Azospira inquinata sp. nov., a nitrate-reducing bacterium of the family Rhodocyclaceae isolated from contaminated groundwater.

Two novel Gram-stain-negative bacterial strains, Azo-3T and Azo-2, were isolated from a toluene-producing enrichment culture that originated from contaminated groundwater at a site in southeast Louisiana (USA). Cells are non-spore forming straight to curved rods with single polar flagella. Strains Azo-3T and Azo-2 are oxidase-positive, catalase-negative, use nitrate and nitrite as electron acceptors, and are able to fix nitrogen. Poly-ß-hydroxybutyrate storage granules are produced. Dominant fatty acids when grown in R2A medium at 37 °C are C16:0, summed feature 3 (C16:1 ω7c and/or C15:0 iso 2OH), C17:0 cyclo and C18:1 ω7c. 16S rRNA gene sequence based phylogenetic analysis indicated that the strains cluster within the family Rhodocyclaceae, class Betaproteobacteria, most closely related to but distinct from type strains of the species Azospira oryzae (96.94% similarity) and Azospira restricta (95.10% similarity). Complete genome sequences determined for strains Azo-3T and Azo-2 revealed DNA G+C content of 62.70 mol%. Genome-wide comparisons based on average nucleotide identity by orthology and estimated DNA-DNA hybridization values combined with phenotypic and chemotaxonomic traits and phylogenetic analysis indicate that strains Azo-3T and Azo-2 represent a novel species within the genus Azospira for which the name Azospira inquinata sp. nov. is proposed. The type strain of Azospira inquinata is Azo-3T (=NRRL B-65590T=DSM 112046T).

Genome Sequence of the Type Strain Azospira restricta SUA2 (DSM 18626).

Azospira restricta SUA2T (DSM 18626) is a Gram-negative-staining bacterium able to fix nitrogen and accumulate polyhydroxybutyrate storage granules. Here, we report the complete genome sequence (3,975,213 bp with 68.64 mol% G+C content), which may prove useful in future efforts to assess the role of Azospira in nutrient cycling.

Bioremediation Strategies Aimed at Stimulating Chlorinated Solvent Dehalogenation Can Lead to Microbially-Mediated Toluene Biogenesis.

In situ bioremediation practices that include subsurface addition of fermentable electron donors to stimulate reductive dechlorination by anaerobic bacteria have become widely employed to combat chlorinated solvent contamination in groundwater. At a contaminated site located near Baton Rouge, Louisiana (USA), toluene was transiently observed in groundwater at concentrations that sometimes far exceeded the US drinking water maximum contaminant level (MCL) of 1 mg/L after a fermentable substrate (agricultural feed grade cane molasses) was injected into the subsurface with the intent of providing electron donors for reductive dechlorination. Here, we present data that demonstrate that indigenous microorganisms can biologically produce toluene by converting phenylacetic acid, phenylalanine, phenyllactate, and phenylpyruvate to toluene. When grown in defined medium with phenylacetic acid at concentrations ≤350 mg/L, the molar ratio between toluene accumulated and phenylacetic acid supplied was highly correlated ( R2 ≥ 0.96) with a toluene yield exceeding 0.9:1. Experiments conducted using 13C labeled compounds (phenylacetic acid-2-13C and l-phenylalanine-3-13C) resulted in production of toluene-α-13C, confirming that toluene was synthesized from these precursors by two independently developed enrichment cultures. Results presented here suggest that monitoring of aromatic hydrocarbons is warranted during enhanced bioremediation activities where electron donors are introduced to stimulate anaerobic biotransformation of chlorinated solvents.

Biofilter treatment of gas phase ß-caryophyllene at an elevated temperature.

