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In this study, the emulsified castor oil (ECO) substrate was developed for a long-term supplement of biodegradable carbon with pH buffering capacity to anaerobically bioremediate trichloroethylene (TCE)-polluted groundwater. The ECO was produced by mixing castor oil, surfactants [sapindales and soya lecithin (SL)], vitamin complex, and a citrate/sodium phosphate dibasic buffer system together for slow carbon release. Results of the emulsification experiments and microcosm tests indicate that ECO emulsion had uniform small droplets (diameter = 539 nm) with stable oil-in-water characteristics. ECO had a long-lasting, dispersive, negative zeta potential (-13 mv), and biodegradable properties (viscosity = 357 cp). Approximately 97% of TCE could be removed with ECO supplement after a 95-day operational period without the accumulation of TCE dechlorination byproducts (dichloroethylene and vinyl chloride). The buffer system could neutralize acidified groundwater, and citrate could be served as a primary substrate. ECO addition caused an abrupt TCE adsorption at the initial stage and the subsequent removal of adsorbed TCE. Results from the next generation sequences and real-time polymerase chain reaction (PCR) indicate that the increased microbial communities and TCE-degrading bacterial consortia were observed after ECO addition. ECO could be used as a pH-control and carbon substrate to enhance anaerobic TCE biodegradation effectively. PRACTITIONER POINTS: Emulsified castor oil (ECO) contains castor oil, surfactants, and buffer for a slow carbon release and pH control. ECO can be a long-term carbon source for trichloroethylene (TCE) dechlorination without causing acidification. TCE removal after ECO addition is due to adsorption and reductive dechlorination mechanisms. Citrate (contained in buffer system) can serve as a primary substrate for TCE dechlorination enhancement. ECO addition causes increased bacterial diversity, Dehalococcoides sp., and hydrogen-producing gene (hydA).

Summary Polybrominated diphenyl ethers (PBDEs), chemicals commonly used as flame-retardants in consumer products, are emerging persistent organic pollutants that are ubiquitous in the environment. In this study, we report the PBDE-respiring isolate - Dehalococcoides mccartyi strain GY50, which debrominates the most toxic tetra- and penta-BDE congeners (∼1.4 µM) to diphenyl ether within 12 days with hydrogen as the electron donor. The complete genome sequence revealed 26 reductive dehalogenase homologous genes (rdhAs), among which three genes (pbrA1, pbrA2, and pbrA3) were highly expressed during PBDE debromination. After ten transfers of GY50 with trichloroethene or 2,4,6-trichlorophenol as the electron acceptor instead of PBDEs, the ssrA-specific genome island (ssrA-GI) containing pbrA1 and pbrA2 was deleted from the genome of strain GY50, leading to two variants (strain GY52 with trichloroethene, strain GY55 with 2,4,6-trichlorophenol) with identically impaired debromination capabilities (debromination of penta-/tetra-BDEs ceased at di-BDE 15). Through analysis of Illumina paired-end sequencing data, we identified read pairs that probably came from variants that contain ssrA-GI deletions, indicating their possible presence in the original strain GY50 culture. The two variant strains provide real-time examples on rapid evolution of organohalide­respiring organisms. As PBDE-respiring organisms, GY50-like strains may serve as key players in detoxifying PBDEs in contaminated environments. This article is protected by copyright. All rights reserved.

Adaptive site management and aggressive bioremediation in the source zone of a complex chlorinated dense nonaqueous phase liquid (DNAPL) site reduced total chlorinated hydrocarbon mass discharge by nearly 80%. Successful anaerobic bioremediation of chlorinated hydrocarbons can be impaired by inadequate concentrations of electron donors, competing electron acceptors, specific inhibitors such as chloroform, and potentially by high contaminant concentrations associated with residual DNAPL. At the study site, the fractured bedrock aquifer was impacted by a mixture of chlorinated solvents and associated daughter products. Concentrations of 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA), 1,1,2-trichloroethane (1,1,2-TCA), and 1,2-dichloroethane (1,2-DCA) were on the order of 100 to 1000 mg/L. Chloroform was present as a co-contaminant and background sulfate concentrations were approximately 400 mg/L. Following propylene glycol injections, concentrations of organohalide-respiring bacteria including Dehalococcoides and Dehalogenimonas spp. increased by two to three orders of magnitude across most of the source area. Statistical analysis indicated that reaching volatile fatty acid concentrations greater than 1000 mg/L and depleting sulfate to concentrations less than 50 mg/L were required to achieve a Dehalococcoides concentration greater than the 104 cells/mL recommended for generally effective reductive dechlorination. In a limited area, chloroform concentrations greater than 5 mg/L inhibited growth of Dehalococcoides populations despite the availability of electron donor and otherwise appropriate geochemical conditions. After implementing a groundwater recirculation system targeting the inhibited area, chloroform concentrations decreased permitting significant increases in concentrations of Dehalococcoides and vinyl chloride reductase gene copies.

Lake Fryxell, McMurdo Dry Valleys, Antarctica contains a constantly cold water column and perennial ice-cover. Although carbon and sulfur cycling in this amictic lake have been studied previously, a paired investigation of 16S rRNA gene based microbial diversity and geochemistry of Lake Fryxell is lacking. Here, we used a combination of radiotracer-based rate measurements, geochemical measurements, and molecular microbial community analysis to investigate the anaerobic oxidation of methane (AOM) and associated processes in Lake Fryxell. The results show that while AOM and sulfate reduction appear coupled in the upper regions of the anoxic water column, in deep anoxic waters, where AOM rates are highest, sulfate is unlikely to be the electron acceptor for AOM. Despite significant rates of AOM in these waters, no putative AOM-associated Archaea or Bacteria were observed. Due to a lack of documented AOM electron acceptors and putative ANMEs, we suggest novel modes of AOM dominate in this extreme environment. First, the notable abundance of the bacterial genus Dehalococcoides suggests that reductive dehalogenation could fuel AOM. Further, taxa of the candidate phylum OP9, the Atribacteria, and the Bathyarchaeota (formerly known as the Miscellaneous Crenarchaeotal Group) both commonly observed at cold methane-seeps globally, may mediate AOM, possibly using humic acids as electron shuttles, in Lake Fryxell.

