Normalized Quantitative PCR Measurements as Predictors for Ethene Formation at Sites Impacted with Chlorinated Ethenes.

Quantitative PCR (qPCR) targeting Dehalococcoides mccartyi ( Dhc) biomarker genes supports effective management at sites impacted with chlorinated ethenes. To establish correlations between Dhc biomarker gene abundances and ethene formation (i.e., detoxification), 859 groundwater samples representing 62 sites undergoing monitored natural attenuation or enhanced remediation were analyzed. Dhc 16S rRNA genes and the vinyl chloride (VC) reductive dehalogenase genes bvcA and vcrA were detected in 88% and 61% of samples, respectively, from wells with ethene. Dhc 16S rRNA, bvcA, vcrA, and tceA (implicated in cometabolic reductive VC dechlorination) gene abundances all positively correlated with ethene formation. Significantly greater ethene concentrations were observed when Dhc 16S rRNA gene and VC RDase gene abundances exceeded 107 and 106 copies L-1, respectively, and when Dhc 16S rRNA- and bvcA + vcrA-to-total bacterial 16S rRNA gene ratios exceeded 0.1%. Dhc 16S rRNA gene-to- vcrA/ bvcA ratios near unity also indicated elevated ethene; however, no increased ethene was observed in 19 wells where vcrA and/or bvcA gene copy numbers exceeded Dhc cell numbers 10- to 10 000-fold. Approximately one-third of samples with detectable ethene lacked bvcA, vcrA, and tceA, suggesting that comprehensive understanding of VC detoxification biomarkers has not been achieved. Although the current biomarker suite is incomplete, the data analysis corroborates the value of the available Dhc DNA biomarkers for prognostic and diagnostic groundwater monitoring at sites impacted with chlorinated ethenes.

Unexpected nondenitrifier nitrous oxide reductase gene diversity and abundance in soils.

Agricultural and industrial practices more than doubled the intrinsic rate of terrestrial N fixation over the past century with drastic consequences, including increased atmospheric nitrous oxide (N(2)O) concentrations. N(2)O is a potent greenhouse gas and contributor to ozone layer destruction, and its release from fixed N is almost entirely controlled by microbial activities. Mitigation of N(2)O emissions to the atmosphere has been attributed exclusively to denitrifiers possessing NosZ, the enzyme system catalyzing N(2)O to N(2) reduction. We demonstrate that diverse microbial taxa possess divergent nos clusters with genes that are related yet evolutionarily distinct from the typical nos genes of denitirifers. nos clusters with atypical nosZ occur in Bacteria and Archaea that denitrify (44% of genomes), do not possess other denitrification genes (56%), or perform dissimilatory nitrate reduction to ammonium (DNRA; (31%). Experiments with the DNRA soil bacterium Anaeromyxobacter dehalogenans demonstrated that the atypical NosZ is an effective N(2)O reductase, and PCR-based surveys suggested that atypical nosZ are abundant in terrestrial environments. Bioinformatic analyses revealed that atypical nos clusters possess distinctive regulatory and functional components (e.g., Sec vs. Tat secretion pathway in typical nos), and that previous nosZ-targeted PCR primers do not capture the atypical nosZ diversity. Collectively, our results suggest that nondenitrifying populations with a broad range of metabolisms and habitats are potentially significant contributors to N(2)O consumption. Apparently, a large, previously unrecognized group of environmental nosZ has not been accounted for, and characterizing their contributions to N(2)O consumption will advance understanding of the ecological controls on N(2)O emissions and lead to refined greenhouse gas flux models.

Identification and environmental distribution of dcpA, which encodes the reductive dehalogenase catalyzing the dichloroelimination of 1,2-dichloropropane to propene in organohalide-respiring chloroflexi.

