (k_cat, ~5.0 s⁻¹). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.     

Dissimilatory Selenate Reduction Potentials in a Diversity of Sediment Types

We measured potential rates of bacterial dissimilatory reduction of ⁷⁵SeO₄²⁻ to ⁷⁵Se⁰ in a diversity of sediment types, with salinities ranging from freshwater (salinity = 1 g/liter) to hypersaline (salinity = 320 g/liter and with pH values ranging from 7.1 to 9.8. Significant biological selenate reduction occurred in all samples with salinities from 1 to 250 g/liter but not in samples with a salinity of 320 g/liter. Potential selenate reduction rates (25 nmol of SeO₄²⁻ per ml of sediment added with isotope) ranged from 0.07 to 22 μmol of SeO₄²⁻ reduced liter⁻¹ h⁻¹. Activity followed Michaelis-Menten kinetics in relation to SeO₄²⁻ concentration (K_m of selenate = 7.9 to 720 μM). There was no linear correlation between potential rates of SeO₄²⁻ reduction and salinity, pH, concentrations of total Se, porosity, or organic carbon in the sediments. However, potential selenate reduction was correlated with apparent K_m for selenate and with potential rates of denitrification (r = 0.92 and 0.81, respectively). NO₃⁻, NO₂⁻, MoO₄²⁻, and WO₄²⁻ inhibited selenate reduction activity to different extents in sediments from both Hunter Drain and Massie Slough, Nev. Sulfate partially inhibited activity in sediment from freshwater (salinity = 1 g/liter) Massie Slough samples but not from the saline (salinity = 60 g/liter) Hunter Drain samples. We conclude that dissimilatory selenate reduction in sediments is widespread in nature. In addition, in situ selenate reduction is a first-order reaction, because the ambient concentrations of selenium oxyanions in the sediments were orders of magnitude less than their K_ms.     

Biomimetic Synthesis of Selenium Nanospheres by Bacterial Strain JS-11 and Its Role as a Biosensor for Nanotoxicity Assessment: A Novel Se-Bioassay

Selenium nanoparticles (Se-NPs) were synthesized by green technology using the bacterial isolate Pseudomonas aeruginosa strain JS-11. The bacteria exhibited significant tolerance to selenite (SeO₃ ²⁻) up to 100 mM concentration with an EC₅₀ value of 140 mM. The spent medium (culture supernatant) contains the potential of reducing soluble and colorless SeO₃ ²⁻ to insoluble red elemental selenium (Se⁰) at 37°C. Characterization of red Se° product by use of UV-Vis spectroscopy, X-ray diffraction (XRD), atomic force microscopy (AFM) and transmission electron microscopy (TEM) with energy dispersive X-ray spectrum (EDX) analysis revealed the presence of stable, predominantly monodispersed and spherical selenium nanoparticles (Se-NPs) of an average size of 21 nm. Most likely, the metabolite phenazine-1-carboxylic acid (PCA) released by strain JS-11 in culture supernatant along with the known redox agents like NADH and NADH dependent reductases are responsible for biomimetic reduction of SeO₃ ²⁻ to Se° nanospheres. Based on the bioreduction of a colorless solution of SeO₃ ²⁻ to elemental red Se⁰, a high throughput colorimetric bioassay (Se-Assay) was developed for parallel detection and quantification of nanoparticles (NPs) cytotoxicity in a 96 well format. Thus, it has been concluded that the reducing power of the culture supernatant of strain JS-11 could be effectively exploited for developing a simple and environmental friendly method of Se-NPs synthesis. The results elucidated that the red colored Se° nanospheres may serve as a biosensor for nanotoxicity assessment, contemplating the inhibition of SeO₃ ²⁻ bioreduction process in NPs treated bacterial cell culture supernatant, as a toxicity end point.     