An enhanced bioremediation pilot test was implemented to study the efficacy of enhancing in situ reductive dechlorination of tetrachloroethene (PCE) in shallow bedrock where some intrinsic degradation to cis-1,2-dichloroethene (cis-1,2-DCE) was observed without further degradation to vinyl chloride or nontoxic ethene. Limited Dehalococcoides spp. cell concentrations were present within the study area prior to the gravity-fed injection of an injectate of fermentable carbon substrates in native anaerobic groundwater. Direct connectivity between the injection well screen and performance monitoring well was evidenced and resulted in the degradation of nearly all PCE to cis-1,2-DCE, significant decrease in pH, and apparent inhibited Dehalococcoides spp. growth in the study area groundwater in the first six months. After 24 months, nearly all cis-1,2-DCE had degraded to nontoxic ethene, pH rebounded to more optimal levels, and abundant growth of Dehalococcoides spp. (6.8E05 cells/mL) and its functional gene expressions responsible for complete dechlorination were evident. The observations indicated initial poor dechlorination within the injection zone did not preclude effective treatment, allowing sufficient monitoring time showed the effective treatment zone (or more-optimal fringe) first moved outward from the injection zone beyond the monitoring point and then receded back toward the point of injection over a period of two years. ©2015 Wiley Periodicals, Inc.

Microbial electric systems (MESs) hold significant promise for the sustainable remediation of chlorinated solvents such as tetrachlorethene (perchloroethylene, PCE). Although the bio-electrochemical potential of some specific bacterial species such as Dehalcoccoides and Geobacteraceae have been exploited, this ability in other undefined microorganisms has not been extensively assessed. Hence, the focus of this study was to investigate indigenous and potentially bio-electrochemically active microorganisms in PCE-contaminated groundwater. Lab-scale MESs were fed with acetate and carbon electrode/PCE as electron donors and acceptors, respectively, under biostimulation (BS) and BS-bioaugmentation (BS-BA) regimes. Molecular analysis of the indigenous groundwater community identified mainly Spirochaetes, Firmicutes, Bacteroidetes, and γ and δ-Proteobacteria. Environmental scanning electron photomicrographs of the anode surfaces showed extensive indigenous microbial colonization under both regimes. This colonization and BS resulted in 100% dechlorination in both treatments with complete dechlorination occurring 4 weeks earlier in BS-BA samples and up to 11.5 μA of current being generated. The indigenous non-Dehalococcoides community was found to contribute significantly to electron transfer with ∼61% of the current generated due to their activities. This study therefore shows the potential of the indigenous non-Dehalococcoides bacterial community in bio-electrochemically reducing PCE that could prove to be a cost-effective and sustainable bioremediation practice.

Introduction Stimulation of microbial populations capable of degrading chlorinated compounds during the implementation of enhanced reductive dechlorination (ERD) is achieved by the addition of a carbon substrate into engineered reactive zones. This can be performed using either continuous or batch injection systems. Biodegradation of chlorinated compounds is facilitated by dehalorespiring bacteria (e.g., Dehalococcoides) that use acetate or hydrogen as an electron donor during the reductive dechlorination process. In most cases, the carbon substrate added to the groundwater must undergo fermentation to produce the electron donors required by the dehalorespirers. However, the addition of a carbon substrate also stimulates the growth of a broad community of organisms beyond the dehalorespiring bacteria, which include fermentative organisms and multiple species of heteroand lithotrophic organisms that can utilize hydrogen and acetate (e.g., acetogens, methanogens). Microbial ecological data from active reductive dehalogenating communities indicate that dehalogenating bacteria make up less than 1% of the total microbial biomass (Lee et al. 2004). If at all possible, designers of in situ ERD systems would prefer electron donor delivery to be selectively available to the dehalogenators alone. However, the complexity of the microbial community that grows in response to carbon substrate addition coupled with the low percentage of dehalogenating bacteria makes the indiscriminate consumption of carbon and the fermentation products by the nontarget organisms unavoidable. This inherent scavenging of electron donors by competing bacteria results in the overwhelming generation and stockpiling of high levels of biomass and subsequent recycling within the reactive zones, which can provide considerable quantities of electron donor to be utilized later by the dehalorespirers. The buildup and subsequent recycling of biomass resulting from multiple carbon injection events has been hypothesized to be a primary factor in preventing rebound of dissolved contaminant levels following completion of the active treatment phase (Adamson and Newell 2009). This mechanism can have a significant advantage for source zone biological treatment where active injection is often most aggressive and biomass growth can extend treatment beyond the active treatment phase. This hypothesis is supported by lab-based studies where the recycling of biomass, or endogenous decay, has been shown to maintain reductive dechlorination processes long after carbon substrate addition was stopped (Sleep et al. 2005). In this paper, we discuss the concept of endogenous decay within in situ reactive zones, evaluate the generation and dissipation of biomass during the active injection phase, and present site data demonstrating how long this enhanced attenuation process is expected to last. We also discuss the practical implications of biomass recycling on the design of in situ ERD systems and how operational management can be used to leverage these mechanisms to improve remedial efficiency.

Reductive dechlorination is a major degradation pathway of chlorinated ethenes in anaerobic subsurface environments, and reactive kinetic models describing the degradation process are needed in fate and transport models of these contaminants. However, reductive dechlorination is a complex biological process, where many microbial populations including dechlorinating, fermentative, methanogenic, iron and sulfate reducing, interact. In this article the modeling approaches and the experimental data needed to calibrate them are reviewed, classified, and discussed. Model approaches considered include first order kinetics, Monod kinetics to describe sequential reductive dechlorination and bacterial growth, and metabolic models which simulate fermentation and redox processes interacting with reductive dechlorination processes. The review shows that the estimated kinetic parameters reported vary over a wide range, and that experimental microbial data are scarce. Very few studies have been performed evaluating the influence of sulfate and iron reduction, and contradictory conclusions on the interaction of redox processes with reductive dechlorination have been reported. The modeling approaches for metabolic reductive dechlorination employing different descriptions of the interaction between redox and dechlorination processes and competition for hydrogen are classified. The current concepts lead to different results, suggesting a need for further investigations on the interactions between the microbial communities performing dechlorination and redox processes, including the establishment of biomarkers quantifying dechlorination, and on geochemical characterization. Finally, the relevance of laboratory data and the development of practical modeling tools for field applications are discussed.