Dehalococcoides mccartyi strains KS and RC grow with 1,2-dichloropropane (1,2-D) as an electron acceptor in enrichment cultures derived from hydrocarbon-contaminated and pristine river sediments, respectively. Transcription, expression, enzymatic, and PCR analyses implicated the reductive dehalogenase gene dcpA in 1,2-D dichloroelimination to propene and inorganic chloride. Quantitative real-time PCR (qPCR) analyses demonstrated a D. mccartyi cell increase during growth with 1,2-D and suggested that both D. mccartyi strains carried a single dcpA gene copy per genome. D. mccartyi strain RC and strain KS produced 1.8 × 10(7) ± 0.1 × 10(7) and 1.4 × 10(7) ± 0.5 × 10(7) cells per µmol of propene formed, respectively. The dcpA gene was identified in 1,2-D-to-propene-dechlorinating microcosms established with sediment samples collected from different geographical locations in Europe and North and South America. Clone library analysis revealed two distinct dcpA phylogenetic clusters, both of which were captured by the dcpA gene-targeted qPCR assay, suggesting that the qPCR assay is useful for site assessment and bioremediation monitoring at 1,2-D-contaminated sites.

Sphaerochaeta multiformis sp. nov., an anaerobic, psychrophilic bacterium isolated from subseafloor sediment, and emended description of the genus Sphaerochaeta.

An anaerobic, psychrophilic bacterium, strain MO-SPC2(T), was isolated from a methanogenic microbial community in a continuous-flow bioreactor that was established from subseafloor sediments collected from off the Shimokita Peninsula of Japan in the north-western Pacific Ocean. Cells were pleomorphic: spherical, annular, curved rod, helical and coccoid cell morphologies were observed. Motility only occurred in helical cells. Strain MO-SPC2(T) grew at 0-17 °C (optimally at 9 °C), at pH 6.0-8.0 (optimally at pH 6.8-7.2) and in 20-40 g NaCl l(-1) (optimally at 20-30 NaCl l(-1)). The strain grew chemo-organotrophically with mono-, di- and polysaccharides. The major end products of glucose fermentation were acetate, ethanol, hydrogen and carbon dioxide. The abundant polar lipids of strain MO-SPC2(T) were phosphatidylglycolipids, phospholipids and glycolipids. The major cellular fatty acids were C14 : 0, C16 : 0 and C16 : 1ω9. Isoprenoid quinones were not detected. The G+C content of the DNA was 32.3 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain MO-SPC2(T) was affiliated with the genus Sphaerochaeta within the phylum Spirochaetes, and its closest relatives were Sphaerochaeta pleomorpha Grapes(T) (88.4 % sequence identity), Sphaerochaeta globosa Buddy(T) (86.7 %) and Sphaerochaeta coccoides SPN1(T) (85.4 %). Based on phenotypic characteristics and phylogenetic traits, strain MO-SPC2(T) is considered to represent a novel species of the genus Sphaerochaeta, for which the name Sphaerochaeta multiformis sp. nov. is proposed. The type strain is MO-SPC2(T) ( = JCM 17281(T) = DSM 23952(T)). An emended description of the genus Sphaerochaeta is also proposed.

Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi.

Six obligately anaerobic bacterial isolates (195(T), CBDB1, BAV1, VS, FL2 and GT) with strictly organohalide-respiring metabolisms were obtained from chlorinated solvent-contaminated aquifers, contaminated and uncontaminated river sediments or anoxic digester sludge. Cells were non-motile with a disc-shaped morphology, 0.3-1 µm in diameter and 0.1-0.2 µm thick, and characteristic indentations on opposite flat sides of the cell. Growth occurred in completely synthetic, reduced medium amended with a haloorganic electron acceptor (mostly chlorinated but also some brominated compounds), hydrogen as electron donor, acetate as carbon source, and vitamins. No other growth-supporting redox couples were identified. Aqueous hydrogen consumption threshold concentrations were <1 nM. Growth ceased when vitamin B(12) was omitted from the medium. Addition of sterile cell-free supernatant of Dehalococcoides-containing enrichment cultures enhanced dechlorination and growth of strains 195 and FL2, suggesting the existence of so-far unidentified stimulants. Dechlorination occurred between pH 6.5 and 8.0 and over a temperature range of 15-35 °C, with an optimum growth temperature between 25 and 30 °C. The major phospholipid fatty acids were 14 : 0 (15.7 mol%), br15 : 0 (6.2 mol%), 16 : 0 (22.7 mol%), 10-methyl 16 : 0 (25.8 mol%) and 18 : 0 (16.6 mol%). Unusual furan fatty acids including 9-(5-pentyl-2-furyl)-nonanoate and 8-(5-hexyl-2-furyl)-octanoate were detected in strains FL2, BAV1 and GT, but not in strains 195(T) and CBDB1. The 16S rRNA gene sequences of the six isolates shared more than 98 % identity, and phylogenetic analysis revealed an affiliation with the phylum Chloroflexi and more than 10 % sequence divergence from other described isolates. The genome sizes and G+C contents ranged from 1.34 to 1.47 Mbp and 47 to 48.9 mol% G+C, respectively. Based on 16S rRNA gene sequence comparisons, genome-wide average nucleotide identity and phenotypic characteristics, the organohalide-respiring isolates represent a new genus and species, for which the name Dehalococcoides mccartyi gen. nov., sp. nov. is proposed. Isolates BAV1 ( = ATCC BAA-2100  = JCM 16839  = KCTC 5957), FL2 ( = ATCC BAA-2098  = DSM 23585  = JCM 16840  = KCTC 5959), GT ( = ATCC BAA-2099  = JCM 16841  = KCTC 5958), CBDB1, 195(T) ( = ATCC BAA-2266(T)  = KCTC 15142(T)) and VS are considered strains of Dehalococcoides mccartyi, with strain 195(T) as the type strain. The new class Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov. are described to accommodate the new taxon.