The antimutagenic effect of selenium on 7,12-dimethylbenz(a)anthracene and metabolites in the amesSalmonella/microsome system

The antimutagenic effect of selenium as sodium selenite, sodium selenate, selenium dioxide, and seleno-methionine was studied in the AmesSalmonella/microsome mutagenicity test using 7,12-dimethylbenz(a)anthracene (DMBA) and some of its metabolites. Selenium (20 ppm) as sodium selenite reduced the number of histidine revertants on plates containing up to 100 μg DMBA/plate. Increasing concentrations of selenium as sodium selenite, sodium selenate, and selenium dioxide up to 40 ppm Se progressively decreased the number of revertants caused by 50 μg DMBA. DMBA and its metabolites 7-hydroxymethyl-12-methylbenz(a)anthracene, 12-hydroxymethyl-7-methylbenz(a)anthracene, and 3-hydroxy-7,12-dimethylbenz(a)anthracene were mutagenic forSalmonella typhimurium TA100 in the presence of an S-9 mixture. Selenium supplementation as Na₂SeO₃ reduced the number of revertants induced by these metabolites to background levels. The antimutagenic effect of inorganic selenium compounds cannot be explained by toxicity of selenium as determined by viability tests withSalmonella typhimurium TA100. Selenium supplementation in all forms examined, except sodium selenate, decreased the rate of spontaneous reversion. Selenium as sodium selenate was slightly mutagenic at concentrations of 4 ppm or less. Higher concentration of Na₂SeO₄ inhibited the mutagenicity of DMBA. The present studies support the anticarcinogenic potential of selenium and indicate that form and concentration are important factors in this trace element's efficacy.     

Fate of selenate and selenite metabolized by Rhodobacter sphaeroides

Cultures of a purple nonsulfur bacterium, Rhodobacter sphaeroides, amended with approximately 1 or approximately 100 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.     

Microbial transformations of selenite by methane-oxidizing bacteria

Methane-oxidizing bacteria are well known for their role in the global methane cycle and their potential for microbial transformation of wide range of hydrocarbon and chlorinated hydrocarbon pollution. Recently, it has also emerged that methane-oxidizing bacteria interact with inorganic pollutants in the environment. Here, we report what we believe to be the first study of the interaction of pure strains of methane-oxidizing bacteria with selenite. Results indicate that the commonly used laboratory model strains of methane-oxidizing bacteria, Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b, are both able to reduce the toxic selenite (SeO₃ ^2?) but not selenate (SeO₄ ^2?) to red spherical nanoparticulate elemental selenium (Se⁰), which was characterized via energy-dispersive X-ray spectroscopy (EDXS), X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS). The cultures also produced volatile selenium-containing species, which suggests that both strains may have an additional activity that can transform either Se⁰ or selenite into volatile methylated forms of selenium. Transmission electron microscopy (TEM) measurements and experiments with the cell fractions cytoplasm, cell wall and cell membrane show that the nanoparticles are formed mainly on the cell wall. Collectively, these results are promising for the use of methane-oxidizing bacteria for bioremediation or suggest possible uses in the production of selenium nanoparticles for biotechnology.

     

Heavy Metal Resistance Patterns of Frankia Strains

The sensitivity of 12 Frankia strains to heavy metals was determined by a growth inhibition assay. In general, all of the strains were sensitive to low concentrations (<0.5 mM) of Ag¹⁺, AsO₂¹⁻, Cd²⁺, SbO₂¹⁻, and Ni²⁺, but most of the strains were less sensitive to Pb²⁺ (6 to 8 mM), CrO₄²⁻ (1.0 to 1.75 mM), AsO₄³⁻ (>50 mM), and SeO₂²⁻ (1.5 to 3.5 mM). While most strains were sensitive to 0.1 mM Cu²⁺, four strains were resistant to elevated levels of Cu²⁺ (2 to 5 mM and concentrations as high as 20 mM). The mechanism of SeO₂²⁻ resistance seems to involve reduction of the selenite oxyanion to insoluble elemental selenium, whereas Pb²⁺ resistance and Cu²⁺ resistance may involve sequestration or binding mechanisms. Indications of the resistance mechanisms for the other heavy metals were not as clear.     