At a low permeability clay till site contaminated with chlorinated ethenes (Gl. Kongevej, Denmark), enhanced reductive dechlorination (ERD) was applied by direct push injection of molasses and dechlorinating bacteria. The performance was investigated by long-term groundwater monitoring, and after 4 years of remediation, the development of degradation in the clay till matrix was investigated by high-resolution subsampling of intact cores. The formation of degradation products, the presence of specific degraders Dehalococcoides spp. with the vinyl chloride (VC) reductase gene vcrA, and the isotope fractionation of trichloroethene, cis-dichloroethene (cis-DCE), and VC showed that degradation of chlorinated ethenes occurred in the clay till matrix as well as in sand lenses, sand stringers, and fractures. Bioactive sections of up to 1.8 m had developed in the clay till matrix, but sections, where degradation was restricted to narrow zones around sand lenses and stringers, were also observed. After 4 years of remediation, an average mass reduction of 24% was estimated. Comparison of the results with model simulation scenarios indicate that a mass reduction of 85% can be obtained within approximately 50 years without further increase in the narrow reaction zones if no donor limitations occur at the site. Long-term monitoring of the concentration of chlorinated ethenes in the underlying chalk aquifer revealed that the aquifer was affected by the more mobile degradation products cis-DCE and VC generated during the remediation by ERD.

This study demonstrated the utility in correlating performance and community structure of a trichloroethene (TCE)-dechlorinating microbial consortium; specifically dechlorinators, fermenters, homoacetogens, and methanogens. Two complementary approaches were applied: predicting trends in the microbial community structure based on an electron balance analysis and experimentally assessing the community structure via pyrosequencing and quantitative polymerase chain reaction (qPCR). Fill-and-draw reactors inoculated with the DehaloR^2 consortium were operated at five TCE-pulsing rates between 14 and 168 µmol/10-day-SRT, amended with TCE every 2 days to give peak concentrations between 0.047 and 0.56 mM (6-74 ppm) and supplied lactate and methanol as sources of e(-) donor and carbon. The complementary approaches demonstrated the same trends: increasing abundance of Dehalococcoides and Geobacter and decreasing abundance of Firmicutes with increasing TCE pulsing rate, except for the highest pulsing rate. Based on qPCR, the abundance of Geobacter and Dehalococcoides decreased for the highest TCE pulsing rate, and pyrosequencing showed this same trend for the latter. This deviation suggested decoupling of Dehalococcoides growth from dechlorination. At pseudo steady-state, methanogenesis was minimal for all TCE pulsing rates. Pyrosequencing and qPCR showed suppression of the homoacetogenic genera Acetobacterium at the two highest pulsing rates, and it was corroborated by a decreased production of acetate from lactate fermentation and increased propionate production. Suppression of Acetobacterium, which can provide growth factors to Dehalococcoides, may have contributed to the decoupling for the highest TCE-pulsing rate.

A study with H(2)-based membrane biofilm reactors (MBfRs) was undertaken to examine the effectiveness of direct H(2) delivery in ex-situ reductive dechlorination of chlorinated ethenes. Trichloroethene (TCE) could be reductively dechlorinated to ethene with up to 95% efficiency as long as the pH-increase effects of methanogens and homoacetogens were managed and dechlorinators were selected for during start-up by creating H(2) limitation. Based on quantitative PCR, the dominant bacterial groups in the biofilm at the end of reactor operation were Dehalococcoides, Geobacter, and homoacetogens. Pyrosequencing confirmed the dominance of the dechlorinators and identified Acetobacterium as the key homoacetogen. Homoacetogens outcompeted methanogens for bicarbonate, based on the effluent concentration of acetate, by suppressing methanogens during batch start-up. This was corroborated by the methanogenesis functional gene mcrA, which was 1-2 orders of magnitude lower than the FTHFS functional gene for homoacetogens. Imaging of the MBfR fibers using scanning electron microscopy showed a distinct Dehalococcoides-like morphology in the fiber biofilm. These results support that direct addition of H(2) can allow for efficient and complete reductive dechlorination, and they shed light into how H(2)-fed biofilms, when operated to manage methanogenic and homoacetogenic activity, can be used for ex-situ bioremediation of chlorinated ethenes.

The bioremediation of chloroethene contaminants in groundwater polluted systems is still a serious environmental challenge. Many previous studies have shown that cooperation of several dechlorinators is crucial for complete dechlorination of trichloroethene to ethene. In the present study, we used an explorative functional DNA microarray (DechloArray) to examine the composition of specific functional genes in groundwater samples in which chloroethene bioremediation was enhanced by delivery of hydrogen-releasing compounds. Our results demonstrate for the first time that complete biodegradation occurs through spatial and temporal variations of a wide diversity of dehalorespiring populations involving both Sulfurospirillum, Dehalobacter, Desulfitobacterium, Geobacter and Dehalococcoides genera. Sulfurospirillum appears to be the most active in the highly contaminated source zone, while Geobacter was only detected in the slightly contaminated downstream zone. The concomitant detection of both bvcA and vcrA genes suggests that at least two different Dehalococcoides species are probably responsible for the dechlorination of dichloroethenes and vinyl chloride to ethene. These species were not detected on sites where cis-dichloroethene accumulation was observed. These results support the notion that monitoring dechlorinators by the presence of specific functional biomarkers using a powerful tool such as DechloArray will be useful for surveying the efficiency of bioremediation strategies.

A numerical reactive transport model was developed to simulate the bioremediation processes in a perchloroethene (PCE) contaminated single fracture system augmented with Dehalococcoides sp. (DHC). The model describes multispecies bioreactive transport processes that include bacterial growth and detachment dynamics, biodegradation of chlorinated species, competitive inhibition of various reactive species, and the loss of daughter products because of back-partitioning effects. Two sets of experimental data, available in the study by Schaefer et al. (2010b), were used to calibrate and test the model. The model was able to simulate both datasets. The simulation results indicated that the yield coefficient and the DHC maximum utilization rate coefficient were the two important process parameters. A detailed sensitivity study was completed to quantify the sensitivity of the model to variations in these two parameter values. The results show that an increase in yield coefficient increases bacterial growth and thus expedites the dechlorination process, whereas an increase in maximum utilization rate coefficient greatly increased dechlorination rates. The proposed model provides a mathematical framework for simulating remediation systems that employ DHC bioaugmentation for restoring chlorinated-solvent contaminated groundwater aquifers.

