Dehalococcoides Ethenogenes Research Articles

A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation

Biological nitrogen fixation (BNF) is the reduction of N2 into NH3 in a group of prokaryotes by an extremely O2-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S2Vred) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S2Vred to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.

Interesting anaerobes in the environment

Prokaryotes (Bacteria and Archaea) have a wide range of capacities to survive by generating energy in environments and situations lacking oxygen, which abound on Earth. Anaerobic metabolic strategies include anaerobic respiration (numerous types – e.g. nitrate reduction – Paracoccus denitrificans; sulfur respiration – Desulfuromonadales; methanogenesis – Methanosarsina spp.; iron reduction – Geobacter spp.; dehalorespiration – Dehalococcoides ethenogenes) and fermentation (sugars converted to simpler organic compounds like acids, gases and alcohols – e.g. Lactobacillus spp.). Relatively novel environmental anaerobic strategies include anaerobic ammonium oxidation (Anammox – e.g. Brocadia spp.) and anaerobic methane oxidation (AMO)1, which is a syntrophic association between anaerobic methanotrophic archaea (ANME) and sulfate-, iron-, manganese- or nitrate-reducing bacteria2. The classic anaerobic synthrophic example is interspecies hydrogen/formate transfer between a hydrogen/formate producing fatty acid oxidising bacterium (the syntroph) and a hydrogen/formate consumer (methanogen or sulfate-reducer)3. Microbes vary in their oxygen tolerance and are described as obligate anaerobes if they are killed by atmospheric levels of oxygen due to the lack of catalase and superoxide dismutase that provide oxygen radical protection.

Comparison of 1,2-dichloroethane, dichloroethene and vinyl chloride carbon stable isotope fractionation during dechlorination by two Dehalococcoides strains

Carbon stable isotope fractionation during 1,2-dichloroethane (1,2-DCA), dichloroethene (DCE) and vinyl chloride (VC) dechlorination was analysed for two Dehalococcoides strains, Dehalococcoides mccartyi strain 195 (formerly Dehalococcoides ethenogenes strain 195) and D. mccartyi strain BTF08, and used to characterize the reaction. The isotope enrichment factors (εC) determined for 1,2-DCA were −30.8 ± 1.3‰ and −29.0 ± 3.0‰ for D. mccartyi strain BTF08 and D. mccartyi strain 195, respectively. Enrichment factors (εC) determined for chlorinated ethenes with strain BTF08 were −28.8 ± 1.5‰ (VC), −30.5 ± 1.5‰ (cis-DCE) and −12.4 ± 1.1‰ (1,1-DCE). Product, ethene, related enrichment factors (εC1,2-DCA-ethene) calculated for 1,2-DCA (−34.1 and −32.3‰ for strain BTF08 and strain 195, respectively) were similar to substrate based enrichment factors (εC1,2-DCA), supporting the hypothesis that ethene is the direct product of 1,2-DCA dichloroelimination but that VC was a side product as result of branching in the reaction.

Soil composition at the aquifer level, groundwater quality and the presence of Dehalococcoides ethenogenes at Dover AFB

Chlorinated solvents are among the most common groundwater pollutants globally. A common method to remediate this groundwater pollutant is through bioremediation. One of the most important bacteria that remediation workers use in this remediation is Dehalococcoides ethenogenes, which are the only dehalogenating bacteria with the capacity to break down these contaminants to the non-chlorinated compound, ethylene. While much is known about these bacteria in the laboratory, little is known about their natural habitat. In this experiment, eight sites on Dover Air Force Base were selected based on the larger CERCLA Closeout project; 2.54 cm wells were installed and groundwater samples were collected. These sites monitored groundwater quality parameters: temperature, pH, dissolved oxygen, and oxidative reduction potential. Additionally, samples were also analyzed for the contaminants: tetrachlorethylene, trichloroethylene, and vinyl chloride. The presence of vinyl chloride is an indicator of Dehalococcoides ethenogenes. Soil samples from the aquifer level were taken from each site as well and soil particle texture was assessed using the Bouyoucos hydrometer method. The objective of this experiment was to determine if there was a relationship between clay particles at the aquifer level, groundwater quality, and the presence of Dehalococcoides ethenogenes.