Experiments were carried out to test the capacity for a laboratory-scale biofilter operated at an elevated temperature level (∼50°C) to remove an air stream containing ß-caryophyllene, a naturally occurring sesquiterpene of environmental concern emitted from wood-related industrial facilities. A water jacket was used to maintain high temperatures in a laboratory-scale biofilter. Inocula, pollutant loading and nutrient supply rate effects were evaluated over 84 days of biofilter operation. The start-up process took over two months when citrus peels were used as inocula while a relatively short start-up period was achieved after introducing forest compost products. While using a sparged-gas bioreactor to cultivate an enrichment culture for 97 days, removal efficiencies in excess of 80% were observed after 18 days. At empty bed contact times of 50 s and at a pollutant loading rate of 3.05 mg C/L/hr, removal efficiency levels reached 90% and the elimination capacity level reached 2.29 mg C/L/hr, corresponding to an elimination capacity of 2.60 mg ß-caryophyllene/L/hr. Collectively, these results demonstrate that ß-caryophyllene can be successfully removed from contaminated air using a biofilter operated at a high temperature (∼50°C), expanding the temperature range within which biofilters are known to biodegrade sesquiterpenes.

Dehalogenimonas formicexedens sp. nov., a chlorinated alkane-respiring bacterium isolated from contaminated groundwater.

A strictly anaerobic, Gram-stain-negative, non-spore-forming bacterium designated NSZ-14T, isolated from contaminated groundwater in Louisiana (USA), was characterized using a polyphasic approach. Strain NSZ-14T reductively dehalogenated a variety of polychlorinated aliphatic alkanes, producing ethene from 1,2-dichloroethane, propene from 1,2-dichloropropane, a mixture of cis- and trans-1,2-dichloroethene from 1,1,2,2-tetrachloroethane, vinyl chloride from 1,1,2-trichloroethane and allyl chloride (3-chloro-1-propene) from 1,2,3-trichloropropane. Formate or hydrogen could both serve as electron donors. Dechlorination occurred between pH 5.5 and 7.5 and over a temperature range of 20-37 °C. Major cellular fatty acids included C18 : 1ω9c, C14 : 0 and C16 : 0. 16S rRNA gene sequence-based phylogenetic analysis indicated that the strain clusters within the class Dehalococcoidia of the phylum Chloroflexi, most closely related to but distinct from type strains of the species Dehalogenimonas alkenigignens (97.63 % similarity) and Dehalogenimonas lykanthroporepellens (95.05 %). A complete genome sequence determined for strain NSZ-14T revealed a DNA G+C content of 53.96 mol%, which was corroborated by HPLC (54.1±0.2 mol% G+C). Genome-wide comparisons based on average nucleotide identity by orthology and estimated DNA-DNA hybridization values combined with phenotypic and chemotaxonomic traits and phylogenetic analysis indicate that strain NSZ-14T represents a novel species within the genus Dehalogenimonas, for which the name Dehalogenimonas formicexedens sp. nov. is proposed. The type strain is NSZ-14T (=HAMBI 3672T=JCM 19277T=VKM B-3058T). An emended description of Dehalogenimonas alkenigignens is also provided.

Genome sequence of the organohalide-respiring Dehalogenimonas alkenigignens type strain (IP3-3(T)).

Dehalogenimonas alkenigignens IP3-3(T) is a strictly anaerobic, mesophilic, Gram negative staining bacterium that grows by organohalide respiration, coupling the oxidation of H2 to the reductive dehalogenation of polychlorinated alkanes. Growth has not been observed with any non-polyhalogenated alkane electron acceptors. Here we describe the features of strain IP3-3(T) together with genome sequence information and its annotation. The 1,849,792 bp high-quality-draft genome contains 1936 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small unit rRNA (16S) locus. The genome contains 29 predicted reductive dehalogenase genes, a large majority of which lack cognate genes encoding membrane anchoring proteins.

Reassessment of PCR primers targeting 16S rRNA genes of the organohalide-respiring genus Dehalogenimonas.

