A Multifunctional Dehalobacter? Tandem Chloroform and Dichloromethane Degradation in a Mixed Microbial Culture.

Chloroform (CF) and dichloromethane (DCM) contaminate groundwater sites around the world but can be cleaned up through bioremediation. Although several strains of Dehalobacter restrictus can reduce CF to DCM and multiple Peptococcaceae can ferment DCM, these processes cannot typically happen simultaneously due to CF sensitivity in the known DCM-degraders or electron donor competition. Here, we present a mixed microbial culture that can simultaneously metabolize CF and DCM and create an additional enrichment culture fed only DCM. Through genus-specific quantitative polymerase chain reaction, we find that Dehalobacter grows while either CF alone or DCM alone is converted, indicating its involvement in both metabolic steps. Additionally, the culture was maintained for over 1400 days without the addition of an exogenous electron donor, and through electron balance calculations, we show that DCM metabolism would produce sufficient reducing equivalents (likely hydrogen) for CF respiration. Together, these results suggest intraspecies electron transfer could occur to continually reduce CF in the culture. Minimizing the addition of electron donor reduces the cost of bioremediation, and "self-feeding" could prolong bioremediation activity long after donor addition ends. Overall, understanding this mechanism informs strategies for culture maintenance and scale-up and benefits contaminated sites where the culture is employed for remediation worldwide.

Investigation of active site amino acid influence on carbon and chlorine isotope fractionation during reductive dechlorination.

Reductive dehalogenases (RDases) are corrinoid-dependent enzymes that reductively dehalogenate organohalides in respiratory processes. By comparing isotope effects in biotically catalyzed reactions to reference experiments with abiotic corrinoid catalysts, compound-specific isotope analysis (CSIA) has been shown to yield valuable insights into enzyme mechanisms and kinetics, including RDases. Here, we report isotopic fractionation (ε) during biotransformation of chloroform (CF) for carbon (εC = -1.52 ± 0.34‰) and chlorine (εCl = -1.84 ± 0.19‰), corresponding to a ΛC/Cl value of 1.13 ± 0.35. These results are highly suppressed compared to isotope effects observed both during CF biotransformation by another organism with a highly similar RDase (>95% sequence identity) at the amino acid level, and to those observed during abiotic dehalogenation of CF. Amino acid differences occur at four locations within the two different RDases' active sites, and this study examines whether these differences potentially affect the observed εC, εCl, and ΛC/Cl. Structural protein models approximating the locations of the residues elucidate possible controls on reaction mechanisms and/or substrate binding efficiency. These four locations are not conserved among other chloroalkane reducing RDases with high amino acid similarity (>90%), suggesting that these locations may be important in determining isotope fractionation within this homologous group of RDases.

Anaerobic Benzene Biodegradation Linked to the Growth of Highly Specific Bacterial Clades.

Reliance on bioremediation to remove benzene from anoxic environments has proven risky for decades but for unknown reasons. Research has revealed a strong link between anaerobic benzene biodegradation and the enrichment of highly specific microbes, including Thermincola in the family Peptococcaceae and the deltaproteobacterial Candidate Sva0485 clade. Using aquifer materials from Canadian Forces Base Borden, we compared five bioremediation approaches in batch microcosms. Under conditions simulating natural attenuation or sulfate biostimulation, benzene was not degraded after 1-2 years of incubation and no enrichment of known benzene-degrading microbes occurred. In contrast, nitrate-amended microcosms reported benzene biodegradation coincident with significant growth of Thermincola spp., along with a functional gene presumed to catalyze anaerobic benzene carboxylation (abcA). Inoculation with 2.5% of a methanogenic benzene-degrading consortium containing Sva0485 (Deltaproteobacteria ORM2) resulted in benzene biodegradation in the presence of sulfate or under methanogenic conditions. The presence of other hydrocarbon co-contaminants decreased the rates of benzene degradation by a factor of 2 to 4. Tracking the abundance of the abcA gene and 16S rRNA genes specific for benzene-degrading Thermincola and Sva0485 is recommended to monitor benzene bioremediation in anoxic groundwater systems to further uncover growth-rate-limiting conditions for these two intriguing phylotypes.