Cultivation-Independent Identification of Candidate Dehalorespiring Bacteria in Tetrachloroethylene Degradation

Tetrachloroethylene (PCE) is one of the major pollutants and is degraded by dissimilation by dehalorespiring bacteria. The dehalorespiring bacteria are anaerobic, and most cannot be cultured by conventional agar plating methods. Therefore, to identify the dehalorespiring bacteria that dissimilatively degrade PCE, a cultivation-independent method is required. To achieve accurate and detailed analysis of the bacteria, we developed a novel stable isotope probing (SIP) method. This technique involves 2 steps, namely, a labeling step, in which a labeled carbon source is incorporated into the sample's DNA, and an analysis step, in which the DNA is isolated, fractionated, and analyzed by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Subsequently, 16S rRNA sequencing and phylogenetic analysis were performed to identify the bacteria. Initially, we examined the effectiveness of this method by using Dehalococcoides ethenogenes 195 consortium as a defined model system. The result indicated the method was able to correctly identify the dehalorespiring bacteria D. ethenogenes 195 from the consortium. Moreover, in an artificially contaminated microcosm experiment, we confirmed that the method was able to identify the indigenous dehalorespiring bacteria Dehalobacter sp. Thus, we concluded that this novel method was a feasible tool to identify dehalorespiring bacteria in natural environments.

EFFECTS OF THE ENVIRONMENTAL STRESS ON TWO FISH POPULATIONS REVEALED BY STATISTICAL AND SPECTRAL ANALYSIS

In the quest for a valid bioaugmenting agent for applications in real sites, an anaerobic mixed culture able to reductively dechlorinate a mixture of C2 chlorinated aliphatic hydrocarbons (CAHs), has been enriched over a period of several years from a groundwater historically contaminated with 1,2-dichloroethane (1,2-DCA) and other C2 CAHs, such as tetrachloroethene (PCE). The culture, named Multidechlorobac, has been analysed for its microbial composition, and the effects of inoculum amount on its dechlorination capabilities have been tested on subcultures prepared in non-sterile conditions, using 1,2-DCA and PCE as final electron acceptors. Lactate, yeast extract and molasses have been used as both carbon and electron sources. Multidechlorobac consisted of both dechlorinators, such as Dehalococcoides ethenogenes strain 195 and Desulfitobacterium sp., and non-dechlorinators belonging to Bacteroidetes, Proteobacteria and Firmicutes. While the former ones are directly involved in the degradation of the pollutants, the latter support the process by providing hydrogen, which acts as electron donor in the dechlorination. The initial bacterial concentration was about 108 cells mL-1, and subcultures were able to degrade the pollutants at 10 % inoculum. Higher dilutions resulted in a loss of dechlorinating capacity, leading to a slower or unsuccessful treatment, even though dechlorination was still observed for 1,2-DCA. Multidechlorobac was effective in the degradation of pollutants, and it was found to be robust and easy to grow, in comparison with pure cultures. The culture might be successfully used as bioaugmenting agent in the clean-up of sites polluted with C2 chlorinated aliphatic hydrocarbons.

Full‐scale bioremediation of a dilute 50‐acre plume

AbstractEnhanced anaerobic dechlorination is being conducted to remediate a 50‐acre groundwater area impacted with chlorinated volatile organic compounds (CVOCs). The plume, which is over 3,000 feet (ft) long, initially contained tetrachloroethene and breakdown products at concentrations of 2 to 3 milligrams per liter. The site's high groundwater flow velocity (greater than 1,000 ft per year) was incorporated into the design to help with amendment distribution. Bioaugmentation was conducted using a mixed culture containing Dehalococcoides ethenogenes. There is evidence that it has migrated to distances exceeding 600 ft. The major benefit of the high groundwater flow velocity is greater areal coverage by the remediation system, but the downside is the difficulty in delivering sufficient donor to create the required anaerobic conditions. Overall performance has been excellent with total CVOC reductions and conversion to ethene of 98 percent within a 25‐acre area downgradient of the treatment transect that has operated the longest. © 2011 Wiley Periodicals, Inc.