Representatives from the genus Dehalogenimonas have the metabolic capacity to anaerobically transform a variety of environmentally important polychlorinated aliphatic compounds. In light of the recent isolation of additional strains, description of a new species, and an expanded number of uncultured DNA sequences, PCR primers and protocols intended to uniquely target members of this organohalide-respiring genus were reevaluated. Nine of fourteen primer combinations reported previously as genus-specific failed to amplify 16S rRNA genes of recently isolated Dehalogenimonas strains. Use of alternative combinations or modified genus-specific primers, however, allowed detection of all presently known Dehalogenimonas strains. Use of a modified primer set in qPCR revealed an approximately two-order of magnitude increase in concentration of Dehalogenimonas 16S rRNA gene copies following subsurface injection of electron donors at a Louisiana Superfund site, demonstrating the utility of the newly developed protocol and suggesting that the genus Dehalogenimonas can respond to biostimulation remediation strategies in a manner similar to that previously reported for other dechlorinating genera such as Dehalococcoides.

Dehalogenimonas lykanthroporepellens BL-DC-9T simultaneously transcribes many rdhA genes during organohalide respiration with 1,2-DCA, 1,2-DCP, and 1,2,3-TCP as electron acceptors.

The genome sequence of the organohalide-respiring bacterium Dehalogenimonas lykanthroporepellensBL-DC-9(T) contains numerous loci annotated as reductive dehalogenase homologous (rdh) genes based on inferred protein sequence identity with functional dehalogenases of other bacterial species. Many of these genes are truncated, lack adjacent regulatory elements, or lack cognate genes coding for membrane-anchoring proteins typical of the functionally characterized active reductive dehalogenases of organohalide-respiring bacteria. To investigate the expression patterns of the rdh genes in D. lykanthroporepellensBL-DC-9(T), oligonucleotide primers were designed to uniquely target 25 rdh genes present in the genome as well as four putative regulatory genes. RNA extracts from cultures of strain BL-DC-9(T) actively dechlorinating three different electron acceptors, 1,2-dichloroethane, 1,2-dichloropropane, and 1,2,3-trichloropropane were reverse-transcribed and subjected to PCR amplification using rdh-specific primers. Nineteen rdh gene transcripts, including 13 full-length rdhA genes, six truncated rdhA genes, and five rdhA genes having cognate rdhB genes were consistently detected during the dechlorination of all three of the polychlorinated alkanes tested. Transcripts from all four of the putative regulatory genes were also consistently detected. Results reported here expand the diversity of bacteria known to simultaneously transcribe multiple rdh genes and provide insights into the transcription factors associated with rdh gene expression.

Substrate interactions in dehalogenation of 1,2-dichloroethane, 1,2-dichloropropane, and 1,1,2-trichloroethane mixtures by Dehalogenimonas spp.

When chlorinated alkanes are present as soil or groundwater pollutants, they often occur in mixtures. This study evaluated substrate interactions during the anaerobic reductive dehalogenation of chlorinated alkanes by the type strains of two Dehalogenimonas species, D. lykanthroporepellens and D. alkenigignens. Four contaminant mixtures comprised of combinations of the chlorinated solvents 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were assessed for each species. Chlorinated solvent depletion and daughter product formation determined as a function of time following inoculation into anaerobic media revealed preferential dechlorination of 1,1,2-TCA over both 1,2-DCA and 1,2-DCP for both species. 1,2-DCA in particular was not dechlorinated until 1,1,2-TCA reached low concentrations. In contrast, both species concurrently dechlorinated 1,2-DCA and 1,2-DCP over a comparably large concentration range. This is the first report of substrate interactions during chlorinated alkane dehalogenation by pure cultures, and the results provide insights into the chlorinated alkane transformation processes that may be expected for contaminant mixtures in environments where Dehalogenimonas spp. are present.

Dehalogenimonas alkenigignens sp. nov., a chlorinated-alkane-dehalogenating bacterium isolated from groundwater.