In the 1960s, trichloroethene (TCE) was used at what is now designated as Installation Restoration Program Site 32 Cluster at Vandenberg Air Force Base to flush missile engines prior to launch and perhaps for other degreasing activities, resulting in releases of TCE to groundwater. The TCE plume extends approximately 1 kilometer from the previous launch facilities beyond the southwestern end of the site. To limit further migration of TCE and chlorinated degradation by-products, an in situ, permeable, reactive bioremediation barrier (biobarrier) was designed as a cost-effective treatment technology to address the TCE plume emanating from the source area. The biobarrier treatment would involve injecting carbon-based substrate and microbes to achieve reductive dechlorination of volatile organic compounds, such as TCE. Under reducing conditions and in the presence of certain dechlorinating microorganisms, TCE degrades to nontoxic ethene in groundwater. To support the design of the full-scale biobarrier, a pilot test was conducted to evaluate site conditions and collect pertinent design data. The pilot test results indicated possible substrate delivery difficulties and a smaller radius of influence than had been estimated, which would be used to determine the final biobarrier well spacing. Based on these results, the full-scale biobarrier design was modified. In January 2010, the biobarrier was implemented at the toe of the source area by adding a fermentable substrate and a dechlorinating microbial culture to the subsurface via an injection well array that spanned the width of the TCE plume. After the injections, the groundwater pH in the injection wells continued to decrease to a level that could be detrimental to the population of Dehalococcoides in the SDC-9TM culture. In addition, 7 months postinjection, the injection wells could not be sampled due to fouling. Cleaning was required to restore their functions. Bioassay and polymerase chain reaction analyses were conducted, as well as titration tests, to assess the need for biobarrier amendments in response to the fouling issues and low pH. Additionally, slug tests were performed on three wells to evaluate possible localized differences in hydraulic conductivity within the biobarrier. Based on the test results, the biobarrier was amended with sodium carbonate and inoculated a second time with SDC-9TM. The aquifer pH was restored, and reductive dechlorination resumed in the treatment zone, evidenced by the reduction in TCE and the increase in degradation products, including ethene. © 2011 Wiley Periodicals, Inc.

A pilot field study was conducted for remediation of a tetrachloroethene (PCE)-contaminated site in Manhattan, Kansas. Prior to the pilot study, the PCE concentration in groundwater at the pilot-study area was about 15 mg/L (91 µM). Nutrient solution comprising soy oil methyl esters (SOME), lactate, and yeast extract was added in the pilot-study area for biostimulation on August 18, 2005 (day 0). Potassium bromide (KBr) was added in the nutrient solution as a tracer. PCE was converted to dichloroethene (DCE) under these conditions. KB-1, a consortium of Dehalococcoides, and a second dose of nutrient solution were added on day 56. After addition of KB-1, both PCE and DCE concentrations decreased. Nutrients were again injected on day 197 (with KBr). On day 348, cheese whey was injected. Soluble nutrients and KB-1 migrated downstream, affecting PCE and DCE concentrations at downgradient wells; however, KB-1 moved slower than the aqueous phase. The total chlorinated ethenes (CEs) decreased by about 80 percent in the pilot-study area due to bioremediation. Biodegradation of CEs continued for several months after the addition of nutrients. A portion of the insoluble SOME was retained in the vicinity of the injection wells and provided a long-time source of nutrients and electron donors that supported degradation of PCE. A mixture of SOME and soluble substrates worked effectively in this study. © 2011 Wiley Periodicals, Inc.

A field demonstration was performed to evaluate the impacts of bioaugmentation dosage for treatment of chlorinated ethenes in a sandy-to-silty shallow aquifer. Specifically, bioaugmentation using a commercially available Dehalococcoides (DHC)-containing culture was performed in three separate groundwater recirculation loops, with one loop bioaugmented with 3.9 × 10 11 DHC, the second loop bioaugmented with 3.9 × 10 12 DHC, and the third loop bioaugmented with 3.9 × 10 13 DHC. Groundwater monitoring was performed to evaluate DHC growth and migration, dechlorination rates, and aquifer geochemistry. The loop inoculated with 3.9 × 10 12 DHC showed slower dechlorination rates and DHC migration/growth compared with the other loops. This relatively poor performance was attributed to low pH conditions. Results for the loops inoculated with 3.9 × 10 11 and 3.9 × 10 13 DHC showed similar timeframes for dechlorination, as evaluated at a monitoring well approximately 10 feet downgradient of the DHC injection well. Application of a recently developed one-dimensional bioaugmentation fate and transport screening model provided a reasonable prediction of the data in these two loops. Overall, these results suggest that increasing bioaugmentation dosage does not necessarily result in decreased dechlorination timeframes in the field. The ability to predict results suggests that modeling potentially can serve as an effective tool for determining bioaugmentation dosage and predicting overall remedial timeframes.

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.

A complete set of mathematically identifiable and meaningful kinetic parameters estimates is needed to accurately describe the activity of individual populations that dehalorespire tetrachloroethene (PCE) and other chlorinated ethenes. These data may be difficult to extract from the literature because kinetic parameter estimates obtained using mixed cultures may reflect the activity of multiple dehalorespiring populations, while those obtained at low initial substrate-to-biomass ratios (S(0)/X(0)) are influenced by culture history and are generally not relevant to other systems. This study focused on estimation of electron donor and acceptor utilization kinetic parameters for the heterotrophic dehalorespirers Desulfuromonas michiganensis strain BB1 and Desulfitobacterium sp. strain PCE1. Electron acceptor utilization kinetic parameters that are identifiable and independent of culture history, i.e., intrinsic, could be estimated at S(0)/X(0) >or= 10, with both concentrations expressed as chemical oxygen demand (COD). However, the parameter estimates did not accurately describe dechlorination kinetics at lower S(0)/X(0) ratios. The maximum specific substrate utilization rates (q(max)) and half-saturation constants (K(S)) for PCE and trichloroethene (TCE) estimated for the two heterotrophic strains are higher than the values reported for Dehalococcoides cultures. These results suggest that the natural niche of Dehalococcoides strains that can metabolize a range of chlorinated ethenes may be to respire dichloroethene and vinyl chloride produced by Desulfuromonas and Desulfitobacterium strains or other populations that dechlorinate PCE and TCE at faster rates. Few data exist on the electron donor utilization kinetics of heterotrophic dehalorespirers. The results of this study suggest that Desulfuromonas and Desulfitobacterium strains should be able to compete for organic electron donors with other heterotrophic populations in the subsurface.