Design and application of an internal amplification control to improve Dehalococcoides mccartyi 16S rRNA gene enumeration by qPCR.

Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of chlorinated ethenes to nontoxic ethene in contaminated aquifers. Enumeration of Dhc biomarker genes using quantitative real-time PCR (qPCR) in groundwater is a key component of site assessment and bioremediation monitoring. Unfortunately, standardized qPCR procedures that recognize impaired measurements due to PCR inhibition, low template DNA concentrations, or analytical error are not available, thus limiting confidence in qPCR data. To improve contemporary approaches for enumerating Dhc in environmental samples, multiplex qPCR assays were designed to quantify the Dhc 16S rRNA gene and one of two different internal amplification controls (IACs): a modified Dhc 16S rRNA gene fragment (Dhc*) and the firefly luciferase gene luc. The Dhc* IAC exhibited competitive inhibition in qPCR with the Dhc 16S rRNA gene template when the ratio of either target was 100-fold greater than the other target. A multiplex qPCR assay with the luc IAC avoided competitive inhibition and accurately quantified Dhc abundances ranging from ∼10 to 10(7) 16S rRNA gene copies per reaction. The addition of ∼10(6) E. coli luc IAC to simulated groundwater amended with the Dhc-containing consortium KB-1 yielded reproducible luc counts after DNA extraction and multiplex qPCR enumeration. The application of the luc IAC assay improved Dhc biomarker gene quantification from simulated groundwater samples and is a valuable approach for "ground truthing" qPCR data obtained in different laboratories, thus reducing ambiguity associated with qPCR enumeration and reproducibility.

Genomic determinants of organohalide-respiration in Geobacter lovleyi, an unusual member of the Geobacteraceae.

