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Resilience and Recovery of Dehalococcoides mccartyi Following Low pH Exposure
Abstract
Bioremediation treatment (e.g. biostimulation) can decrease groundwater pH with consequences for Dehalococcoides mccartyi (Dhc) reductive dechlorination activity. To explore the pH resilience of Dhc, the Dhc-containing consortium BDI was exposed to pH 5.5 for up to 40 days. Following 8- and 16-day exposure periods to pH 5.5, dechlorination activity and growth recovered when returned to pH 7.2; however, the ability of the culture to dechlorinate VC to ethene was impaired (i.e. decreased rate of VC transformation). Dhc cells exposed to pH 5.5 for 40 days did not recover the ethene-producing phenotype upon transfer to pH 7.2 even after 200 days of incubation. When returned to pH 7.2 conditions after an 8-, a 16- and a 40-day low pH exposure, tceA and vcrA genes showed distinct fold increases, suggesting Dhc strain-specific responses to low pH exposure. Furthermore, an independent survey of Dhc biomarker genes in groundwater samples revealed the average abundances of Dhc 16S rRNA, tceA and vcrA genes in pH 4.5-6 groundwater were significantly lower (p-value < 0.05) than in pH 6-8.3 groundwater. Overall, the results of the laboratory study and the assessment of field data demonstrate that sustained Dhc activity should not be expected in low pH groundwater, and the duration of low pH exposure affects the ability of Dhc to recover activity at circumneutral pH.
... 7−10 A particular challenge is sites with low pH groundwater because the known Dehalococcoidia do not grow under acidic pH conditions. 8,11 The pH of groundwater depends largely on the chemical composition of the local bedrock and soil, and moderately acidic pH (e.g., 5.0−6.0) is commonly observed. 12−15 Although microbial PCE and TCE reductive dechlorination to cis-1,2-dichloroethene at low pH has been demonstrated, sustained in situ reductive dechlorination to ethene in low pH groundwater has not been observed, presumably because organohalide-respiring Dehalococcoidia cannot grow at pH < 5.5. ...
... 12−15 Although microbial PCE and TCE reductive dechlorination to cis-1,2-dichloroethene at low pH has been demonstrated, sustained in situ reductive dechlorination to ethene in low pH groundwater has not been observed, presumably because organohalide-respiring Dehalococcoidia cannot grow at pH < 5.5. 8,9,11 Aggravating the pH problem is the release of protons during reductive dechlorination, which can contribute to further acidification. 16 Engineering approaches for buffering groundwater aquifers exist; however, the biogeochemical and hydrological complexity of in situ subsurface environments limits their implementation. ...
... The new findings indicate that the stimulation of acidophilic methanotrophs can achieve detoxification at low-pH VCcontaminated sites, where the reductive dechlorination process has stalled due to unfavorable conditions. 8,11 The enzymes catalyzing CH 4 to CH 3 OH oxidation and cometabolic oxidation of chlorinated ethenes in proteobacterial methanotrophs are pMMO and sMMO. 23 Copper is a key regulator of pMMO and sMMO expression in methanotrophs and has a crucial but not fully understood role in pMMO activation. ...
Remediation of toxic chlorinated ethenes via microbial reductive dechlorination can lead to ethene formation; however, the process stalls in acidic groundwater, leading to the accumulation of carcinogenic vinyl chloride (VC). This study explored the feasibility of cometabolic VC degradation by moderately acidophilic methanotrophs. Two novel isolates, Methylomonas sp. strain JS1 and Methylocystis sp. strain MJC1, were obtained from distinct alpine peat bogs located in South Korea. Both isolates cometabolized VC with CH 4 as the primary substrate under oxic conditions at pH at or below 5.5. VC cometabolism in axenic cultures occurred in the presence (10 μM) or absence (<0.01 μM) of copper, suggesting that VC removal had little dependence on copper availability, which regulates expression and activity of soluble and particulate methane monooxygenases in methanotrophs. The model neutrophilic methanotroph Methylosinus trichosporium strain OB3b also grew and cometabolized VC at pH 5.0 regardless of copper availability. Bioaugmentation of acidic peat soil slurries with methanotroph isolates demonstrated enhanced VC degradation and VC consumption below the maximum concentration level of 2 μg L −1. Community profiling of the microcosms suggested species-specific differences, indicating that robust bioaugmentation with methanotroph cultures requires further research.