Transformation of Chlorofluorocarbons Investigated via Stable Carbon Compound-Specific Isotope Analysis.

Compound-specific isotope analysis (CSIA) is a valuable tool in contaminant remediation studies. Chlorofluorocarbons (CFCs) are ozone-depleting substances previously thought to be persistent in groundwater under most geochemical conditions but more recently have been found to (bio)transform in some laboratory experiments. To date, limited applications of CSIA to CFCs have been undertaken. Here, biotransformation-associated carbon isotope enrichment factors, εC,bulk for CFC-113 (εC,bulk = -8.5 ± 0.4‰) and CFC-11 (εC,bulk = -14.5 ± 1.9‰), were determined. δ13C signatures of pure-phase CFCs and hydrochlorofluorocarbons were measured to establish source signatures. These findings were applied to investigate potential in situ CFC transformation in groundwater at a field site, where carbon isotope fractionation of CFC-11 suggests naturally occurring biotransformation by indigenous microorganisms. The maximum extent of CFC-11 transformation is estimated to be up to 86% by an approximate calculation using the Rayleigh concept. CFC-113 δ13C values in contrast were not resolvably different from pure-phase sources measured to date, demonstrating that CSIA can aid in identifying which compounds may, or may not, be undergoing reactive processes at field sites. Science and public attention remains focused on CFCs, as unexplained source inputs to the atmosphere have been recently reported, and the potential for CFC biotransformation in surface and groundwaters remains unclear. This study proposes δ13C CSIA as a novel application to study the fate of CFCs in groundwater.

Chlorinated Electron Acceptor Abundance Drives Selection of Dehalococcoides mccartyi (D. mccartyi) Strains in Dechlorinating Enrichment Cultures and Groundwater Environments.

Dehalococcoides mccartyi (D. mccartyi) strains differ primarily from one another by the number and identity of the reductive dehalogenase homologous catalytic subunit A (rdhA) genes within their respective genomes. While multiple rdhA genes have been sequenced, the activity of the corresponding proteins has been identified in only a few cases. Examples include the enzymes whose substrates are groundwater contaminants such as trichloroethene (TCE), cis-dichloroethene (cDCE) and vinyl chloride (VC). The associated rdhA genes, namely tceA, bvcA, and vcrA, along with the D. mccartyi 16S rRNA gene are often used as biomarkers of growth in field samples. In this study, we monitored an additional 12 uncharacterized rdhA sequences identified in the metagenome in the mixed D. mccartyi-containing culture KB-1 to monitor population shifts in more detail. Quantitative PCR (qPCR) assays were developed for 15 D. mccartyi rdhA genes and used to measure population diversity in 11 different sub-cultures of KB-1, each enriched on different chlorinated ethenes and ethanes. The proportion of rdhA gene copies relative to D. mccartyi 16S rRNA gene copies revealed the presence of multiple distinct D. mccartyi strains in each culture, many more than the two strains inferred from 16S rRNA analysis. The specific electron acceptor amended to each culture had a major influence on the distribution of D. mccartyi strains and their associated rdhA genes. We also surveyed the abundance of rdhA genes in samples from two bioaugmented field sites (Canada and United Kingdom). Growth of the dominant D. mccartyi strain in KB-1 was detected at the United Kingdom site. At both field sites, the measurement of relative rdhA abundances revealed D. mccartyi population shifts over time as dechlorination progressed from TCE through cDCE to VC and ethene. These shifts indicate a selective pressure of the most abundant chlorinated electron acceptor, as was also observed in lab cultures. These results also suggest that reductive dechlorination at contaminated sites is brought about by multiple strains of D. mccartyi whether or not the site is bioaugmented. Understanding the driving forces behind D. mccartyi population selection and activity is improving predictability of remediation performance at chlorinated solvent contaminated sites.

Use of statistical tools to evaluate the reductive dechlorination of high levels of TCE in microcosm studies.