Dehalogenase gene detection and microbial diversity of a chlorinated hydrocarbon contaminated site

In recent years large quantities of mixtures of chlorinated hydrocarbons have accumulated in the environment due to the widespread use and production of these compounds. Microbes have been found to demonstrate a widespread and diverse potential to adapt to the dechlorination of such compounds. Therefore the aim of this study was to investigate the presence and diversity of reductive and hydrolytic dehalogenase genes in a site contaminated with a mixture of chlorinated hydrocarbons. Primers targeting reductive and hydrolytic bacterial dehalogenase genes were designed. In addition, DGGE analysis was performed in order to determine the presence of any known dehalogenase-producing organisms. Total DNA isolated from borehole water samples was used as the template for the amplification reactions. All PCR products obtained with the reductive and hydrolytic gene primers, as well as the dominant bands present on the DGGE gel were cloned and sequenced. Sequencing of the individual amplicons revealed significant identities to the tceA gene of Dehalococcoides ethenogenes 195, the vcrA gene of Dehalococcoides sp. VS as well as the dhlA and dhlB genes of Xanthobacter autotrophicus GJ10. DGGE analysis indicated a high level of commonality with the different sampling times and depths. However, sequence analysis revealed that 66% of the cloned fragments showed significant (95–99%) identity with uncultured microorganisms. Phylogenetic analysis of the sequences revealed that the DGGE clones clustered into two groups when compared to known bacteria having hydrocarbon degradative capabilities. This indicated that the sequences of the clones were diverse when compared to known microorganisms. This diversity represents a largely untapped genetic pool that can be exploited for the discovery of novel biocatalysts that can be employed in bioremediation. In addition, the presence of both hydrolytic and reductive dehalogenases provided strong evidence that bacteria capable of dehalogenation of chlorinated hydrocarbons may be present in sites contaminated with these compounds.

Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases

The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas "Dehalococcoides"-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of "Dehalococcoides ethenogenes" strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidated.

The effect of co-substrate activation on indigenous and bioaugmented PCB dechlorinating bacterial communities in sediment microcosms

Microbial reductive dechlorination by members of the phylum Chloroflexi, including the genus Dehalococcoides, may play an important role in natural detoxification of highly chlorinated environmental pollutants, such as polychlorinated biphenyls (PCBs). Previously, we showed the increase of an indigenous bacterial population belonging to the Pinellas subgroup of Dehalococcoides spp. in Anacostia River sediment (Washington DC, USA) microcosms treated with halogenated co-substrates ("haloprimers"), tetrachlorobenzene (TeCB), or pentachloronitrobenzene (PCNB). The PCNB-amended microcosms exhibited enhanced dechlorination of weathered PCBs, while TeCB-amended microcosms did not. We therefore developed and used different phylogenetic approaches to discriminate the effect of the two different haloprimers. We also developed complementary approaches to monitor the effects of haloprimer treatments on 12 putative reductive dehalogenase (rdh) genes common to Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain CBDB1. Our results indicate that 16S rRNA gene-based phylogenetic analyses have a limit in their ability to distinguish the effects of two haloprimer treatments and that two of rdh genes were present in high abundance when microcosms were amended with PCNB, but not TeCB. rdh gene-based phylogenetic analysis supports that these two rdh genes originated from the Pinellas subgroup of Dehalococcoides spp., which corresponds to the 16S rRNA gene-based phylogenetic analysis.

Carbon isotope fractionation during dechlorination of 1,2,3,4-tetrachlorodibenzo- p-dioxin by a Dehalococcoides-containing culture

Carbon isotope fractionation was observed during dechlorination of 1,2,3,4-tetrachlorodibenzo- p-dioxin (1,2,3,4-TeCDD) by a mixed culture containing Dehalococcoides ethenogenes strain 195. Fractionation was examined when 1,2,3,4-TeCDD was added as the only chlorinated compound and when 1,2,3,4-TeCDD was added with a known growth substrate, tetrachloroethene (PCE). The 1,2,3,4-TeCDD was dechlorinated to 1,2,4-trichlorodibenzo- p-dioxin (1,2,4-TrCDD) which was enriched in 13C relative to 1,2,3,4-TeCDD with isotope separation factors, ε C, of 1.3 ± 0.2‰ and 1.7 ± 0.4‰ (average ± 95% confidence interval (CI)) in cultures with and without PCE, respectively. The 1,2,4-TrCDD was further dechlorinated to 1,3-dichlorodibenzo- p-dioxin (1,3-DCDD) which was depleted in 13C relative to 1,2,4-TrCDD with ε C of −2.4 ± 0.4‰ and −2.9 ± 0.8‰ (average ± 95% CI) in cultures with and without PCE, respectively. This demonstrates carbon isotope fractionation during sequential reductive dechlorination of PCDDs, where isotope fractionation during dechlorination of the intermediate was substantial and a 13C depleted lightly chlorinated PCDD congener was ultimately formed during dechlorination of more highly chlorinated PCDD congeners. Despite reproducible, statistically significant differences between isotope compositions of the parent, 1,2,3,4-TeCDD and daughter, 1,2,4-TrCDD and 1,3-DCDD congeners in triplicate bottles of both treatments, fractionation factors for 1,2,3,4-TeCDD could not be determined for all replicates by regression analysis of the plot of the Rayleigh equation. It is possible that dissolution of 1,2,3,4-TeCDD imposed a kinetic limitation on dechlorination, thus masking isotope fractionation during its dechlorination.