... strain Viet1) were unable to dechlorinate PCE when concentrations exceeded 540 mM (Amos et al. 2007(Amos et al. , 2008, MRD of PCE near the aqueous solubility (C sat,PCE z 1200 mM) has been reported in column (Isalou et al., 1998), aquifer cell (C apiro et al., 2015), and pilot (Adamson et al., 2003) studies. The underlying reasons for these discrepancies are unclear, but geochemical conditions such as dissolved oxygen (Amos et al., 2008;Heavner et al., 2018), temperature (Bradley et al., 2005;Fletcher et al., 2011;Macbeth et al., 2012;Truex et al., 2007), pH (Yang et al. 2017a(Yang et al. , 2017b, contaminant concentration gradients (Amos et al., 2008;Behrens et al., 2008;Cope and Hughes, 2001;Sleep et al., 2006), or biofilms/microenvironments (Costerton et al., 1995;Lens et al., 1993) can have strainspecific impacts, leading to variability in microbial growth and dechlorination activity. ...
... The vcrA and bvcA genes are often monitored in groundwater via quantitative real-time polymerase chain reaction (qPCR) analysis because of their association with ethene formation (Holmes et al., 2006;Lee et al., 2008;Müller et al., 2004;Scheutz et al., 2008). Several studies have demonstrated that Dhc strains harboring the vcrA and bvcA genes appear to have different responses to geochemical conditions Heavner et al., 2018;van der Zaan et al., 2010;Yan et al., 2015;Yang et al. 2017aYang et al. , 2017b. For example, shifts in the relative abundance of Dhc strains occurred during ISTT at a site in Fort Lewis, WA, wherein heating from ambient temperature (~12 C) to 33 C caused a relative increase of the bvcA gene versus the vcrA gene (Macbeth et al., 2012). ...
... This apparent correlation between increased bvcA gene abundance and ethene production at elevated temperatures is consistent with data obtained at the field scale (Macbeth et al., 2012). These findings support the mounting volume of evidence demonstrating that, despite coding for enzymes with seemingly identical function (i.e., cis-DCE and VC reduction), Dhc strains carrying vcrA versus bvcA genes are not equivalent in terms of in situ dechlorination performance under different environmental conditions Heavner et al., 2018;van der Zaan et al., 2010;Yan et al., 2015;Yang et al. 2017aYang et al. , 2017b. The recognition that functionally equivalent (i.e., cis-DCE-to-ethene-dechlorinating) Dhc strains respond differently to environmental parameters is relevant, and suggests that further optimization of bioaugmentation consortia for site-specific treatment is possible. ...
Coupling in situ thermal treatment (ISTT) with microbial reductive dechlorination (MRD) has the potential to enhance contaminant degradation and reduce cleanup costs compared to conventional standalone remediation technologies. Impacts of low-temperature ISTT on Dehalococcoides mccartyi (Dhc), a relevant species in the anaerobic degradation of cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC) to nontoxic ethene, were assessed in sand-packed columns under dynamic flow conditions. Dissolved tetrachloroethene (PCE; 258 ± 46 μM) was introduced to identical columns bioaugmented with the PCE-to-ethene dechlorinating consortium KB-1®. Initial column temperatures represented a typical aquifer (15 °C) or a site undergoing low-temperature ISTT (35 °C), and were subsequently increased to 35 and 74 °C, respectively, to assess temperature impacts on reductive dechlorination activity. In the 15 °C column, PCE was transformed primarily to cis-DCE (159 ± 2 μM), which was further degraded to VC (164 ± 3 μM) and ethene (30 ± 0 μM) within 17 pore volumes (PVs) after the temperature was increased to 35 °C. Regardless of the initial column temperature, ethene constituted >50 mol% of effluent degradation products in both columns after 73-74 PVs at 35 °C, indicating that MRD performance was greatly improved under low-temperature ISTT conditions. Increasing the temperature of the column initially at 35 °C resulted in continued VC and ethene production until a temperature of approximately 43 °C was reached, at which point Dhc activity substantially decreased. The abundance of the vcrA reductive dehalogenase gene exceeded that of the bvcA gene by 1-2.5 orders of magnitude at 15 °C, but this relationship inversed at temperatures >35 °C, suggesting Dhc strain-specific responses to temperature. These findings demonstrate improved MRD performance with low-temperature thermal treatment and emphasize potential synergistic effects at sites undergoing ISTT.