A large, multi-laboratory microcosm study was performed to select amendments for supporting reductive dechlorination of high levels of trichloroethylene (TCE) found at an industrial site in the United Kingdom (UK) containing dense non-aqueous phase liquid (DNAPL) TCE. The study was designed as a fractional factorial experiment involving 177 bottles distributed between four industrial laboratories and was used to assess the impact of six electron donors, bioaugmentation, addition of supplemental nutrients, and two TCE levels (0.57 and 1.90 mM or 75 and 250 mg/L in the aqueous phase) on TCE dechlorination. Performance was assessed based on the concentration changes of TCE and reductive dechlorination degradation products. The chemical data was evaluated using analysis of variance (ANOVA) and survival analysis techniques to determine both main effects and important interactions for all the experimental variables during the 203-day study. The statistically based design and analysis provided powerful tools that aided decision-making for field application of this technology. The analysis showed that emulsified vegetable oil (EVO), lactate, and methanol were the most effective electron donors, promoting rapid and complete dechlorination of TCE to ethene. Bioaugmentation and nutrient addition also had a statistically significant positive impact on TCE dechlorination. In addition, the microbial community was measured using phospholipid fatty acid analysis (PLFA) for quantification of total biomass and characterization of the community structure and quantitative polymerase chain reaction (qPCR) for enumeration of Dehalococcoides organisms (Dhc) and the vinyl chloride reductase (vcrA) gene. The highest increase in levels of total biomass and Dhc was observed in the EVO microcosms, which correlated well with the dechlorination results.

Pathway-dependent isotope fractionation during aerobic and anaerobic degradation of monochlorobenzene and 1,2,4-trichlorobenzene.

Stable carbon isotope fractionation is a valuable tool for monitoring natural attenuation and to establish the fate of groundwater contaminants. In this study, we measured carbon isotope fractionation during aerobic and anaerobic degradation of two chlorinated benzenes: monochlorobenzene (MCB) and 1,2,4-trichlorobenzene (1,2,4-TCB). MCB isotope fractionation was measured in anaerobic methanogenic microcosms, while 1,2,4-TCB isotope experiments were carried out in both aerobic and anaerobic microcosms. Large isotope fractionation was observed in both the anaerobic microcosm experiments. Enrichment factors (ε) for anaerobic reductive dechlorination of MCB and 1,2,4-TCB were -5.0‰ ± 0.2‰ and -3.0‰ ± 0.4‰, respectively. In contrast, no significant isotope fractionation was found during aerobic microbial degradation of 1,2,4-TCB. The cleavage of a C-Cl σ bond occurs during anaerobic reductive dechlorination of MCB and 1,2,4-TCB, while no σ bond cleavage is involved during aerobic degradation via dioxygenase. The difference in isotope fractionation for aerobic versus anaerobic biodegradation of MCB and 1,2,4-TCB can be explained by the difference in the initial step of aerobic versus anaerobic biodegradation pathways.

Chloroform respiration to dichloromethane by a Dehalobacter population.

Chloroform (CF), or trichloromethane, is an ubiquitous environmental pollutant because of its widespread industrial use, historically poor disposal and recalcitrance to biodegradation. Chloroform is a potent inhibitor of metabolism and no known organism uses it as a growth substrate. We discovered that CF was rapidly and sustainably dechlorinated in the course of investigating anaerobic reductive dechlorination of 1,1,1-trichloroethane in a Dehalobacter-containing culture. Like 1,1,1-trichloroethane dechlorination in this culture, CF dechlorination was a growth-linked respiratory process, requiring H(2) as an electron donor and CF as an electron acceptor. Moreover, the same specific reductive dehalogenase likely catalyzed both reactions. This Dehalobacter population appears specialized for substrates with three halogen substituents on the same carbon atom, with widespread implications for bioremediation.

Microbial oxidation of 1,2-dichloroethane under anoxic conditions with nitrate as electron acceptor in mixed and pure cultures.

