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A Gram-stain-negative, non-endospore-producing, short-rod strain, KNDSS-Mac4T, was isolated from a downstream sediment sample of the river Ganges, Kanpur, India and studied by using the polyphasic taxonomic approach. 16S rRNA gene sequence analysis uncovered that the strain had similarity to species of the genus Thauera and formed a distinct phylogenetic cluster with Thauera humireducens KACC16524T. However, KNDSS-Mac4T showed closest phylogenetic affiliation to Thauera aminoaromatica DSM 14742T with 16S rRNA gene sequence similarity of 98.7 % followed by Thauera phenylacetica DSM 14743T (98.6 %), Thauera chlorobenzoica (98.2 %), T. humireducens KACC16524T (98.2 %), Thauera selenatis ATCC 55363T (98.2 %) and Thauera mechernichensis DSM 12266T (98.0 %). The digital DNA-DNA hybridization and average nucleotide identity values between strain KNDSS-Mac4T and the two most closely related taxa, T. aminoaromatica DSM 14742T and T. phenylacetica DSM 14743T, were 26.0, 26.7 and 84.0, 84.3 % respectively. Major lipids present were phosphatidylglycerol, three unknown aminophospholipids, phosphatidylmethylethanolamine, two unidentified lipids and Q-8 as the only ubiquonone. The major cellular fatty acids present were C16 : 1 ω6c/C16 : 1ω7c and C16 : 0. The DNA G+C content of strain KNDSS-Mac4T was 65.9 %. Based on data from phenotypic tests and the genotypic differences of strain KNDSS-Mac4T from its closest phylogenetic relatives, it is evident that this isolate should be regarded as a new species. It is proposed that strain KNDSS-Mac4T should be classified in the genus Thauera as a novel species, Thauerapropionica sp. nov. The type strain is KNDSS-Mac4T (=KCTC 52820T=VTCC-B-910017T).

We designed a primer set to target selenate reductase (SerA) for detecting selenate reducing bacteria (SeRB). Our serA gene-based PCR primer set has high specificity in that it and positively amplified some SeRB, but not denitrifying bacteria (DB). Phylogenetic analysis of serA clone sequences of environmental samples from selenate-reducing membrane biofilm reactor (MBfR) biofilms showed that these sequences were closely grouped and had high similarity to selenate reductase gene sequences from SeRB Thauera selenatis and DB Dechloromonas; however, they were distant to other genes from dimethylsulfoxide (DMSO) enzyme family. Constructing a standard curve targeting the serA gene, we found that the good linearity for the qPCR assay when applied it to quantify SeRB in MBfR biofilms, and the gene copies of SeRB correlated well to the selenate removal percentages. Our results demonstrated the feasibility of using the serA gene-based PCR primer set to detect and quantify SeRB in environmental samples.

A heterotrophic bacterial strain TL1T capable of aerobic denitrification was previously enriched in continuous culture from a landfill leachate treatment plant and isolated as a pure culture. The taxonomic position of this isolate within the beta-subclass of the Proteobacteria was determined by 16S rDNA sequence analysis and by conventional taxonomy including substrate spectrum, quinone type (ubiquinone Q-8) and cellular fatty acid composition. Detection of the specific polyamine 2-hydroxyputrescine supports the membership of strain TL1T in the beta-subclass of the Proteobacteria. The results of 16S rDNA sequencing showed that the strain clustered with, but was separate from, Thauera aromatica and Thauera selenatis. DNA-DNA hybridization experiments indicated that the new isolate represents a new species of the genus, for which the name Thauera mechernichensis is proposed; the type strain is DSM 12266T.

It is critical that an inexpensive electrondonor/carbon-source be found for selenium bioremediation using the selenate-respiring bacterium, Thauera selenatis. Since acetate is a preferred substrate for growth of this organism, a method was developed for fermenting the lactose in whey to large amounts of acetate. Indigenous whey microorganisms fermented the whey lactose in this manner when grown in continuous culture at a very slow dilution rate (D = 0.05 h-1). The successful use of the fermented whey lactose as the carbon-source/electron-donor feed for a laboratory-scale selenium-bioremediation reactor system, inoculated with T. selenatis, treating selenium-contaminated drainage water was also demonstrated. Selenium oxyanions and nitrate were reduced by 98%.