The fate and persistence of chlorinated organics in the environment have been a concern for the past 50 years. Industrialization and extensive agricultural activities have led to the accumulation of these pollutants in the environment, while their adverse impact on various ecosystems and human health also became evident. This review provides an update on the current knowledge of specialized anaerobic bacteria, namely 'Dehalococcoides' spp., which are dedicated to the transformation of various chlorinated organic compounds via reductive dechlorination. Advances in microbiology and molecular techniques shed light into the diversity and functioning of Dehalococcoides spp. in several different locations. Recent genome sequencing projects revealed a large number of genes that are potentially involved in reductive dechlorination. Molecular approaches towards analysis of diversity and expression especially of reductive dehalogenase-encoding genes are providing a growing body of knowledge on biodegradative pathways active in defined pure and mixed cultures as well as directly in the environment. Moreover, several successful field cases of bioremediation strengthen the notion of dedicated degraders such as Dehalococcoides spp. as key players in the restoration of contaminated environments

In situ bioremediation of industrial chlorinated solvents, such as trichloroethene (TCE), is typically accomplished by providing an organic electron donor to naturally occurring dechlorinating populations. In the present study, we show that TCE dechlorinating bacteria can access the electrons required for TCE dechlorination directly from a negatively polarized (-450 mV vs. SHE) carbon paper electrode. In replicated batch experiments, a mixed dechlorinating culture, also containing Dehalococcoides spp., dechlorinated TCE to cis-dichloroethene (cis-DCE) and lower amounts of vinyl chloride (VC) and ethene using the polarized electrode as the sole electron donor. Conversely, neither VC nor ethene formation occurred when a pure culture of the electro-active microorganism Geobacter lovleyi was used, under identical experimental conditions. Cyclic voltammetry tests, carried out on the filter-sterilized supernatant of the mixed culture revealed the presence of a self-produced redox mediator, exhibiting a midpoint potential of around -400 mV (vs. SHE). This yet unidentified redox-active molecule appeared to be involved in the extracellular electron transfer from the electrode to the dechlorinating bacteria. The ability of dechlorinating bacteria to use electrodes as electron donors opens new perspectives for the development of clean, versatile, and efficient bioremediation systems based on a controlled subsurface delivery of electrons in support of biodegradative metabolisms and provides further evidence on the possibility of using conductive materials to manipulate and control a range of microbial bioprocesses.

BACKGROUND: A bioelectrochemical process for trichloroethene (TCE) dechlorination was developed. In this new process, a solid-state electrode polarized to −450 mV versus the standard hydrogen electrode (SHE), in combination with a redox mediator (i.e., methyl viologen, MV) is employed as an electron donor for the microbial reductive dechlorination of TCE. In this study we compared the performance of the process with the redox mediator immobilized at the surface of electrodes or dissolved in the bulk liquid, using both a culture highly enriched in Desulfitobacterium spp., capable of dechlorinating TCE to cis-dichloroethene (cis-DCE), and a culture highly enriched in Dehalococcoides spp. capable of dechlorinating cis-DCE to ethene. RESULTS: Short-term potentiostatic (−450 mV versus SHE) experiments showed that TCE or cis-DCE was dechlorinated both in the presence of soluble (500 µmol L−1) and immobilized MV. However, TCE or cis-DCE dechlorination rates with MV-modified electrodes were remarkably lower than with soluble MV. Both cultures produced significant amounts of H2 in the presence of electrically reduced, soluble MV, whereas no H2 was produced when the mediator was immobilized at the electrode surface, regardless of the potential applied to the electrode, in the range −425 to −500 mV versus SHE. CONCLUSIONS: The process, operated with modified electrodes, supports the microbial dechlorination of TCE to ethene. Immobilization not only allows retention of the mediator within the system, but also increases process efficiency by preventing bioelectrochemical H2 formation. On the other hand, strategies to increase dechlorination rates with modified electrodes need to be developed. Copyright © 2009 Society of Chemical Industry

Summary Enrichment cultures capable of reductively dechlorinating 1,2,4-trichlorodibenzo-p-dioxin (1,2,4-TrCDD) were shown to dechlorinate 1,2,3-trichlorobenzene (1,2,3-TrCB) to 1,3-dichlorobenzene. To test if this activity can be used to enrich for dioxin-dechlorinating bacteria, a two-liquid phase cultivation with 200 mM 1,2,3-TrCB dissolved in hexadecane was established. During the dechlorination of 1,2,3-TrCB, the number of 1,2,4-TrCDD-dechlorinating bacteria increased by four orders of magnitude, eventually accounting for 11% of the total cell number. Characterization of the bacterial communities of the initial dioxin-dechlorinating culture and of the trichlorobenzene enrichments by restriction fragment length polymorphism (RFLP) analysis of cloned 16S rRNA genes revealed a proportional increase of nine different sequence types, one representing a Dehalococcoides strain. Inhibition of methanogens further enhanced the rate of chlorobenzene dehalogenation and also resulted in a rapid dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin that was applied via a hexadecane phase. The further enrichment was monitored by terminal RFLP, quantitative real-time PCR and microscopy, and aimed at the reduction of the accompanying non-dehalogenating populations by using different combinations of electron donors and the application of antibiotics. Hydrogen as the sole electron donor proved to be less efficient due to the co-enrichment of acetogens. The novel Dehalococcoides strain DCMB5 was enriched up to 50% by the cultivation with organic acids, hydrogen and vancomycin, and was finally purified by conventional isolation techniques.