... capable of Lee et al. 2006). Methanotrophs are also less vulnerable to adverse changes to environmental conditions than VC-respiring Dehalococcoides spp., which are easily inactivated by O 2 exposure or modest changes to pH or salinity (Amos et al. 2008;Islam et al. 2016;Matturro et al. 2016;Yang et al. 2017a;Ho et al. 2018). Methanotrophic degradation of chlorinated ethenes have been examined only at the neutral-pH conditions; however, diverse groups of acidophilic and halophilic methanotrophs harboring pMMO and/or sMMO have been isolated, suggesting that acidophilic or halophilic biological degradation of chlorinated ethenes, including VC, may be feasible (Kip et al. 2011;Semrau 2011). ...
... No VC-to-ethene reduction activity has been observed in acidic VC-to-ethene reduction or saline environments to date (Kittelmann and Friedrich 2008;Yang et al. 2017b). Reductive dehalogenation of chlorinated ethenes at pH 5.5 had resulted in permanent stoichiometric accumulation of VC in D. mccartyi cultures, suggesting complete inactivation of VC reductase and potential VC accumulation from PCE or TCE in contaminated acidic soil (Yang et al. 2017a). Biostimulation or, if necessary, bioaugmentation of acidophilic or halophilic methanotrophs may be a plausible alternative for removal of residual VC from such environments recalcitrant to the reductive dechlorination approach. ...
Methanotrophs are Microorganisms that are able to utilize Methane as the electron donor and carbon source. For long, Methanotrophs have been widely studied for their application in Environmental biotechnology, due mainly to the exclusive ownership of the unique Enzymes that mediate Oxidation of Methane to Methanol, namely the Particulate methane monooxygenases (pMMO) and soluble methane monooxygenases Soluble methane monooxygenase (sMMO). Utilizing these Methane monooxygenases, Methanotrophs are capable of Co-oxidizing a broad range of Organic pollutants including Chlorinated ethenes. Thus, Methanotrophs have long been studied and utilized as Biocatalysts for In situBioremediation of soil and aquatic environments contaminated with these Xenobiotic compounds. Due to the growing concerns in anthropogenically induced Climate change and Global warming, Methanotrophs have increasingly gained attention also for greenhouse gas mitigation purposes. Active Methane removal using MethanotrophicBiofilters of diverse configurations have proven to be effective for treatments of gases with relatively high Methane concentrations, e.g., landfill Landfill gases (LFG) and animal husbandry tank exhausts. Furthermore, improving the atmospheric Methane sink capability of agricultural soils has been one of the foremost foci of climate-smart Climate-smart soils research. This chapter provides an extensive overview of scientific and engineering breakthroughs geared towards practical applications of Methanotroph biotechnology in managing impending environmental problems.
... Hydrogenolysis of chloroethenes can decrease the pH of bicarbonate buffered medium from pH 7.1 to 4.9 (Adamson, Lyon and Hughes 2004). Low pH (< pH 6.0) has been shown to have a reversible deleterious effect on ORB (Yang et al. 2017a), and ORB exhibit differential tolerance to low pH. For example, in a tetrachloroethene (PCE) dehalogenating consortium, Sulfurospirillum populations dominated Dehalococcoides mccartyi populations at pH 5.0 and 5.5 while the latter were more abundant at pH 6.0 and 7.5 (Lacroix et al. 2014). ...