BACKGROUND: Geobacter lovleyi is a unique member of the Geobacteraceae because strains of this species share the ability to couple tetrachloroethene (PCE) reductive dechlorination to cis-1,2-dichloroethene (cis-DCE) with energy conservation and growth (i.e., organohalide respiration). Strain SZ also reduces U(VI) to U(IV) and contributes to uranium immobilization, making G. lovleyi relevant for bioremediation at sites impacted with chlorinated ethenes and radionuclides. G. lovleyi is the only fully sequenced representative of this distinct Geobacter clade, and comparative genome analyses identified genetic elements associated with organohalide respiration and elucidated genome features that distinguish strain SZ from other members of the Geobacteraceae. RESULTS: Sequencing the G. lovleyi strain SZ genome revealed a 3.9 Mbp chromosome with 54.7% GC content (i.e., the percent of the total guanines (Gs) and cytosines (Cs) among the four bases within the genome), and average amino acid identities of 53-56% compared to other sequenced Geobacter spp. Sequencing also revealed the presence of a 77 kbp plasmid, pSZ77 (53.0% GC), with nearly half of its encoded genes corresponding to chromosomal homologs in other Geobacteraceae genomes. Among these chromosome-derived features, pSZ77 encodes 15 out of the 24 genes required for de novo cobalamin biosynthesis, a required cofactor for organohalide respiration. A plasmid with 99% sequence identity to pSZ77 was subsequently detected in the PCE-dechlorinating G. lovleyi strain KB-1 present in the PCE-to-ethene-dechlorinating consortium KB-1. Additional PCE-to-cis-DCE-dechlorinating G. lovleyi strains obtained from the PCE-contaminated Fort Lewis, WA, site did not carry a plasmid indicating that pSZ77 is not a requirement (marker) for PCE respiration within this species. Chromosomal genomic islands found within the G. lovleyi strain SZ genome encode two reductive dehalogenase (RDase) homologs and a putative conjugative pilus system. Despite the loss of many c-type cytochrome and oxidative-stress-responsive genes, strain SZ retained the majority of Geobacter core metabolic capabilities, including U(VI) respiration. CONCLUSIONS: Gene acquisitions have expanded strain SZ's respiratory capabilities to include PCE and TCE as electron acceptors. Respiratory processes core to the Geobacter genus, such as metal reduction, were retained despite a substantially reduced number of c-type cytochrome genes. pSZ77 is stably maintained within its host strains SZ and KB-1, likely because the replicon carries essential genes including genes involved in cobalamin biosynthesis and possibly corrinoid transport. Lateral acquisition of the plasmid replicon and the RDase genomic island represent unique genome features of the PCE-respiring G. lovleyi strains SZ and KB-1, and at least the latter signifies adaptation to PCE contamination.

Unexpected specificity of interspecies cobamide transfer from Geobacter spp. to organohalide-respiring Dehalococcoides mccartyi strains.

Dehalococcoides mccartyi strains conserve energy from reductive dechlorination reactions catalyzed by corrinoid-dependent reductive dehalogenase enzyme systems. Dehalococcoides lacks the ability for de novo corrinoid synthesis, and pure cultures require the addition of cyanocobalamin (vitamin B(12)) for growth. In contrast, Geobacter lovleyi, which dechlorinates tetrachloroethene to cis-1,2-dichloroethene (cis-DCE), and the nondechlorinating species Geobacter sulfurreducens have complete sets of cobamide biosynthesis genes and produced 12.9 ± 2.4 and 24.2 ± 5.8 ng of extracellular cobamide per liter of culture suspension, respectively, during growth with acetate and fumarate in a completely synthetic medium. G. lovleyi-D. mccartyi strain BAV1 or strain FL2 cocultures provided evidence for interspecies corrinoid transfer, and cis-DCE was dechlorinated to vinyl chloride and ethene concomitant with Dehalococcoides growth. In contrast, negligible increase in Dehalococcoides 16S rRNA gene copies and insignificant dechlorination occurred in G. sulfurreducens-D. mccartyi strain BAV1 or strain FL2 cocultures. Apparently, G. lovleyi produces a cobamide that complements Dehalococcoides' nutritional requirements, whereas G. sulfurreducens does not. Interestingly, Dehalococcoides dechlorination activity and growth could be restored in G. sulfurreducens-Dehalococcoides cocultures by adding 10 µM 5',6'-dimethylbenzimidazole. Observations made with the G. sulfurreducens-Dehalococcoides cocultures suggest that the exchange of the lower ligand generated a cobalamin, which supported Dehalococcoides activity. These findings have implications for in situ bioremediation and suggest that the corrinoid metabolism of Dehalococcoides must be understood to faithfully predict, and possibly enhance, reductive dechlorination activities.

Dichloromethane fermentation by a Dehalobacter sp. in an enrichment culture derived from pristine river sediment.

Dichloromethane (DCM) as the sole substrate supported growth of a Dehalobacter sp. in an enrichment culture derived from noncontaminated river sediment. DCM was not reductively dechlorinated, and acetate was produced, indicating DCM fermentation and further suggesting Dehalobacter growth is not limited to organohalide respiration.

Sphaerochaeta globosa gen. nov., sp. nov. and Sphaerochaeta pleomorpha sp. nov., free-living, spherical spirochaetes.