Aerobic, Selenium-Utilizing Bacillus Isolated from Seeds of Astragalus crotalariae

Bacillus sp. strain SS, an aerobic, gram-positive sporeformer, was isolated from seeds of Astragalus crotalariae, a selenium-accumulating plant. This bacillus grew in a nutrient broth (containing beef extract and peptone) if the medium was supplemented with high concentrations of selenium. Concentrations of Na₂SeO₃ that supported growth ranged from 3 to 100 mM. After 24 h of growth, the culture developed a deep red color characteristic of elemental selenium. When selenium was provided in the form of selenate, the pattern of growth showed a prolonged lag period, from 24 to 48 h. Final growth remained below that of cells cultured in the presence of selenite, and only a light red color developed. Concentrations of selenate below 40 mM failed to support growth. Tellurate, though not tellurite, could replace selenite, but only over a narrow concentration range, 5 to 10 mM. By 24 h, the typical black color of elemental tellurium developed. Bacillus sp. strain SS grew also in brain heart infusion broth and Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) without the addition of selenium or tellurium compounds. When added to these media, 50 mM selenite was tolerated and metabolized by the organism. The crucial distinction between this bacillus and other selenium-tolerant organisms (e.g., Salmonella) remains: under certain conditions, growth requirements of Bacillus sp. strain SS are fulfilled by selenium (and tellurium) compounds.     

Response to selenium by callus cultures derived from astragalus species

Callus cultures were obtained from five selenium accumulator and three nonaccumulator species of Astragalus. Their morphological characteristics and their growth responses to light, sucrose, kinetin, and 2,4-dichlorophenoxyacetic acid are described. Calluses derived from accumulator species characteristically retained their tolerance to high concentrations of selenate and selenite, whereas calluses derived from nonaccumulator species were markedly inhibited by these two forms of selenium. Competition between sulfate and selenate was demonstrated. The two types of calluses could not be distinguished on the basis of ⁷⁵Se-labeled selenate or selenite uptake. Neutron activation analysis failed to show differences in selenium content between the two types of calluses grown on media to which no selenium had been added.     

Pseudomonas seleniipraecipitans Proteins Potentially Involved in Selenite Reduction

Pseudomonas seleniipraecipitans grows in the presence of high levels of selenite and selenate and reduces both oxyanions to elemental selenium (Se⁰), a property that may make P. seleniipraecipitans useful as an inoculant for biobarriers designed to remove selenite or selenate from ground or surface waters. An earlier study showed that P. seleniipraecipitans nitrate reductase reduced selenate to Se⁰, but failed to identify the protein(s) involved in selenite reduction. This study used ammonium sulfate precipitation, hydrophobic interaction chromatography, and native PAGE to isolate two electrophoretic gel regions, identified as bands A and B that showed selenite-reductase-activity. Proteomics was used to identify the proteins present in those regions. Glutathione reductase (GR) was detected in the A-band; based on this information, Saccharomyces cerevisiae GR, obtained from a commercial source, was evaluated and found to have selenite-reductase-activity, confirming that GR can reduce selenite to Se⁰. Proteomics was also used to detect the proteins present in the B-band and thioredoxin reductase (ThxR) was detected as a B-band protein; based on this information, E. coli ThxR, obtained from a commercial source, was evaluated and found to have selenite-reductase-activity, confirming that ThxR can reduce selenite to elemental selenium. Thus, evidence presented in this study shows that S. cerevisiae GR and E. coli ThxR can reduce SeO₃ ^2? to Se⁰ and strongly suggests that P. seleniipraecipitans GR and ThxR can also reduce SeO₃ ^2? to Se⁰.     