Thauera selenatis is one of two isolated bacterial species that can obtain energy by respiring anaerobically with selenate as the terminal electron acceptor. The reduction of selenate to selenite is catalyzed by a selenate reductase, previously shown to be located in the periplasmic space of the cell. This study describes the purification of the enzyme from T. selenatis grown anaerobically with selenate. The enzyme is a trimeric alphabetagamma complex with an apparent Mr of 180,000. The alpha, beta, and gamma subunits are 96 kDa, 40 kDa, and 23 kDa, respectively, in size. The selenate reductase contains molybdenum, iron, and acid-labile sulfur as prosthetic group constituents. UV-visible absorption spectroscopy also revealed the presence of one cytochrome b per alphabetagamma complex. The Km for selenate was determined to be 16 microM, and the Vmax was 40 micromol/min/mg of protein. The enzyme is specific for the reduction of selenate; nitrate, nitrite, chlorate, and sulfate were not reduced at detectable rates. These studies constitute the first description of a selenate reductase, which represents a new class of enzymes. The significance of this enzyme in relation to cell growth and energy generation is discussed.

This report describes a simple method for the bioremediation of selenium from agricultural drainage water. A medium-packed pilot-scale biological reactor system, inoculated with the selenate-respiring bacterium Thauera selenatis, was constructed at the Panoche Water District, San Joaquin Valley, Calif. The reactor was used to treat drainage water (7.6 liters/min) containing both selenium and nitrate. Acetate (5 mM) was the carbon source-electron donor reactor feed. Selenium oxyanion concentrations (selenate plus selenite) in the drainage water were reduced by 98%, to an average of 12 (plusmn) 9 (mu)g/liter. Frequently (47% of the sampling days), reactor effluent concentrations of less than 5 (mu)g/liter were achieved. Denitrification was also observed in this system; nitrate and nitrite concentrations in the drainage water were reduced to 0.1 and 0.01 mM, respectively (98% reduction). Analysis of the reactor effluent showed that 91 to 96% of the total selenium recovered was elemental selenium; 97.9% of this elemental selenium could be removed with Nalmet 8072, a new, commercially available precipitant-coagulant. Widespread use of this system (in the Grasslands Water District) could reduce the amount of selenium deposited in the San Joaquin River from 7,000 to 140 lb (ca. 3,000 to 60 kg)/year.

Selenium-oxyanion-containing wastewater, with levels of selenite as high as 3690 μg Se/l and very low levels of selenate, was treated in a laboratory-scale biological reactor system inoculated with the selenate-respiring bacterium Thauera selenatis. The wastewater contained selenite that had been removed from refinery effluent wastewater using iron-coprecipitation followed by selenite release to yield a more concentrated selenium-containing wastewater. The reactor system consisted of recycling sludge-blanket (500 ml; 200 g sand) and fluidized-bed reactors (500 ml; 150 g sand). The flow rate through the reactor system was 3.5 ml/min. The carbon source fed into the reactor was acetate (3 mM); nitrate was also present (3 mM). The selenium oxyanion levels in the wastewater were reduced by 95%. T. selenatis was the only selenate-reducing bacterium detected in the reactor system and it presumably reduced a portion of the selenate present in the water to selenite. The selenite present in the water, and that formed by selenate reduction, was reduced both by the Thauera and by a population of denitrifying bacteria also present in high numbers in the reactor system.

The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrying toluene degraders. In order to identify and group members of the genus Azoarcus, phylogenetic analysis based on partial sequences of 16S rRNA genes (16S rDNAs) is proposed. 16S rRNA-targeted PCR using specific primers to exclude amplification in the majority of other members of the beta subclass of the class Proteobacteria was combined with direct sequencing of the PCR products. Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp. sequences and for analysis of the complete 16S rDNA sequence. These three species formed a phylogenetically coherent group with representatives of two other Azoarcus species which were subjected to 16S rRNA sequencing in this study. This group was related to Rhodocyclus purpureus and Thauera selenatis. New isolates and also sequences of so far uncultured bacteria from roots of Kallar grass were assigned to the genus Azoarcus as well. Also, strains degrading monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates. All representative members of the five species harboring rhizospheric bacteria were able to form N2O from nitrate and showed anaerobic growth on malic acid with nitrate but not on toluene. In order to visualize different Azoarcus spp. by whole-cell in situ hybridizations, we generated 16S rRNA-targeted, fluorescent probes by in vitro transcription directly from PCR products which spanned the variable region V2. Hybridization was species specific for Azoarcus communis and Azoarcus indigens.(ABSTRACT TRUNCATED AT 250 WORDS)