Free-living bacteria with spherical cells 0.5-2.5 µm in diameter were isolated from freshwater sediment. 16S rRNA gene sequence analysis placed the new isolates within the phylum Spirochaetes ('spirochaetes'). The isolates never displayed a helical morphology or motility. Growth occurred in the presence of 100 mg ampicillin l(-1) in complex and defined mineral salts medium amended with vitamins, yeast extract and monosaccharides, disaccharides or soluble starch as fermentable substrates. Two distinct isolates, designated Buddy(T) and Grapes(T), exhibited doubling times of 21±2 and 15±1 h in glucose-amended medium and grew at 15-37 and 15-30 °C. Optimum growth was observed between 25 and 30 °C and pH 6.5-7.5, with no growth below pH 5 or above pH 10. Hexose and pentose fermentation yielded ethanol, acetate and formate as major end products. Growth was strictly fermentative and anaerobic, but the isolates tolerated brief oxygen exposure. Nitrate, sulfate, thiosulfate and carbon dioxide were not used as electron acceptors, but soluble Fe(III) was reduced to Fe(II) in glucose-amended medium. The DNA G+C base contents of isolates Buddy(T) and Grapes(T) were 45.5-46.4 and 47.0-49.2 mol%, respectively. Phospholipid fatty acid (PLFA) profiles contained large proportions of C(14:0) and C(16:0) straight-chain saturated fatty acids; C(16:1)ω7c and C(16:1)ω9c dominated the mono-unsaturated PLFAs in isolate Grapes(T), whereas isolate Buddy(T) also possessed C(18:1)ω5c, C(18:1)ω7c and C(18:1)ω9c fatty acids. Branched monoenoic acids accounted for up to 12.4 and 30% of the total PLFA in isolates Grapes(T) and Buddy(T), respectively. Based on their unique morphological features and the phylogenetic distance from their closest relatives, we propose the new genus, Sphaerochaeta gen. nov., to accommodate the new isolates within the novel species Sphaerochaeta globosa sp. nov. (type strain Buddy(T) =DSM 22777(T) =ATCC BAA-1886(T)) and Sphaerochaeta pleomorpha sp. nov. (type strain Grapes(T) =DSM 22778(T) =ATCC BAA-1885(T)). Sphaerochaeta globosa is the type species of the genus.

Localized plasticity in the streamlined genomes of vinyl chloride respiring Dehalococcoides.

Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism.

Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic bacterium.

Tetrachloroethene (PCE) and trichloroethene (TCE) are ideal solvents for numerous applications, and their widespread use makes them prominent groundwater pollutants. Even more troubling, natural biotic and abiotic processes acting on these solvents lead to the accumulation of toxic intermediates (such as dichloroethenes) and carcinogenic intermediates (such as vinyl chloride). Vinyl chloride was found in at least 496 of the 1,430 National Priorities List sites identified by the US Environmental Protection Agency, and its precursors PCE and TCE are present in at least 771 and 852 of these sites, respectively. Here we describe an unusual, strictly anaerobic bacterium that destroys dichloroethenes and vinyl chloride as part of its energy metabolism, generating environmentally benign products (biomass, ethene and inorganic chloride). This organism might be useful for cleaning contaminated subsurface environments and restoring drinking-water reservoirs.

Resolution of culture Clostridium bifermentans DPH-1 into two populations, a Clostridium sp. and tetrachloroethene-dechlorinating Desulfitobacterium hafniense strain JH1.

Clostridium bifermentans strain DPH-1 reportedly dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene. Cultivation-based approaches resolved the DPH-1 culture into two populations: a nondechlorinating Clostridium sp. and PCE-dechlorinating Desulfitobacterium hafniense strain JH1. Strain JH1 carries pceA, encoding a PCE reductive dehalogenase, and shares other characteristics with Desulfitobacterium hafniense strain Y51.

Environmental distribution of the trichloroethene reductive dehalogenase gene (tceA) suggests lateral gene transfer among Dehalococcoides.

The trichloroethene reductive dehalogenase gene (tceA) of Dehalococcoides spp. was detected in 12 of 21 trichloroethene-to-ethene dechlorinating enrichment cultures established from aquifer and sediment samples collected from diverse geographic locations in the USA. Analysis of the tceA chromosomal regions indicated that the tceA genes shared greater than 95% sequence identity, and all shared identical tceAB spacer sequences and tceB genes downstream of tceA. A putative transposable element (PTE) was present 1077 bp downstream of the tceB stop codon in three of eight chromosomal regions analyzed. Sequence identity was interrupted downstream of tceB and upstream or downstream of the PTE, suggesting that intrachromosomal or interchromosomal transfer of tceAB had occurred.