Tetrachloroethene (PCE)- and trichloroethene (TCE)-impacted sites pose significant challenges even when site characterization activities indicate that biodegradation has occurred naturally. Although site-specific, regulatory, and economic factors play roles in the remedy-selection process, the application of molecular biological tools to the bioremediation field has streamlined the assessment of remedial alternatives and allowed for detailed evaluation of the chosen remedial technology. The case study described here was performed at a PCE-impacted site at which reductive dechlorination of PCE and TCE had led to accumulation of cis-dichlorethene (cis-DCE) with concentrations ranging from approximately 10 to 100 mg/L. Bio-Trap® samplers and quantitative polymerase chain reaction (qPCR) enumeration of Dehalococcoides spp. were used to evaluate three remedial options: monitored natural attenuation, biostimulation with HRC®, and biostimulation with HRC-S®. Dehalococcoides populations in HRC-S-amended Bio-Traps deployed in impacted wells were on the order of 103 to 104 cells/bead but were below detection limits in most unamended and HRC-amended Bio-Traps. Thus the in situ Bio-Trap study identified biostimulation with HRC-S as the recommended approach, which was further evaluated with a pilot study. After the pilot HRC-S injection, Dehalococcoides populations increased to 106 to 107 cells/bead, and concentrations of cis-DCE and vinyl chloride decreased with concurrent ethene production. Based on these results, a full-scale HRC-S injection was designed and implemented at the site. As with the pilot study, full-scale HRC-S injection promoted growth of Dehalococcoides spp. and stimulated reductive dechlorination of the daughter products cis-DCE and vinyl chloride. © 2008 Wiley Periodicals, Inc.

Competitive PCR and denaturing HPLC analyses together with an assay detecting potential polychlorinated biphenyl (PCB) dechlorinating activities were combined with physical-chemical site characterizations to identify factors affecting the reductive dechlorination of PCBs in the three historically impacted sediments: Grasse and Buffalo Rivers, NY and Anacostia River, DC. In Grasse River sediment an in situ enriched population of Dehalococcoides phylotypes was abundant in high numbers together with a relatively high dechlorination activity and a high concentration of congeners containing unflanked chlorine substitutions. In contrast microbial communities in Anacostia and Buffalo Rivers sediments consisted of similar total numbers of putative dechlorinating bacteria, but the populations consisted of more diverse putative dechlorinating phylotypes and were associated with lower dechlorination activities and higher concentrations of flanked congeners. Differences observed in the PCB dechlorination activity were not influenced by the chemical PCB availability in spiked sediment or physical sediment characteristics, but were consistent with the concentration of PCBs and total organic carbon in the native sediment. Application of molecular methods for selective detection of indigenous microbial dechlorinating communities combined with assessment of the dechlorinating activity and analysis of the in situ congener profiles provided a comprehensive approach for characterization and identification of sites that are amenable to bioremediation, which is essential for the development of in situ treatment strategies.

An in situ bioremediation (ISB) pilot study, using whey powder as an electron donor, is being performed at Site 19, Edwards Air Force Base, California, to treat groundwater contaminated with trichloroethene (TCE) via anaerobic reductive dechlorination. Challenging site features include a fractured granitic aquifer, complex geochemistry, and limited biological capacity for reductive dechlorination. ISB was conducted in two phases with Phase I including one-and-a-half years of biostimulation only using whey powder and Phase II including biostimulation with buffered whey powder and bioaugmentation. Results of Phase I demonstrated effective distribution of whey during injections resulting in depletion of high concentrations of sulfate and methanogenesis, but acid production due to whey fermentation and limited buffering capacity of the aquifer resulted in undesirable impacts to pH. In addition, cis-1,2-dichloroethene (cis-1,2-DCE) stall was observed, which correlated to the unsuccessful growth of native Dehalococcoides populations. Therefore, Phase II included the successful buffering of whey powder using bicarbonate, which mitigated negative pH effects. In addition, bioaugmentation resulted in successful transport of Dehalococcoides populations to greater than 50 feet away from the injection point four months after inoculation. A concomitant depletion of accumulated cis-1,2-DCE was observed at all wells affected by bioaugmented Dehalococcoides. © 2008 Wiley Periodicals, Inc.

The ability of bioremediation to treat a source area containing trichloroethene (TCE) present as dense nonaqueous phase liquid (DNAPL) was assessed through a laboratory study and a pilot test at Launch Complex 34, Cape Canaveral Air Force Center. The results of microcosm testing indicate that the indigenous microbial community was capable of dechlorinating TCE to ethene if amended with electron donor; however, bioaugmentation with a dechlorinating culture (KB-1; SiREM, Guelph, Ontario, Canada) significantly increased the rate of ethene formation. In microcosms, the activity of the dechlorinating organisms in KB-1 was not inhibited at initial TCE concentrations as high as 2 mM. The initially high TCE concentration in ground water (1.2 mM or 155 mg/L) did not inhibit reductive dechlorination, and at the end of the study, the average concentration of ethene (2.4 mM or 67 mg/L) was in stoichiometric excess of this initial TCE concentration. The production of ethene in stoichiometric excess in comparison to the initial TCE concentration indicates that the bioremediation treatment enhanced the removal of TCE mass (either sorbed to soil or present as DNAPL). Detailed soil sampling indicated that the bioremediation treatment removed greater than 98.5% of the initial TCE mass. Confirmatory ground water samples collected 22 months after the bioremediation treatment indicated that chloroethene concentrations had continued to decline in the absence of further electron donor addition. The results of this study confirm that dechlorination to ethene can proceed at the high TCE concentrations often encountered in source areas and that bioremediation was capable of removing significant TCE mass from the test plot, suggesting that enhanced bioremediation is a potentially viable remediation technology for TCE source areas. Dehalococcoides abundance increased by 2 orders of magnitude following biostimulation and bioaugmentation. Some figures in this paper are available in color in the online version of the paper.