Two strictly anaerobic bacterial strains, designated IP3-3(T) and SBP-1, were isolated from groundwater contaminated by chlorinated alkanes and alkenes at a Superfund Site located near Baton Rouge, Louisiana (USA). Both strains reductively dehalogenate a variety of polychlorinated aliphatic alkanes, including 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane and 1,2,3-trichloropropane, when provided with hydrogen as the electron donor. To clarify their taxonomic position, strains IP3-3(T) and SBP-1 were characterized using a polyphasic approach. Both IP3-3(T) and SBP-1 are mesophilic, non-spore-forming, non-motile and Gram-stain-negative. Cells of both strains are irregular cocci with diameters of 0.4-1.1 µm. Both are resistant to ampicillin and vancomycin. The genomic DNA G+C contents of strains IP3-3(T) and SBP-1 are 55.5±0.4 and 56.2±0.2 mol% (HPLC), respectively. Major cellular fatty acids include C18 : 1ω9c, C16 : 0, C14 : 0 and C16 : 1ω9c. 16S rRNA gene sequence based phylogenetic analyses indicated that the strains cluster within the phylum Chloroflexi most closely related to but distinct from the species Dehalogenimonas lykanthroporepellens (96.2 % pairwise similarity) and Dehalococcoides mccartyi (90.6 % pairwise similarity). Physiological and chemotaxonomic traits as well as phylogenetic analysis support the conclusion that these strains represent a novel species within the genus Dehalogenimonas for which the name Dehalogenimonas alkenigignens sp. nov. is proposed. The type strain is IP3-3(T) ( = JCM 17062(T) = NRRL B-59545(T)).

Removal of the sesquiterpene ß-caryophyllene from air via biofiltration: performance assessment and microbial community structure.

Experiments were conducted in a laboratory-scale biofilter to assess the ability of a fixed-film biological process to treat an air stream containing ß-caryophyllene, a sesquiterpene emitted by a variety of conifer trees as well as industrial wood processing operations. Treatment performance was evaluated under a variety of pollutant loading conditions and nutrient supply rates over an operational period lasting more than 240 days. At empty bed contact times (EBCTs) as low as 10 s and daily average pollutant loading rate as high as 24.2 g C/(m(3) h) (grams pollutant measured as carbon per cubic meter packed bed volume per hour), removal efficiencies in excess of 95 % were observed when sufficient nutrients were supplied. Results demonstrate that, as with biofilters treating other compounds, biofilters treating ß-caryophyllene can experience local nutrient limitations that result in diminished performance. The biofilter successfully recovered high removal efficiency within a few days after resumption of pollutant loading following a 14-day interval of no contaminant loading. Construction of a 16S rRNA gene library via pyrosequencing revealed the presence of a high proportion of bacteria clustering within the genera Gordonia (39.7 % of the library) and Rhodanobacter (37.6 %). Other phylotypes detected at lower relative abundances included Pandoraea (6.2 %), unclassified Acetobacteraceae (5.5 %), Dyella (3.3 %), unclassified Xanthomonadaceae (2.6 %), Mycobacterium (1.8 %), and Nocardia (0.6 %). Collectively, results demonstrate that ß-caryophyllene can be effectively removed from contaminated gas streams using biofilters.

Dehalogenimonas spp. can Reductively Dehalogenate High Concentrations of 1,2-Dichloroethane, 1,2-Dichloropropane, and 1,1,2-Trichloroethane.

The contaminant concentrations over which type strains of the species Dehalogenimonas alkenigignens and Dehalogenimonas lykanthroporepellens were able to reductively dechlorinate 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were evaluated. Although initially isolated from an environment with much lower halogenated solvent concentrations, D. alkenigignens IP3-3T was found to reductively dehalogenate chlorinated alkanes at concentrations comparable to D. lykanthroporepellens BL-DC-9T. Both species dechlorinated 1,2-DCA, 1,2-DCP, and 1,1,2-TCA present at initial concentrations at least as high as 8.7, 4.0, and 3.5 mM, respectively. The ability of Dehalogenimonas spp. to carry out anaerobic reductive dechlorination even in the presence of high concentrations of chlorinated aliphatic alkanes has important implications for remediation of contaminated soil and groundwater.

Complete genome sequence of Dehalogenimonas lykanthroporepellens type strain (BL-DC-9(T)) and comparison to "Dehalococcoides" strains.