Enrichment, cultivation, and detection of reductively dechlorinating bacteria.

Strategies and procedures for enriching, isolating, and cultivating reductively dechlorinating bacteria that use chloroorganic compounds as metabolic electron acceptors from environmental samples are described. Further, nucleic acid-based approaches used to detect and quantify dechlorinator (i.e., Dehalococcoides)-specific genes are presented.

Denitrification versus respiratory ammonification: environmental controls of two competing dissimilatory NO3(-)/NO2(-) reduction pathways in Shewanella loihica strain PV-4.

Denitrification and respiratory ammonification are two competing, energy-conserving NO3(-)/NO2(-) reduction pathways that have major biogeochemical consequences for N retention, plant growth and climate. Batch and continuous culture experiments using Shewanella loihica strain PV-4, a bacterium possessing both the denitrification and respiratory ammonification pathways, revealed factors that determine NO3(-)/NO2(-) fate. Denitrification dominated at low carbon-to-nitrogen (C/N) ratios (that is, electron donor-limiting growth conditions), whereas ammonium was the predominant product at high C/N ratios (that is, electron acceptor-limiting growth conditions). pH and temperature also affected NO3(-)/NO2(-) fate, and incubation above pH 7.0 and temperatures of 30 °C favored ammonium formation. Reverse-transcriptase real-time quantitative PCR analyses correlated the phenotypic observations with nirK and nosZ transcript abundances that decreased up to 1600-fold and 27-fold, respectively, under conditions favoring respiratory ammonification. Of the two nrfA genes encoded on the strain PV-4 genome, nrfA0844 transcription decreased only when the chemostat reactor received medium with the lowest C/N ratio of 1.5, whereas nrfA0505 transcription occurred at low levels (≤3.4 × 10(-2) transcripts per cell) under all growth conditions. At intermediate C/N ratios, denitrification and respiratory ammonification occurred concomitantly, and both nrfA0844 (5.5 transcripts per cell) and nirK (0.88 transcripts per cell) were transcribed. Recent findings suggest that organisms with both the denitrification and respiratory ammonification pathways are not uncommon in soil and sediment ecosystems, and strain PV-4 offers a tractable experimental system to explore regulation of dissimilatory NO3(-)/NO2(-) reduction pathways.

Comparing on-site to off-site biomass collection for Dehalococcoides biomarker gene quantification to predict in situ chlorinated ethene detoxification potential.

Biostimulation and bioaugmentation have emerged as constructive remedies for chlorinated ethene-contaminated aquifers, and a link between Dehalococcoides (Dhc) bacteria and chlorinated ethene detoxification has been established. To quantify Dhc biomarker genes, groundwater samples are shipped to analytical laboratories where biomass is collected on membrane filters by vacuum filtration for DNA extraction and quantitative real-time PCR analysis. This common practice was compared with a straightforward, on-site filtration approach to Sterivex cartridges. In initial laboratory studies with groundwater amended with known amounts of Dhc target cells, Sterivex cartridges yielded one-third of the total DNA and 9-18% of the Dhc biomarker gene copies compared with vacuum filtration. Upon optimization, DNA yields increased to 94 +/- 38% (+/-SD, n = 10), and quantification of Dhc biomarker genes exceeded the values obtained with the vacuum filtration procedure up to 5-fold. Both methods generated reproducible results when volumes containing >10(4) total Dhc target gene copies were collected. Analysis of on-site and off-site biomass collection procedures corroborated the applicability of the Sterivex cartridge for Dhc biomarker quantification in groundwater. Ethene formation coincided with Dhc cell titers of >2 x 10(6) L(-1) and high (i.e., >10(5)) abundance of the vinyl chloride reductive dehalogenase genes vcrA and/or bvcA; however, high Dhc cell titers alone were insufficient to predict ethene formation. Further, ethene formation occurred at sites with high Dhc cell titers but low or no detectable vcrA or bvcA genes, suggesting that other, not yet identified vinyl chloride reductive dehalogenases contribute to ethene formation. On-site biomass collection with Sterivex cartridges avoids problems associated with shipping groundwater and has broad applicability for biomarker monitoring in aqueous samples.