An anaerobic microcosm set up with aquifer material from a 1,1,2,2-tetrachloroethane (TeCA) contaminated site and amended with butyrate showed a complete TeCA dechlorination to ethene. A structure analysis of the microbial community was performed by fluorescence in situ hybridization (FISH) with already available and on purpose designed probes from sequences retrieved through 16S rDNA clone library construction. FISH was chosen as identification tool to evaluate in situ whether the retrieved sequences belong to primary bacteria responsible for the biodegradative reactions. FISH probes identified up to 80% of total bacteria and revealed the absence or the marginal presence of known TeCA degraders and the abundance of two well-known H(2)-utilizing halorespiring bacteria, Sulfurospirillum (32.4 +/- 8.6% of total bacteria) and Dehalococcoides spp. (14.8 +/- 2.8), thereby providing a strong indication of their involvement in the dechlorination processes. These results were supported by the kinetic and thermodynamic analysis which provided indications that hydrogen was the actual electron donor for TeCA dechlorination. The specific probes, developed in this study, for known dechlorinators (i.e., Geobacter, Dehalobacter, and Sulfurospirillum species) represent a valuable tool for any future in situ bioremediation study as well as a quick and specific investigation tool for tracking their distribution in the field.

Compound specific isotope analysis (CSIA) was used to investigate biodegradation of trichloroethene (TCE) and methyl tert-butyl ether (MTBE) at contaminated field sites in Alaska and New York State, respectively. At both sites, geochemical conditions and the presence of metabolic intermediates (cis-1-2-dichloroethene and tert-butyl alcohol [TBA]) suggested the potential for biodegradation of TCE and MTBE, respectively. Given that in both cases these metabolic intermediates could also have been present as cocontaminants in the source zone, CSIA was undertaken to evaluate the possibility of in situ biodegradation. At the TCE-contaminated field site in Alaska, d 13 C values of TCE in ground water determined in this study showed no evidence of biodegradation (mean d 13 Co f� 27.0 6 1.0& for nine wells), and quantitative-polymerase chain reaction analyses of ground water from four wells found no evidence of dechlorinator Dehalococcoides sp. at this site. At the MTBE-contaminated field site in New York, TBA was present in the ground water but was not present in gasoline sampled from underground storage tanks (UST) on-site, suggesting that at this site, TBA was potentially a metabolite of MTBE biodegradation rather than a cocontaminant. However, at all sampling times and locations, d 13 C and d 2 H values of MTBE in ground water were within range of published values for undegraded MTBE in gasoline. While the occurrence of a small extent of in situ MTBE biodegradation cannot be ruled out, the findings suggest that it is more likely that multiple gasoline spills occurred through time, and while present day USTs do not contain TBA as a cocontaminant, gasoline spilled at the site in the past may have. At both contaminated field sites, CSIA, chemical, and microbiological lines of evidence suggest that biodegradation was not a significant attenuation process. The results of these two studies underscore the need for an integrated approach to site assessment that draws on measurements of metabolic intermediates, analysis of stable isotopes, and microbial evidence to give a reliable assessment of in situ biodegradation at contaminated field sites.

We conducted a field test to investigate whether ground water transfer from one site (showing complete natural reductive dechlorination of chlorinated ethenes to ethene) could induce full reductive dechlorination at another site polluted with tetrachloroethene and its partial dechlorination products trichloroethene and cis-dichloroethene (cDCE). Addition of electron donor (lactate) at the test site established low redox conditions but did not stimulate further dechlorination past cDCE. After transferring 2 m 3 of ground water from the first site to the test site, full dechlorination commenced and high levels of ethene were measured to distances up to 6 m downstream of the injection location within 7 months. Ground water samples from monitoring wells were analyzed before and after inoculation of the test site for the presence of Dehalococcoides species (16S ribosomal RNA) and vinyl chloride reductase (vCRA) genes using the polymerase chain reaction. These tests showed that Dehalococcoides species were present both before and after ground water transfer, while vCRA genes were detected at the test site only after ground water transfer. The vCRA genes were detected in ground water samples collected 6 m downstream of the injection locations 7 months after ground water transfer, suggesting that the microorganisms carrying the dehalogenase genes were effectively transported in the aquifer.

EOS, or emulsified oil substrate, was used to stimulate anaerobic biodegradation of trichloroethene (TCE) and tetrachloroethene (PCE) at a former Army-owned manufacturing facility located in the Piedmont area of North Carolina. Previous use of chlorinated solvents at the facility resulted in soil and groundwater impacts. Ten years of active remediation utilizing soil vacuum extraction and air sparging (SVE/AS) were largely ineffective in reducing the TCE/PCE plume. In 2002, the Army authorized preparation of an amended Remedial Action Plan (RAP) to evaluate in situ bioremediation methods to remediate TCE in groundwater. The RAP evaluated eight groundwater remediation technologies and recommended EOS as the preferred bioremediation alternative for the site. Eight wells were drilled within the 100 × 100 feet area believed to be the primary source area for the TCE plume. In a first injection phase, dilute EOS emulsion was injected into half of the wells. Distribution of the carbon substrate through the treatment zone was enhanced by pumping the four wells that were not injected and recirculating the extracted water through the injection wells. The process was repeated in a second phase that reversed the injection/extraction well pairs. Overall, 18,480 pounds of EOS were injected and 163,000 gallons of water were recirculated through the source area. Anaerobic groundwater conditions were observed shortly after injection with a corresponding decrease in both PCE and TCE concentrations. Dissolved oxygen, oxidation-reduction potential, and sulfate concentrations also decreased after injection, while TCE-degradation products, ferrous iron, and methane concentrations increased. The reduction in TCE allowed the Army to meet the groundwater remediation goals for the site. Approximately 18 months after injection, eight wells were innoculated with a commercially prepared dechlorinating culture (KB-1) in an attempt to address lingering cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC) that continued to be observed in some wells. Dehalococcoides populations increased slightly post-bioaugmentation. Both cis-DCE and VC continue to slowly decrease. © 2007 Wiley Periodicals, Inc.