Reduction of Selenite by Azospirillum brasilense with the Formation of Selenium Nanoparticles

The ability to reduce selenite (SeO₃ ^2?) ions with the formation of selenium nanoparticles was demonstrated in Azospirillum brasilense for the first time. The influence of selenite ions on the growth of A. brasilense Sp7 and Sp245, two widely studied wild-type strains, was investigated. Growth of cultures on both liquid and solid (2 % agar) media in the presence of SeO₃ ^2? was found to be accompanied by the appearance of the typical red colouration. By means of transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS) and X-ray fluorescence analysis (XFA), intracellular accumulation of elementary selenium in the form of nanoparticles (50 to 400 nm in diameter) was demonstrated for both strains. The proposed mechanism of selenite-to-selenium (0) reduction could involve SeO₃ ^2? in the denitrification process, which has been well studied in azospirilla, rather than a selenite detoxification strategy. The results obtained point to the possibility of using Azospirillum strains as endophytic or rhizospheric bacteria to assist phytoremediation of, and cereal cultivation on, selenium-contaminated soils. The ability of A. brasilense to synthesise selenium nanoparticles may be of interest to nanobiotechnology for “green synthesis” of bioavailable amorphous red selenium nanostructures.     

Formate Dehydrogenase of Clostridium thermoaceticum: Incorporation of Selenium-75, and the Effects of Selenite, Molybdate, and Tungstate on the Enzyme

The formation of the nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase in Clostridium thermoaceticum is stimulated by the presence of molybdate and selenite in the growth medium. The highest formate dehydrogenase activity was obtained with 2.5 × 10⁻⁴ M Na₂MoO₄ and 5 × 10⁻⁵ Na₂SeO₃. Tungstate but not vanadate could replace molybdate and stimulate the formation of formate dehydrogenase. Tungstate stimulated activity more than molybdate, and in combination with molybdate the stimulation of formation of formate dehydrogenase was additive. Formate dehydrogenase was isolated from cells grown in the presence of Na₂⁷⁵SeO₂, and a correlation was observed between bound ⁷⁵Se and enzyme activity.     

Identification and Characterization of an Aeromonas salmonicida (syn Haemophilus piscium) Strain That Reduces Selenite to Elemental Red Selenium

A bacterium that reduces toxic and mobile selenite to insoluble elemental selenium (Se⁰) was isolated from a laboratory scale permeable reactive biobarrier. Biochemical tests and 16S rRNA gene sequence alignment identified the isolate as Aeromonas salmonicida. Two colony types were isolated, one more resistant to selenite than the other. Both grew on agar plates containing 16 mM selenite, although the colony diameter was reduced to 8% of controls with the small colony type and to 18% with the large colony type. Further study was done with the large colony type. In anaerobic culture, this bacterium was able to use nitrate as a term electron acceptor but not selenate or selenite. In aerobic culture, when no nitrate was present, early log phase cells removed selenite at a rate of 2.6 ± 0.42 μmol SeO₃⁻²/mg protein/day. Reduction was retarded by 25 mM nitrate. Mutants with a diminished ability to reduce selenite to Se⁰ also had a reduced ability to reduce nitrate to nitrous oxide. This bacterium, or perhaps its enzymes or DNA, might be used to remove selenite from contaminated groundwaters.     

Biochemical and Biophysical Characterization of the Selenium-binding and Reducing Site in Arabidopsis thaliana Homologue to Mammals Selenium-binding Protein 1

The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO₃²⁻) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys²¹ and Cys²² as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO₃²⁻ to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism.     