Oxygen effect on Dehalococcoides viability and biomarker quantification.

Oxygen-sensitive Dehalococcoides bacteria play crucial roles in detoxification of chlorinated contaminants (e.g., chlorinated ethenes), and bioremediation monitoring relies on quantification of Dehalococcoides DNA and RNA biomarkers. To explore the effects of oxygen on Dehalococcoides activity, viability, and biomarker quantification, batch experiments with a tetrachloroethene-to-ethene dechlorinating consortium (Bio-Dechlor INOCULUM [BDI]) harboring multiple Dehalococcoides strains were performed to quantify the effects of < or = 4 mg/L dissolved oxygen. Oxygen inhibited reductive dechlorination, and only incomplete dechlorination to vinyl chloride (VC) occurred following oxygen consumption and extended incubation periods (89 days). Following 30 days of oxygen exposure and subsequent oxygen removal (i.e., reversibility experiments), all trichloroethene- (TCE-) fed cultures dechlorinated TCE to VC, but VC dechlorination to ethene occurred in only one out of fourteen replicates. These results suggest that Dehalococcoides strains respond differently to oxygen exposure, and strains catalyzing the VC-to-ethene dechlorination step are more susceptible to oxygen inhibition. Quantitative real-time PCR (qPCR) analysis detected a 1-1.5 order-of-magnitude decrease in the number of Dehalococcoides biomarker genes (i.e., 16S rRNA gene and the reductive dehalogenase [RDase] genes tceA, vcrA, bvcA) in the oxygen-amended cultures, but qPCR analysis failed to distinguish viable, dechlorinating from irreversibly inhibited (nonviable) Dehalococcoides cells. Reverse transcriptase qPCR (RT-qPCR) detected Dehalococcoides gene transcripts in the oxygen-amended, non-dechlorinating cultures, and biomarker transcription did not always correlate with dechlorination (in)activity. Enhanced molecular tools that complement existing protocols and provide quantitative information on the viability and activity of the Dehalococcoides population are desirable.

The Dehalococcoides population in sediment-free mixed cultures metabolically dechlorinates the commercial polychlorinated biphenyl mixture aroclor 1260.

Microbial reductive dechlorination of commercial polychlorinated biphenyl (PCB) mixtures (e.g., Aroclors) in aquatic sediments is crucial to achieve detoxification. Despite extensive efforts over nearly two decades, the microorganisms responsible for Aroclor dechlorination remained elusive. Here we demonstrate that anaerobic bacteria of the Dehalococcoides group derived from sediment of the Housatonic River (Lenox, MA) simultaneously dechlorinate 64 PCB congeners carrying four to nine chlorines in Aroclor 1260 in the sediment-free JN cultures. Quantitative real-time PCR showed that the Dehalococcoides cell titer in JN cultures amended with acetate and hydrogen increased from 7.07 x 10(6) +/- 0.42 x 10(6) to 1.67 x 10(8) +/- 0.04 x 10(8) cells/ml, concomitant with a 64.2% decrease of the PCBs with six or more chlorines in Aroclor 1260. No Dehalococcoides growth occurred in parallel cultures without PCBs. Aroclor 1260 dechlorination supported the growth of 9.25 x 10(8) +/- 0.04 x 10(8) Dehalococcoides cells per mumol of chlorine removed. 16S rRNA gene-targeted PCR analysis of known dechlorinators (i.e., Desulfitobacterium, Dehalobacter, Desulfuromonas, Sulfurospirillum, Anaeromyxobacter, Geobacter, and o-17/DF-1-type Chloroflexi organisms) ruled out any involvement of these bacterial groups in the dechlorination. Our results suggest that the Dehalococcoides population present in the JN cultures also catalyzes in situ dechlorination of Aroclor 1260 in the Housatonic River. The identification of Dehalococcoides organisms as catalysts of extensive Aroclor 1260 dechlorination and our ability to propagate the JN cultures under defined conditions offer opportunities to study the organisms' ecophysiology, elucidate nutritional requirements, identify reductive dehalogenase genes involved in PCB dechlorination, and design molecular tools required for bioremediation applications.