The removal of pollutants from the environment has been declared a priority by a number of Environmental Protection Agencies (Roze et al., 2009). A great number of aerobic pathways have been deciphered and their relevance in microbiology and biotechnology has been reviewed several times (Garmendia et al., 2008; Siezen and Galardini, 2008; Govantes et al., 2008; Atlas and Bragg, 2009). In the area of biodegradation the role anaerobes and fungi play in removal of pollutants is of mounting interest. Microbial Biotechnology is publishing a number of new titles in this area, and here we have extracted some of the main conclusions. Tas and colleagues (2009) have dealt with mineralization of polychlorinated chemicals, which are harmful contaminants due to their persistence and their chronic toxicity to living organisms. Dehalococcoides spp. can anaerobically transform chlorinated xenobiotics to less‐ or even non‐noxious derivatives via reductive dechlorination. Tas and colleagues (2009) have reviewed the biology of this genus, focusing on its genetic peculiarities, its variability and, of course, its biodegradative properties. Dehalococcoides can replace chlorine by hydrogen atoms in recalcitrant halogenated compounds, using them as electron acceptors during anaerobic respiration. More than 100 16S rRNAs from environmental Dehalococcoides spp., are available, most of them corresponding to uncultured strains. In addition to the standard problems of cultivating anaerobic microbes, these coccoids usually grow in microbial communities where they can find a H2 supplier needed for thriving. The full genome sequences of several Dehalococcoides strains show that they have very small genomes which are highly similar. Moreover, they exhibit a large number of putative dehalogenase‐encoding genes (rdh), reaching up to 1.7% of the coding sequences in Dehalococcoides sp. Further work, combining transcriptional and proteomic techniques, will identify which proteins are really essential for the degradation of polychorinated xenobiotics. Jeon and colleagues (2009) also report in Microbial Biotechnology issues related with the attack of halogenated chemicals. They detail the discovery of four HAD (Halodehalogenases) defluorinases from different microbial genomes. Some of these dehalogenases have enhanced activities and this appears to arise from their sequence diversity (less than 30% sequence identity for HADs) (Prudnikova et al., 2009; Rye et al., 2009). The set of new dehalogenase were elucidated via biochemical characterization of 163 potential dehalogenases from the sequenced genomes of five common soil bacteria. Their discovery and characterization will be imperative to the future use of these enzymes in the biodegradation of halogenated chemicals. Another area of interest is the anaerobic degradation of monoaromatic compounds such as benzene, toluene, ethylbenzene and the xylene isomers (BTEX; Dou et al., 2008a; Wolicka et al., 2009). Anaerobic BTEX degradation has been shown to occur under denitrifying, sulfate‐reducing, iron‐reducing, manganese‐reducing and methanogenic conditions (Dou et al., 2008a,b; Barton and Fauque, 2009). These activities are of the relevance in removal of pollutants from contaminated aquifers and soils, and they are considered an important remediation strategy for hydrocarbon‐contaminated sites. New approaches based on isotopes are being taken, in fact, recently, compound‐specific isotope analysis was successfully used to distinguish between the effects of non‐degradative processes of mass loss such as sorption, volatilization, and dilution and those of biodegradation for aromatic hydrocarbons in the field (Fischer et al., 2008; Vogt et al., 2008). Compound‐specific isotope analysis is based on the fact that, in most chemical reactions, lighter isotopomers react faster than heavier ones, leading to a kinetic isotope effect. Herrmann and colleagues (2008) in Environmental Microbiology Reports, suggest that two‐dimensional isotope fractionation analyses are a valuable tool for identifying and monitoring anaerobic biodegradation of xylene isomers. They explored the carbon and hydrogen isotope fractionation of benzylsuccinate synthase (Bss)‐initiated degradation pathways for xylene isomers in order to obtain further information on the variability of isotope fractionation processes associated with Bss that might be important for the assessment of anaerobic degradation of xylene and toluene in the environment. The use of combined carbon and hydrogen isotope fractionation analyses may therefore be useful to monitor anaerobic xylene degradation at contaminated sites; this sort of technology will allow invaluable in situ monitoring of bioremediation processes.

The widespread global distribution of chlorinated hydrocarbons, their high lipophilicity and their recalcitrance have contributed to their importance as environmental toxicants. Their metabolism under oxic and anoxic conditions mediated by prokaryotic and eukaryotic cells is discussed. Various aerobic bacteria are able to use chlorinated hydrocarbons as the sole source of carbon and energy and some anaerobic bacteria can use some of these as an artificial electron acceptor in reductive dechlorination. Liver enzymes are responsible for the formation of hydrophilic metabolites ready for excretion which often lead to highly reactive and potentially toxic intermediates. Whereas fungi, especially ligninolytic ones, usually only exhibit ‘side activities’ for chlorinated hydrocarbons. The capabilities of bacteria had led to the development of various bioremediation processes. Both, successes and failures within these processes are known. Therefore, current research aims at a better understanding of global community interactions. Key Concepts: Chlorinated hydrocarbons have been produced by the chemical industry since nearly a decade in large amounts, but they can also be observed as natural compounds, sometimes exceeding the industrially produced amounts. Even though toxicological properties have pushed the chlorochemistry into the focus of considerable debate and governmental regulatory action, chlorinated hydrocarbons remain essential for certain applications. The aerobic metabolism of chlorinated hydrocarbons by bacteria has been studied in detail and an immense amount of information is available on pathways, enzymes and genes involved in the mineralisation of chloroaromatics. The anaerobic degradation of chlorinated hydrocarbons is due to the capablity of anaerobic bacteria to use them in anaerobic respiration, which results in dechlorination. Dehalococcoides organisms are the most versatile reductive dehalogenators as being capable to dehalogenate chlorinated dioxins, biphenyls, benzenes and vinyl chloride, among others. Microbial activities have been widespread used for bioremediation purposes through natural attenuation, biostimulation and bioaugmentation. The poor understanding of the functioning of the complex microbial activities in situ made bioremediation efforts quite unreliable. The rapid development of molecular techniques in recent years allows immense insights into the processes in situ, but also on the overall physiology of biocatalysts. Keywords: biodegradation; bioremediation; chlorochemistry; dechlorination; toxicology

Fractionation of stable carbon isotopes upon degradation of trichlorobenzenes was studied under aerobic and anaerobic conditions. Mineralization of 1,2,4-trichlorobenzene by the aerobic strain Pseudomonas sp. P51 which uses a dioxygenase for the initial enzymatic reaction was not accompanied by a significant isotope fractionation. In contrast, reductive dehalogenation by the anaerobic strain Dehalococcoides sp. strain CBDB1 revealed average isotope enrichment factors (ε) between -3.1 and -3.7 for 1,2,3- and 1,2,4-trichlorobenzene, respectively. The significant isotope fractionation during reductive dehalogenation would allow tracing the in situ biodegradation of halogenated benzenes in contaminated anoxic aquifers, whereas the lack of isotope fractionation during aerobic transformation limits the use of this approach in oxic environments. © 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.

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.