Many organisms have been found to readily oxidize the prevalent contaminant 1,2-dichloroethane (1,2-DCA) to CO2 under aerobic conditions. Some organisms have also been isolated that can reduce 1,2-DCA to ethene via dihaloelimination under anaerobic, fermentative conditions. However, none have been described that can metabolize 1,2-DCA under anoxic, nitrate-reducing conditions. In microcosms prepared from aquifer material and groundwater samples from a contaminated site in eastern Louisiana, USA, 1,2-DCA was observed to degrade with nitrate as the terminal electron acceptor. Nitrate-dependent enrichment cultures were developed from these microcosms that sustained rapid 1,2-DCA degradation rates of up to 500 microM day(-1). This degradation was tightly coupled to complete reduction of nitrate via nitrite to nitrogen gas. A novel 1,2-DCA-degrading organism belonging to the Betaproteobacteria (affiliated with the genus Thauera) was isolated from this enrichment culture. However, degradation rates were much slower in cultures of the isolate than observed in the parent mixed culture. Complete mineralization of 1,2-DCA to CO2 was linked to cell growth and to nitrate reduction in both enrichment and isolated cultures. Monochloroacetate, a putative metabolite of 1,2-DCA degradation, could also be mineralized by these cultures.

Comparison of anaerobic dechlorinating enrichment cultures maintained on tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride.

An anaerobic mixed microbial culture was enriched from soil and groundwater taken from a site contaminated with trichloroethene (TCE). This enrichment culture was divided into four subcultures amended separately with either perchloroethene (PCE), TCE, cis-dichloroethene (cDCE) or vinyl chloride (VC). In each of the four subcultures, the chlorinated ethenes were rapidly, consistently, and completely converted to ethene at rates of 30-50 micromol/l of culture per day, or an average 160 micro-electron equivalents/l of culture per day. These cultures were capable of sustained and rapid dechlorination of VC, and could not dechlorinate 1,2-dichloroethane, differentiating them from Dehalococcoides ethenogenes, the only known isolate capable of complete dechlorination of PCE to ethene. Chloroform (CF) and 1,1,1-trichloroethane, frequent groundwater co-contaminants with TCE and PCE, inhibited chlorinated ethene dechlorination. Most strongly inhibited was the final conversion of VC to ethene, with complete inhibition occurring at an aqueous CF concentration of 2.5 microM. Differences in rates and community composition developed between the different subcultures, including the loss of the VC enrichment culture's ability to dechlorinate PCE. Denaturing gradient gel electrophoresis of amplified bacterial 16S rRNA gene fragments identified three different DNA sequences in the enrichment cultures, all phylogenetically related to D. ethenogenes. Based on the PCR-DGGE results and substrate utilization patterns, it is apparent that significant mechanistic differences exist between each step of dechlorination from TCE to ethene, especially for the last important dechlorination step from VC to ethene.

Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene.

A laboratory microcosm study and a pilot scale field test were conducted to evaluate biostimulation and bioaugmentation to dechlorinate tetrachloroethene (PCE) to ethene at Kelly Air Force Base. The site groundwater contained about 1 mg/L of PCE and lower amounts of trichloroethene (TCE) and cis-1,2-dichloroethene (cDCE). Laboratory microcosms inoculated with soil and groundwater from the site exhibited partial dechlorination of TCE to cDCE when amended with lactate or methanol. Following the addition of a dechlorinating enrichment culture, KB-1, the chlorinated ethenes in the microcosms were completely converted to ethene. The KB-1 culture is a natural dechlorinating microbial consortium that contains phylogenetic relatives of Dehalococcoides ethenogenes. The ability of KB-1 to stimulate biodegradation of chlorinated ethenes in situ was explored using a closed loop recirculation cell with a pore volume of approximately 64,000 L The pilot test area (PTA) groundwater was first amended with methanol and acetate to establish reducing conditions. Under these conditions, dechlorination of PCE to cDCE was observed. Thirteen liters of the KB-1 culture were then injected into the subsurface. Within 200 days, the concentrations of PCE, TCE, and cis-1,2-DCE within the PTA were all below 5 microg/L, and ethene production accounted for the observed mass loss. The maximum rates of dechlorination estimated from field date were rapid (half-lives of a few hours). Throughout the pilot test period, groundwater samples were assayed for the presence of Dehalococcoides using both a Dehalococcoides-specific PCR assay and 16S rDNA sequence information. The sequences detected in the PTA after bioaugmentation were specific to the Dehalococcoides species in the KB-1 culture. These sequences were observed to progressively increase in abundance and spread downgradient within the PTA. These results confirm that organisms in the KB-1 culture populated the PTA aquifer and contributed to the stimulation of dechlorination beyond cDCE to ethene.