Dehalogenimonas lykanthroporepellens is the type species of the genus Dehalogenimonas, which belongs to a deeply branching lineage within the phylum Chloroflexi. This strictly anaerobic, mesophilic, non spore-forming, Gram-negative staining bacterium was first isolated from chlorinated solvent contaminated groundwater at a Superfund site located near Baton Rouge, Louisiana, USA. D. lykanthroporepellens was of interest for genome sequencing for two reasons: (a) an unusual ability to couple growth with reductive dechlorination of environmentally important polychlorinated aliphatic alkanes and (b) a phylogenetic position that is distant from previously sequenced bacteria. The 1,686,510 bp circular chromosome of strain BL-DC-9(T) contains 1,720 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small subunit rRNA (16S) locus.

Actinomyces naturae sp. nov., the first Actinomyces sp. isolated from a non-human or animal source.

Three facultatively anaerobic, Gram-positive staining, rod-shaped, non-spore forming, flagellated bacterial strains, BL-75, BL-79(T) and BL-104, were isolated from chlorinated solvent-contaminated groundwater. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed them to represent a distinct lineage within the genus Actinomyces with sequence identities in the range of <88-95.4% with previously described Actinomyces species. The strains were oxidase and catalase negative. Nitrate was not reduced. Esculin was hydrolyzed. Growth occurred in the temperature range of 20-43°C (optimum 30-37°C) and pH range 4.5-9.0 (optimum pH 6.5). Substrates supporting growth included various mono-, di-, and tri-saccharides. The end products of glucose fermentation were acetate, lactate, succinate and formate. Fermentative growth was observed in the presence of near saturation concentrations of perchloroethene (PCE) and toluene and in the presence of 1,2-dichloroethane and 1,1,2-trichloroethane at concentrations up to at least 24.4 mM and 11.2 mM, respectively. The dominant cellular fatty acids when grown in peptone/yeast extract/glucose (PYG) medium were C(18:1) ω9c, C(16:0), and C(14:0). The peptidoglycan was found to contain the amino acids alanine, glutamic acid, lysine, and ornithine at approximate molar ratios of 1.7 Ala: 2.3 Glu: 1.3 Lys: 1.0 Orn. The cell wall sugars were found to include rhamnose and mannose. The polar lipids were found to include diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phospholipid (PL), phosphoglycolipids (PGL), and glycolipids (GL). The main respiratory quinone of strain BL-79(T) was MK-9(H(4)), with minor components MK-10(H(4)) and MK-8(H(4)). The DNA mol% G+C content of the type strain is 69.8%. On the basis of phylogenetic and phenotypic characteristics, these strains could be differentiated from previously described species of the genus Actinomyces. Strains BL-75, BL-79(T) and BL-104 are designated as a novel species, for which the name Actinomyces naturae sp. nov. is proposed. This is the first Actinomyces species isolated from an environmental rather than human or animal sources. The type strain of Actinomyces naturae is BL-79(T) (= CCUG 56698(T) = NRRL B-24670(T)).

Pelosinus defluvii sp. nov., isolated from chlorinated solvent-contaminated groundwater, emended description of the genus Pelosinus and transfer of Sporotalea propionica to Pelosinus propionicus comb. nov.