During selenate respiration by Thauera selenatis , the reduction of selenate results in the formation of intracellular selenium (Se) deposits that are ultimately secreted as Se nanospheres of approximately 150 nm in diameter. We report that the Se nanospheres are associated with a protein of approximately 95 kDa. Subsequent experiments to investigate the expression and secretion profile of this protein have demonstrated that it is up-regulated and secreted in response to increasing selenite concentrations. The protein was purified from Se nanospheres, and peptide fragments from a tryptic digest were used to identify the gene in the draft T. selenatis genome. A matched open reading frame was located, encoding a protein with a calculated mass of 94.5 kDa. N-terminal sequence analysis of the mature protein revealed no cleavable signal peptide, suggesting that the protein is exported directly from the cytoplasm. The protein has been called Se factor A (SefA), and homologues of known function have not been reported previously. The sefA gene was cloned and expressed in Escherichia coli , and the recombinant His-tagged SefA purified. In vivo experiments demonstrate that SefA forms larger (approximately 300 nm) Se nanospheres in E. coli when treated with selenite, and these are retained within the cell. In vitro assays demonstrate that the formation of Se nanospheres upon the reduction of selenite by glutathione are stabilized by the presence of SefA. The role of SefA in selenium nanosphere assembly has potential for exploitation in bionanomaterial fabrication.

Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (E(m) + 234 +/- 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified ( approximately 24 and approximately 6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c(4) family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 +/- 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c(4) is a novel route for a member of the DMSO reductase family of molybdoenzymes.

A number of approaches have been used to show that a recently isolated selenate-respiring bacterium, Thauera selenatis, is able to synthesize both a selenate reductase (SR) and a nitrate reductase (NR). (i) The pH optimum of the SR was found to be 6.0; that of the NR was 7.0. (ii) The presence of nitrate did not inhibit selenate reduction in selenate-grown cells. (iii) In cell extracts, the highest SR or NR activity was observed in cells grown with the respective electron acceptor. (iv) Mutants that were unable to grow with nitrate as the terminal electron acceptor and lacked NR activity were isolated; these mutants grew normally with selenate and synthesized SR. (v) The SR was found in the periplasmic space of the cell, whereas the NR was present in the cytoplasmic membrane. A hypothetical electron transport system involving the SR is described.

Inhibitory effects of selenite and selenate towards hydrogenotrophic and acetoclastic methanogenesis were evaluated in anaerobic toxicity assays. The 50% inhibitory concentration (IC50) of both selenium oxyanions was below 6.1 X 10(-5) M in hydrogenotrophic assays, whereas acetoclastic methanogens were less inhibited: IC50 = 8.3 x 10(-5) M and 5.5 x 10(-4) M for selenite and selenate, respectively. Selenite completely inhibits methanogenesis from both substrates tested at concentrations >= 10(-3) M selenite, while only marginal methanogenic activities occur at equimolar concentrations of selenate. Selenite becomes irreversibly inhibitory upon a single exposure, whereas selenate inhibits methanogens upon repeated exposure. Consequently, methane recovery can be seriously hampered or even impossible during anaerobic treatment of highly selenium contaminated waste streams.

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, andSelenihalanaerobacter 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.

Bacterial strain T1, originally isolated by P. J. Evans on the basis of its capacity for toluene degradation under denitrifying conditions, has been classified as Thauera aromatica. In a comprehensive study of strains of this species, it was found that the cells have a different type of flagellar insertion from that of cells of the type species of the genus, Thauera selenatis, suggesting the convenience of an emendation of the description of the genus Thauera. Further studies on a larger collection of strains with the above characteristics may serve in the future as the basis for the creation of a new generic designation.