Field Evidence for Co-Metabolism of Trichloroethene Stimulated by Addition of Electron Donor to Groundwater

For more than 10 years, electron donor has been injected into the Snake River aquifer beneath the Test Area North site of the Idaho National Laboratory for the purpose of stimulating microbial reductive dechlorination of trichloroethene (TCE) in groundwater. This has resulted in significant TCE removal from the source area of the contaminant plume and elevated dissolved CH(4) in the groundwater extending 250 m from the injection well. The delta(13)C of the CH(4) increases from -56 per thousand in the source area to -13 per thousand with distance from the injection well, whereas the delta(13)C of dissolved inorganic carbon decreases from 8 per thousand to -13 per thousand, indicating a shift from methanogenesis to methane oxidation. This change in microbial activity along the plume axis is confirmed by PhyloChip microarray analyses of 16S rRNA genes obtained from groundwater microbial communities, which indicate decreasing abundances of reductive dechlorinating microorganisms (e.g., Dehalococcoides ethenogenes) and increasing CH(4)-oxidizing microorganisms capable of aerobic co-metabolism of TCE (e.g., Methylosinus trichosporium). Incubation experiments with (13)C-labeled TCE introduced into microcosms containing basalt and groundwater from the aquifer confirm that TCE co-metabolism is possible. The results of these studies indicate that electron donor amendment designed to stimulate reductive dechlorination of TCE may also stimulate co-metabolism of TCE.

Dechlorinating and Iron Reducing Bacteria Distribution in a TCE‐Contaminated Aquifer

Dehalorespiration bioremediation has been considered for chlorinated compound removal from two trichloroethene contaminated groundwater plumes in the OU 5 area of Hill Air Force Base, Utah. The distributions and population densities of the 16S rRNA genes of Bacteria, Dehalococcoides ethenogenes, Desulfuromonas michiganensis, Geobacter spp. and Rhodoferax ferrireducens‐like bacteria, as well as the functional genes trichloroethene reductive dehalogenase (tceA) and vinyl chloride reductase (vcrA) were determined in contaminated aquifer material samples. The influence of aquifer physical and chemical properties, including iron availability, on these distributions was evaluated. Twenty aquifer cores were collected. DNA was extracted and analyzed using real‐time quantitative polymerase chain reaction (RT‐qPCR) to quantify the gene densities. Dehalococcoides population densities were low and unevenly distributed. D. michiganensis was found in 12 cores while Geobacter spp. were found in 8 cores. Rhodoferax ferrireducens‐like bacteria were widely distributed. The vcrA gene distribution was relatively uniform and broad but the tceA gene was detectable in only 2 cores. Gene distribution was not related to core clusters derived from physical/chemical characteristics.

Assessment and monitoring tools for aerobic bioremediation of vinyl chloride in groundwater

AbstractDirect aerobic biodegradation of vinyl chloride (VC) offers a remedial solution for persistent vinyl chloride plumes that are not amenable to the anaerobic process of reductive dechlorination because of either prevailing geochemical conditions or the absence of active Dehalococcoides ethenogenes. However, tools are needed to evaluate and optimize aerobic VC bioremediation. This article describes the development and testing of two techniques—a microbiological tool and a molecular tool—for this purpose. Both methods are based on detection of bacteria that can use vinyl chloride and ethene as growth substrates in the presence of oxygen. The microbiological tool is an activity assay that indicates whether bacteria capable of degrading ethene under aerobic conditions are present in a groundwater sample. This activity assay gave positive results in the area of active VC degradation of an aerobic VC bioremediation test site. A rapid semiquantitative genetic assay was also developed. This molecular tool, based on polymerase chain reaction (PCR) detection of a gene involved in the metabolism of both ethene and VC, revealed the presence of potential VC degraders in an enrichment culture and site groundwater. These tools could provide a basis for judging the potential of aerobic VC degradation by ethenotrophs at other sites in addition to offering a mechanism for treatment monitoring and system optimization. © 2009 Wiley Periodicals, Inc.