Two anaerobic bacterial strains, designated SHI-1(T) and SHI-2, were isolated from chlorinated solvent-contaminated groundwater. They were found to be identical in phenotypic properties and shared high (98.5-99.8 %) pairwise 16S rRNA gene sequence similarity. Multiple 16S rRNA genes were found to be present in the isolates as well as Pelosinus fermentans DSM 17108(T) and Sporotalea propionica DSM 13327(T). Strains SHI-1(T) and SHI-2 could be differentiated from their closest phylogenetic relatives, P. fermentans DSM 17108(T) and S. propionica DSM 13327(T), on the basis of their phenotypic and phylogenetic properties. The isolates were Gram-negative, spore-forming, motile rods with peritrichous flagella. Growth occurred at 10-42 °C and pH 5.5-8.5. Fermentative growth was observed on Casamino acids, fructose, fumarate, glucose, glycerol, pyruvate and yeast extract. The major organic acids produced from glucose and glycerol fermentation were propionate and acetate. The major organic acids produced from fermentation of fumarate were propionate, acetate and succinate. The major cellular fatty acids were summed feature 4 (consisting of C(15:1)ω8c and/or C(15:2)), summed feature 8 (consisting of C(17:1)ω8c and/or C(17:2)) and C(14:0) dimethyl aldehyde. The polar lipids comprised aminophospholipids, including phosphatidylethanolamine and phosphatidylserine, and an unknown phospholipid. The genomic DNA G+C content was 39.2 mol%. We propose that strains SHI-1(T) and SHI-2 are assigned to a novel species of the genus Pelosinus, with the name Pelosinus defluvii sp. nov. (type strain SHI-1(T) = NRRL Y-59407(T) = LMG 25549(T)). The description of the genus Pelosinus is emended. We also propose the transfer of S. propionica to the genus Pelosinus as Pelosinus propionicus comb. nov. (type strain TmPN3(T) = DSM 13327(T) = ATCC BAA-626(T)), on the basis of phylogenetic, chemotaxonomic and phenotypic properties.

Effects of water vapor on activated carbon load equalization of gas phase toluene.

Experimental testing and numerical simulations were conducted to assess the effects of elevated water vapor concentrations on the ability of granular activated carbon (GAC) to achieve load equalization of dynamically varying gas-phase toluene concentrations. Columns packed with Calgon BPL 4 x 6 mesh GAC were subjected to intermittent (8 h/day) toluene loading in air streams containing up to 90% relative humidity. Influent toluene concentrations ranged from 100 to 1000 ppm(v), and GAC column empty bed residence times ranged from 1.5 to 10 s. In comparison to load equalization performance achieved with dry air, high relative humidity improved load attenuation at high influent toluene concentration (e.g., 1000 ppm(v)) but decreased the degree of load attenuation at low influent toluene concentration (e.g., 100 ppm(v)). Model simulations conducted using a pore and surface diffusion model were in good general agreement with experimental observations. Collectively, results demonstrate that GAC columns can be of practical benefit as passively-operated load equalization devices even in the case of high relative humidity. Such systems may prove useful as a pre-treatment process for biofilters and other air pollution control devices that would otherwise be subjected to wide variation in contaminant loading.

Clostridium hydrogeniformans sp. nov. and Clostridium cavendishii sp. nov., hydrogen-producing bacteria from chlorinated solvent-contaminated groundwater.

Four hydrogen-producing, aerotolerant, anaerobic bacterial strains isolated from chlorinated solvent-contaminated groundwater were characterized using a polyphasic approach. Three of the strains, designated BL-18, BL-19 and BL-20(T), were found to be identical in 16S rRNA gene sequences and in phenotypic properties. Cells of these strains are Gram-positive-staining, spore-forming, motile rods with peritrichous flagella. Growth occurred at 15-40 degrees C, pH 5.0-10.0 and at NaCl concentrations up to 5 % (w/v). Acid was produced in fermentation of cellobiose, fructose, galactose (weak), glucose, maltose and salicin. Products of fermentation in PYG medium were acetate, butyrate, ethanol, formate, carbon dioxide and hydrogen. Dominant cellular fatty acids when grown in PYG medium were C(13 : 0) iso, C(16 : 0), C(13 : 0) anteiso, C(15 : 0) iso and C(15 : 0) anteiso. The genomic DNA G+C content was 30.4 mol%. These isolates can be differentiated from their closest phylogenetic relative, the cluster I Clostridium species Clostridium frigidicarnis (97.2 % similar to the type strain in 16S rRNA gene sequence), on the basis of phenotypic and chemotaxonomic properties. The other strain characterized in this study, BL-28(T), was Gram-positive-staining with spore-forming, rod-shaped cells. Growth occurred at 15-46 degrees C, pH 6.0-8.5 and at NaCl concentrations up to 3 % (w/v). Acid was produced from cellobiose, dextran, fructose (weak), glucose, maltose, salicin and trehalose. End products of PYG fermentation included acetate, butyrate, pyruvate, carbon dioxide and hydrogen. Dominant cellular fatty acids from cells grown in PYG medium at 30 degrees C were C(14 : 0), C(14 : 0) dimethyl aldehyde, C(16 : 0) and C(12 : 0). The DNA G+C content was 28.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BL-28(T) falls within cluster I of the genus Clostridium, but with