Author(s): Kausch, Matteo; Ng, Peter; Ha, Juyoung; Pallud, Celine | Abstract: Given the role of Se as both an environmental contaminant and a micronutrient, the microbial reduction and subsequent sequestration of bioavailable Se in soils are of great ecological interest. Primary particles in surface soils are typically bound into loosely packed, microporous aggregates, which may be critical spatial units in determining the fate of Se in soils. Surrounded by macropores where preferential flow rapidly advects dissolved compounds, soil aggregates are domains of slow diffusive transport where spatial variations in chemical concentrations and biogeochemical reactions can prevail. We conducted a series of controlled flow-through experiments utilizing three-dimensional, artificial soil aggregates (2.5-cm i.d.) surrounded by a macropore. Aggregates were composed of either quartz sand or ferrihydrite-coated sand inoculated with one of two Se-reducing bacteria (Thauera selenatis or Enterobacter cloacae SLD1a-1). Selenite export rates varied between 0.02 ± 0.01 and 3.4 ± 0.2 nmol h−1 g−1 as a function of aeration condition and input solution composition (higher SeO4 2− or C-source concentrations led to higher SeO3 2− export). Oxic input conditions significantly decreased Se reduction; however, the detection of SeO3 2− in effluent samples indicates the occurrence of anoxic microzones within aggregates. Furthermore, we found that solid-phase concentrations of reduced Se increased toward the core of aggregates and are estimated to at least double within the first millimeter into the aggregate under all conditions investigated. This indicates that concentrations of reduced Se may generally be expected to increase with distance from the advection boundary (macropore) inside aggregates, which would imply that soils with larger aggregates retain more Se.

This report describes the use of a new selenate-respiring bacterium, Thauera selenatis, for the bioremediation of selenium (Se, as selenate) in drainage water from the Westlands Water District, San Joaquin Valley. The organism respires selenate anaerobically using acetate as the preferred electron donor. The reduction of selenate is not inhibited by nitrate; both electron acceptors are reduced concomitantly. T. selenatis was inoculated into, and was maintained in, a biological reactor system for anaerobic treatment of selenate-nitrate containing drainage water; a population of denitrifying bacteria was also present. When the pH of inflowing water was 6.9, and 2 mm acetate plus 0.56 mm ammonium chloride were fed into the reactor, selenate/selenite levels were reduced from 350–450 μg Se/l to 5.39±3.6 μg Se/l. The final product of selenate reduction was elemental Se. Analysis of reactor contents revealed that T. selenatis was the only selenate-respiring organism present in the system. Nitrate in the drainage water was also reduced in the reactor system by 98%. The lab-scale biological reactor system consisted of recycled sludge-blanket (1 l; 400 g sand) and fluidized-bed (1 l; 300 g sand) reactors. At a system flow rate of 6.5 ml/min, the retention time was 140 min.

A recently isolated, selenate-respiring microorganism (strain AXT [T = type strain]) was classified by using a polyphasic approach in which both genotypic and phenotypic characteristics were determined. Strain AXT is a motile, gram-negative, rod-shaped organism with a single polar flagellum. On the basis of phenotypic characteristics, this organism can be classified as a Pseudomonas sp. However, a comparison of the 16S rRNA sequence of strain AXT with the sequences of other organisms indicated that strain AXT is most similar to members of the beta subclass (level of similarity, 86.8%) rather than to members of the gamma subclass (level of similarity, 80.2%) of the Proteobacteria. The presence of the specific polyamine 2-hydroxyputrescine and the presence of a ubiquinone with eight isoprenoid units in the side chain (ubiquinone Q-8) excluded strain AXT from the authentic genus Pseudomonas and allowed placement in the beta subclass of the Proteobacteria. Within the beta subclass, strain AXT is related to Iodobacter fluvatile. The phylogenetic distance (level of similarity, less than 90%), as well as a lack of common phenotypic characteristics between these organisms, prevents classification of strain AXT as a member of the genus Iodobacter. In addition, strain AXT possesses a unique mechanism for anaerobic respiration, which allows it to utilize selenate as an electron acceptor without interference by nitrate. Therefore, we propose that strain AXT should be the first member of a new genus and species, Thauera selenatis.

The high levels of selenium (selenate, selenite) in agricultural drainage water in the San Joaquin Valley of California, which have led to environmental problems, might be lowered if the selenate/selenite could be reduced to elemental insoluble selenium [1]. Two organisms have been newly isolated which do this in anaerobic coculture. One, a strictly anaerobic, Gram-positive rod, reduces selenite to elemental selenium. The other, a Pseudomonas species, was shown to respire selenate to selenite. Cells grown anaerobically in Minimal Medium on acetate plus selenate oxidized 14C-acetate to 14CO2 with concomitant reduction of selenate to selenite and small amounts of elemental selenium.