Evidence for Nitrogen Fixation by “ Dehalococcoides ethenogenes ” Strain 195

Genome annotation of the chlorinated ethene-respiring "Dehalococcoides ethenogenes" strain 195 indicated the presence of a complete nitrogenase operon. Here, results from long-term growth experiments, gene expression, and (15)N(2)-isotope measurements confirm that strain 195 is capable of fixing atmospheric dinitrogen when a defined fixed-nitrogen source such as ammonium is unavailable.

PCB dechlorination enhancement in Anacostia River sediment microcosms

In situ treatment of PCB contaminated sediments via microbial dechlorination is a promising alternative to dredging, which may be reserved for only the most contaminated areas. Reductive dechlorination of low levels of weathered PCB mixtures typical of urban environments may occur at slow rates. Here, we report that biostimulation and bioaugmentation enhanced dechlorination of low concentration (2.1 mg PCBs/kg dry weight) historical PCBs in microcosms prepared with Anacostia River, Washington, DC, sediment. Treatments included electron donors butyrate, lactate, propionate and acetate (1 mM each); alternate halogenated electron acceptors (haloprimers) tetrachlorobenzene (TeCB, 25 μM), pentachloronitrobenzene (PCNB, 25 μM), or 2,3,4,5,6-PCB (PCB116, 2.0 μM); and/or bioaugmentation with a culture containing Dehalococcoides ethenogenes strain 195 (3 × 10 6 cells/mL). Dechlorination rates were enhanced in microcosms receiving bioaugmentation, PCNB and PCNB plus bioaugmentation, compared to other treatments. Microcosm subcultures generated after 415 days and spiked with PCB116 showed sustained capacity for dechlorination of PCB116 in PCNB, PCNB plus bioaugmentation, and TeCB treatments, relative to other treatments. Analysis of Chloroflexi 16S rRNA genes showed that TeCB and PCNB increased native Dehalococcoides spp. from the Pinellas subgroup; however this increase was correlated to enhanced dechlorination of low concentration weathered PCBs only in PCNB-amended microcosms. D. ethenogenes strain 195 was detected only in bioaugmented microcosms and decreased over 281 days. Bioaugmentation with D. ethenogenes strain 195 increased PCB dechlorination rates initially, but enhanced capacity for dechlorination of a model congener, PCB116, after 415 days occurred only in microcosms with enhanced native Dehalococcoides spp.

A Dehalococcoides-containing co-culture that dechlorinates tetrachloroethene to trans-1,2-dichloroethene

In the microbial reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), dechlorinators usually produce cis-1,2-dichloroethene (cis-DCE) as the predominant product or an intermediate. This study shows that dechlorination of PCE and TCE can also lead to the generation of trans-1,2-dichloroethene (trans-DCE) by a co-culture MB. During its enrichment process, the ratio of trans- to cis-DCE increased from 1.4 (+/-0.1):1-3.7 (+/-0.4):1, whereas the TCE reductive dechlorination rate went up from approximately 26.2 to approximately 68.8 micromol l(-1) day(-1). PCR-denaturing gradient gel electrophoresis (PCR-DGGE) revealed that the increased ratio of trans- /cis-DCE was well correlated with the increased proportions of Dehalococcoides and the disappearance of Desulfuromonas during the enrichment process. As shown by PCR-DGGE, similar Dehalococcoides species were consistently present in another three sediment-free cultures with various trans- /cis-DCE ratios. The 16S rRNA gene sequence of this Dehalococcoides sp. in co-culture MB is 100% identical (over 1489 bp) to that of Dehalococcoides ethenogenes strain 195 (CP000027), which belongs to the Cornell subgroup of the Dehalococcoides cluster. The other bacterium in this co-culture MB was a Sedimentibacter species, which showed no PCE or TCE dechlorination activity. Results from this study show that microbial dechlorination of chloroethenes by this particular subgroup of Dehalococcoides could result in significant accumulation of trans-DCE in the environment if no trans-DCE dechlorinators coexist in the contaminated sites.