Detection and quantification of Dehalogenimonas and "Dehalococcoides" populations via PCR-based protocols targeting 16S rRNA genes.

Members of the haloalkane dechlorinating genus Dehalogenimonas are distantly related to "Dehalococcoides" but share high homology in some variable regions of their 16S rRNA gene sequences. In this study, primers and PCR protocols intended to uniquely target Dehalococcoides were reevaluated, and primers and PCR protocols intended to uniquely target Dehalogenimonas were developed and tested. Use of the genus-specific primers revealed the presence of both bacterial groups in groundwater at a Louisiana Superfund site.

Dehalogenimonas lykanthroporepellens gen. nov., sp. nov., a reductively dehalogenating bacterium isolated from chlorinated solvent-contaminated groundwater.

Two recently reported bacterial strains that are able to reductively dehalogenate polychlorinated aliphatic alkanes, including 1,2,3-trichloropropane, 1,2-dichloropropane, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane and 1,2-dichloroethane, were further characterized to clarify their taxonomic position. The two strains, designated BL-DC-8 and BL-DC-9(T), were mesophilic, non-spore-forming, non-motile, Gram-negative staining and strictly anaerobic. Cells were irregular cocci, 0.3-0.6 mum in diameter. The two strains were resistant to ampicillin and vancomycin. Hydrogen was utilized as an electron donor. The genomic DNA G+C content of strains BL-DC-8 and BL-DC-9(T) was 54.0 and 53.8 mol%, respectively. The major cellular fatty acids were C(18 : 1)omega9c, C(16 : 1)omega9c, C(16 : 0) and C(14 : 0). Phylogenetic analyses based on 16S rRNA gene sequences indicated that the strains cluster within the phylum Chloroflexi, but are related only distantly to all recognized taxa in the phylum. Morphological, physiological and chemotaxonomic traits as well as phylogenetic analysis support the conclusion that these two strains represent a novel species of a new genus in the phylum Chloroflexi, for which the name Dehalogenimonas lykanthroporepellens gen. nov., sp. nov. is proposed. The type strain of Dehalogenimonas lykanthroporepellens is BL-DC-9(T) (=ATCC BAA-1523(T) =JCM 15061(T)).

Production of hydrogen by Clostridium species in the presence of chlorinated solvents.

Although anaerobic bioremediation of chlorinated organic contaminants in the environment often requires exogenous supply of hydrogen as an electron donor, little is known about the ability of hydrogen-producing bacteria to grow in the presence of chlorinated solvents. In this study, 18 Clostridium strains including nine uncharacterized isolates originating from chlorinated solvent contaminated groundwater were tested to determine their ability to fermentatively produce hydrogen in the presence of three common chlorinated aliphatic groundwater contaminants: 1,2-dichloroethane (DCA), 1,1,2-trichloroethane (TCA), and tetrachloroethene (PCE). All strains produced hydrogen in the presence of at least 7.4 mM DCA, 2.4 mM TCA, and 0.31 mM PCE. Some strains produced hydrogen in media containing concentrations as high as 29.7 mM DCA, 9.8 mM TCA, and 1.1 mM PCE. None of the strains biotransformed chlorinated solvents under the conditions tested. Results demonstrate that many Clostridium species are chlorinated solvent tolerant, producing hydrogen even in the presence of high concentrations of DCA, TCA, and PCE. These findings have important implications for bioremediation of contaminated soil and groundwater.