Isolation, Growth, and Metabolism of an Obligately Anaerobic, Selenate-Respiring Bacterium, Strain SES-3

A gram-negative, strictly anaerobic, motile vibrio was isolated from a selenate-respiring enrichment culture. The isolate, designated strain SES-3, grew by coupling the oxidation of lactate to acetate plus CO₂ with the concomitant reduction of selenate to selenite or of nitrate to ammonium. No growth was observed on sulfate or selenite, but cell suspensions readily reduced selenite to elemental selenium (Se⁰). Hence, SES-3 can carry out a complete reduction of selenate to Se⁰. Washed cell suspensions of selenate-grown cells did not reduce nitrate, and nitrate-grown cells did not reduce selenate, indicating that these reductions are achieved by separate inducible enzyme systems. However, both nitrate-grown and selenate-grown cells have a constitutive ability to reduce selenite or nitrite. The oxidation of [¹⁴C]lactate to ¹⁴CO₂ coupled to the reduction of selenate or nitrate by cell suspensions was inhibited by CCCP (carbonyl cyanide m-chlorophenylhydrazone), cyanide, and azide. High concentrations of selenite (5 mM) were readily reduced to Se⁰ by selenate-grown cells, but selenite appeared to block the synthesis of pyruvate dehydrogenase. Tracer experiments with [⁷⁵Se]selenite indicated that cell suspensions could achieve a rapid and quantitative reduction of selenite to Se⁰. This reduction was totally inhibited by sulfite, partially inhibited by selenate or nitrite, but unaffected by sulfate or nitrate. Cell suspensions could reduce thiosulfate, but not sulfite, to sulfide. These results suggest that reduction of selenite to Se⁰ may proceed, in part, by some of the components of a dissimilatory system for sulfur oxyanions.     

Reduction of Selenite and Detoxification of Elemental Selenium by the Phototrophic Bacterium Rhodospirillum rubrum

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1.5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.     

A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite

The measurement of elemental selenium (Se⁰) is needed to assess the rate and magnitude of bacteria reduction of selenite or selenate. We have developed a spectrophotometric method for the measurement Se⁰ that is rapid and can be employed to measure the quantity of Se⁰ produced by bacterial cultures. This method employs the use of 1 M Na₂S to convert the insoluble elemental selenium to a red-brown solution and with this method there is a direct correlation between concentration of elemental selenium and the absorption at 500 nm. To demonstrate the utility of this assay, we have followed the reduction of selenite to Se⁰ by Moraxella bovis, and by bacterial consortia in soil and water samples.     

Cytotoxicity Associated with Trichloroethylene Oxidation in Burkholderia cepacia G4

The effects of trichloroethylene (TCE) oxidation on toluene 2-monooxygenase activity, general respiratory activity, and cell culturability were examined in the toluene-oxidizing bacterium Burkholderia cepacia G4. Nonspecific damage outpaced inactivation of toluene 2-monooxygenase in B. cepacia G4 cells. Cells that had degraded approximately 0.5 μmol of TCE (mg of cells⁻¹) lost 95% of their acetate-dependent O₂ uptake activity (a measure of general respiratory activity), yet toluene-dependent O₂ uptake activity decreased only 35%. Cell culturability also decreased upon TCE oxidation; however, the extent of loss varied greatly (up to 3 orders of magnitude) with the method of assessment. Addition of catalase or sodium pyruvate to the surfaces of agar plates increased enumeration of TCE-injured cells by as much as 100-fold, indicating that the TCE-injured cells were ultrasensitive to oxidative stress. Cell suspensions that had oxidized TCE recovered the ability to grow in liquid minimal medium containing lactate or phenol, but recovery was delayed substantially when TCE degradation approached 0.5 μmol (mg of cells⁻¹) or 66% of the cells' transformation capacity for TCE at the cell density utilized. Furthermore, among B. cepacia G4 cells isolated on Luria-Bertani agar plates from cultures that had degraded approximately 0.5 μmol of TCE (mg of cells⁻¹), up to 90% were Tol⁻ variants, no longer capable of TCE degradation. These results indicate that a toxicity threshold for TCE oxidation exists in B. cepacia G4 and that once a cell suspension has exceeded this toxicity threshold, the likelihood of reestablishing an active, TCE-degrading biomass from the cells will decrease significantly.