Investigation of Carbon Metabolism in “ Dehalococcoides ethenogenes ” Strain 195 by Use of Isotopomer and Transcriptomic Analyses

Members of the genus "Dehalococcoides" are the only known microorganisms that can completely dechlorinate tetrachloroethene and trichloroethene to the innocuous end product, ethene. This study examines the central metabolism in "Dehalococcoides ethenogenes" strain 195 via (13)C-labeled tracer experiments. Supported by the genome annotation and the transcript profile, isotopomer analysis of key metabolites clarifies ambiguities in the genome annotation and identifies an unusual biosynthetic pathway in strain 195. First, the (13)C-labeling studies revealed that strain 195 contains complete amino acid biosynthesis pathways, even though current genome annotation suggests that several of these pathways are incomplete. Second, the tricarboxylic acid cycle of strain 195 is confirmed to be branched, and the Wood-Ljungdahl carbon fixation pathway is shown to not be functionally active under our experimental conditions; rather, CO(2) is assimilated via two reactions, conversion of acetyl-coenzyme A (acetyl coenzyme A [acetyl-CoA]) to pyruvate catalyzed by pyruvate synthase (DET0724-0727) and pyruvate conversion to oxaloacetate via pyruvate carboxylase (DET0119-0120). Third, the (13)C-labeling studies also suggested that isoleucine is synthesized from acetyl-CoA and pyruvate via citramalate synthase (CimA, EC 2.3.1.182), rather than from the common pathway via threonine ammonia-lyase (EC 4.3.1.19). Finally, evidence is presented that strain 195 may contain an undocumented citrate synthase (>95% Re-type stereospecific), i.e., a novel Re-citrate synthase that is apparently different from the one recently reported in Clostridium kluyveri.

Modeling the Effects of Microbial Competition and Hydrodynamics on the Dissolution and Detoxification of Dense Nonaqueous Phase Liquid Contaminants

The real significance and engineering potential for bioenhanced dissolution of chlorinated ethene dense nonaqueous phase liquid (DNAPL) contaminants are currently not well understood, in part because they can be influenced by a complex set of factors, including microbial competition for growth substrates. Mathematical simulations were performed to evaluate the effects of competition between Dehalococcoides ethenogenes and Desulfuromonas michiganensis for the electron acceptor tetrachloroethene (PCE) on the distribution of dehalorespirers, PCE dissolution, and the extent of PCE detoxification. The modeling results demonstrate that the outcome of competition between populations for growth substrates can have a significant impact on bioenhancement and, thus, on DNAPL source zone longevity and identify the key factors in determining the outcome of competition and its effects on DNAPL dissolution. The potential for bioenhancement is greatest at lower groundwater velocities. At higher velocities, kinetic properties play a key role in determining which population dominates and where, and the amount of bioenhancement that is realized. Engineered bioremediation techniques that maintain multiple dehalorespiring populations may offer the best approach for optimizing the twin cleanup goals of reduced source zone longevity and complete detoxification while maximizing the utilization of added electron donors.

Quantitative discrimination of 16 S rRNA genes of Dehalococcoides species by MagSNiPer, a quantitative single‐nucleotide‐polymorphism genotyping method

Previously we developed MagSNiPer, an SNP (single nucleotide polymorphism) genotyping method. In the present paper we show development of an automated system for MagSNiPer, namely MagSNiPer Station, and its application for quantitative discrimination of Dehalococcoides species, which perform anaerobic dechlorination of chloroethenes. MagSNiPer Station is equipped with a thermal cycler, a tip stand, a microtitre-plate automated stacker, an eight-channel tip dispenser, a magnetic separation unit for Magtration technology, and a chemiluminescence detector. It can automatically perform all processes required for SNP genotyping by MagSNiPer. A primer was designed for discriminating single nucleotide difference between 16 S rRNA genes of Dehalococcoides ethenogenes and Dehalococcoides BAV1. Chemiluminescence intensities for the 16 S rRNA genes obtained by MagSNiPer were proportional to their quantity. MagSNiPer analysis of 16 S rRNA genes amplified on the DNA purified from groundwater gave a ratio of these two 16 S rRNA genes similar to that obtained by cloning and sequencing. MagSNiPer is much easier, more rapid and more cost-effective than conventional sequencing. Compared with denaturing gradient-gel electrophoresis, MagSNiPer has the advantage of being quantitative. Therefore, by applying MagSNiPer at several sites where single base differences exist among Dehalococcoides species, it is possible to analyse Dehalococcoides consortia with ease, yielding useful information on anaerobic bioremediation of chloroethenes.