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A Gram-stain negative, short rod-shaped and non-motile bacterial strain ZV1CT capable of degrading phenol was isolated from a wastewater treatment system of Huafu mustard tuber salinity preservation factory in Chongqing, China. Aerobic growth was observed at 20–42 °C (optimum, 30 °C) and at pH 5–10 (optimum, pH 8). Cells tolerated NaCl concentrations of 0–2% (w/v) (optimum, 0%). The major respiratory quinone is ubiquinone Q-8 and the major cellular fatty acids are C16:1 ω7c /C16:1 ω6c and C16:0. The 16S rRNA gene sequence of stain ZV1CT is phylogenetically related to the 16S rRNA genes of the type strains of Thauera species (similarity: 96.6–97.7%). The genome of strain ZV1CT was sequenced and the size of the genome is 3.68 Mb. The genomic DNA G+C content is 68.2 mol %. Strain ZV1CT exhibited whole-genome average nucleotide identity values of 82.3, 81.5 and 80.9% with respect to Thauera phenylacetica B4PT, Thauera aminoaromatica S2T and Thauera selenatis AXT, respectively. Accordingly, the genome-to-genome distances between strain ZV1CT and the type strains ranged from 21.5 to 31.3%. Based on the results of this study, it is proposed that strain ZV1CT represents a novel species of the genus Thauera, for which the name Thauera phenolivorans is proposed. The type strain is ZV1CT (=CGMCC 1.15497 = NCBR 112379).

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).

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%.

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.

A Gram-negative, rod-shaped, non-spore-forming bacterium, designated SgZ-1T, was isolated from the anode biofilm of a microbial fuel cell. The strain had the ability to grow under anaerobic condition via the oxidation of various organic compounds coupled to the reduction of anthraquione-2,6-disulfonate (AQDS) to anthrahydroquinone-2,6-disulfonate (AHQDS). Growth occurred in TSB in the presence of 0–5.5 % (w/v) NaCl (optimum 0–1 %), at 10–45 °C (optimum 25–37 °C) and at pH 6.0–10.0 (optimum 8.0–8.5). Based on 16S rRNA gene sequence similarity, strain SgZ-1T belonged to the genus Thauera . The highest level of 16S rRNA gene sequences similarity (96.7 %) was found to be with Thauera aminoaromatica S2 T and Thauera selenatis AXT, and lower values were obtained when compared with other recognized Thauera species. Chemotaxonomic analysis revealed that strain SgZ-1T contained Q-8 as the predominant quinone, and putrescine and 2-hydroxyputrescine as the major polyamines. The major cellular fatty acids (>5 %) were C16 : 1ω6c and/or C16 : 1ω7c (44.6 %), C16 : 0 (18.8 %), and C18 : 1ω6c and/or C18 : 1ω7c (12.7 %). Based on its phenotypic and phylogenetic properties, chemotaxonomic analysis and the results of physiological and biochemical tests, strain SgZ-1T ( = KACC 16524T = CCTCC M 2011497T) was designated the type strain of a novel species of the genus Thauera , for which the name Thauera humireducens sp. nov. was proposed.

Thauera selenatis was grown anaerobically in minimal medium with either selenate or nitrate as the terminal electron acceptor and acetate as the carbon source and electron donor. The molar cell protein yields, YM-protein (selenate) and YM-protein (nitrate), were found to be 7.8 g cell protein/mol selenite formed and 7.5 g cell protein/mol nitrite formed, respectively. These values represent YM values of 57 and 55 g (dry weight)/mol acetate when selenate or nitrate was the electron acceptor, respectively. Based upon a calculated YATP value of 10.0 g (dry weight) cells/mol ATP, for growth on acetate in inorganic salts, growth with selenate as the terminal electron acceptor theoretically yielded 5.7 ATP/acetate oxidized, and 5.5 ATP when nitrate was the terminal electron acceptor. The results support the conclusion that energy is conserved via electron transport phosphorylation when selenate or nitrate reduction are the terminal electron acceptors during anaerobic growth with acetate.

Selenate reductase (SER) from Thauera selenatis is a member of a distinct class of the TAT-translocated type II molybdoenzymes and is closely related to a group of thermostable nitrate reductases (pNAR) found in hyperthermophilic archaea. In the present study the thermostable and thermo-active properties of SER, isolated with either molybdenum (Mo) or tungsten (W) at the active site, are reported. Results show that the purified Mo–SER complex is stable and active upon heat-shock incubation for 10 min at temperatures up to 60 °C. At temperatures greater than 65 °C all three subunits (SerABC) are readily denatured. The optimum temperature for maximum activity recorded was also determined to be 65 °C. T. selenatis can grow readily on a tungstate rich medium up to concentrations of 1 mM. SER isolated from periplasmic fractions from cells grown on 1 mM tungstate displayed selenate reductase activities with a 20-fold reduction in Vmax (0.01 μmol [S]/min/mg) and a 23-fold increase in substrate binding affinity (Km 0.7 μM). The thermo-stability and pH dependence of W–SER was shown to be similar to that observed for Mo–SER. By contrast, the optimum reaction temperature for W–SER exceeded the maximum temperature tested (>80 °C). The combined data from the kinetic analysis and thermal activity profiles provide evidence that W can substitute for Mo at the active site of SER and retain detectable selenate reductase activity. It is argued that despite the similarity in their catalytic and electron conducting subunits, the presence of a membrane anchor in the archaeal pNAR system appears pivotal to the enhanced hyperthermostability. The fact that Mo–SER is thermostable up to 65 °C however, could be advantageous when designing selenate contamination remediation strategies.

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.

Selenate reductase from Thauera selenatis was crystallized using ammonium sulfate as a precipitant. Crystals of selenate reductase belong to the space group C2, with unit-cell parameters a = 116.9, b = 67.5, c = 186.7 A, beta = 90 degrees. Native data to 2.1 A resolution have been collected and a heavy-atom derivative has been identified following soaking of the crystals in a solution of trimethyl lead acetate.

Anaerobic degradation of alkylbenzenes with side chains longer than that of toluene was studied in freshwater mud samples in the presence of nitrate. Two new denitrifying strains, EbN1 and PbN1, were isolated on ethylbenzene and n-propylbenzene, respectively. For comparison, two further denitrifying strains, ToN1 and mXyN1, were isolated from the same mud with toluene and m-xylene, respectively. Sequencing of 16SrDNA revealed a close relationship of the new isolates to Thauera selenatis. The strains exhibited different specific capacities for degradation of alkylbenzenes. In addition to ethylbenzene, strain EbN1 utilized toluene, but not propylbenzene. In contrast, propylbenzene-degrading strain PbN1 did not grow on toluene, but was able to utilize ethylbenzene. Strain ToN1 used toluene as the only hydrocarbon substrate, whereas strain mXyN1 utilized both toluene and m-xylene. Measurement of the degradation balance demonstrated complete oxidation of ethylbenzene to CO2 by strain EbN1. Further characteristic substrates of strains EbN1 and PbN1 were 1-phenylethanol and acetophenone. In contrast to the other isolates, stain mXyN1 did not grow on benzyl alcohol. Benzyl alcohol (also m-methyl-benzyl alcohol) was even a specific inhibitor of toluene and m-xylene utilization by strain mXyN1. None of the strains was able to grow on any of the alkylbenzenes with oxygen as electron acceptor. However, polar aromatic compounds such as benzoate were utilized under both oxic and anoxic conditions. All four isolates grew anaerobically on crude oil. Gas chromatographic analysis of crude oil after growth of strain ToN1 revealed specific depletion of toluene.

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.