... Organohalide respiration is an acidification reaction that can decrease pH in aquifers with low-buffering capacity (Adamson, Lyon and Hughes 2004), changing the composition of microbial communities (Yang et al. 2017a;Yang et al. 2017b) and even impeding bioremediation. An acidotolerant Desulfitobacterium sp. ...
A Desulfitobacterium sp. strain AusDCA of the Peptococcaceae family capable of respiring 1,2-dichloroethane (1,2-DCA) to ethene anaerobically with ethanol or hydrogen as electron donor at pH 5.0 with optimal range between pH 6.5-7.5 was isolated from an acidic aquifer near Sydney, Australia. Strain AusDCA is distant (94% nucleotide identity) from its nearest phylogenetic neighbor, D. metallireducens, and could represent a new species. Reference gene-based quantification of growth indicated a doubling time of 2 days in cultures buffered at pH 7.2, and a yield of 7.66 (± 4.0) × 106 cells µmol-1 of 1,2-DCA. A putative 1,2-DCA reductive dehalogenase was translated from a dcaAB locus and had high amino acid identity (97.3% for DcaA and 100% for DcaB) to RdhA1B1 of the 1,2-DCA respiring Dehalobacter strain WL. Proteomic analysis confirmed DcaA expression in the pure culture. Dehalogenation of 1,2-DCA (1.6 mM) was observed in batch cultures established from groundwater at pH 5.5 collected 38 days after in situ bioaugmentation but not in cultures established with groundwater collected at the same time from wells not receiving bioaugmentation. Overall, strain AusDCA can tolerate lower pH than previously characterized organohalide respiring bacteria and remained viable in groundwater at pH 5.5.
... pH is another parameter impacting the microbial reductive dechlorination process. D. mccartyi strains perform reductive dechlorination between pH 6.5 and pH 8.0 (15), but sustained (i.e., growth-linked) organohalide respiration activity is not expected at pH values below 5.5 (56,57). Dehalogenimonas lykanthroporepellens strains BL-DC-9 T and BL-DC-8 and Dehalogenimonas alkenigignens strain IP3-3 T exhibited reductive dechlorination in the pH range of 6.0 to 8.0 but not at pH values of #5.5 or $8.5 (27,28,30). ...
Dehalococcoides mccartyi strains harboring vinyl chloride (VC) reductive dehalogenase (RDase) genes are keystone bacteria for VC detoxification in groundwater aquifers, and bioremediation monitoring regimens focus on D. mccartyi biomarkers. We isolated a novel anaerobic bacterium, "Candidatus Dehalogenimonas etheniformans" strain GP, capable of respiratory dechlorination of VC to ethene. This bacterium couples formate and hydrogen (H2) oxidation to the reduction of trichloro-ethene (TCE), all dichloroethene (DCE) isomers, and VC with acetate as the carbon source. Cultures that received formate and H2 consumed the two electron donors concomitantly at similar rates. A 16S rRNA gene-targeted quantitative PCR (qPCR) assay measured growth yields of (1.2 ± 0.2) × 108 and (1.9 ± 0.2) × 108 cells per μmol of VC dechlorinated in cultures with H2 or formate as electron donor, respectively. About 1.5-fold higher cell numbers were measured with qPCR targeting cerA, a single-copy gene encoding a putative VC RDase. A VC dechlorination rate of 215 ± 40 μmol L-1 day-1 was measured at 30°C, with about 25% of this activity occurring at 15°C. Increasing NaCl concentrations progressively impacted VC dechlorination rates, and dechlorination ceased at 15 g NaCl L-1. During growth with TCE, all DCE isomers were intermediates. Tetrachloroethene was not dechlorinated and inhibited dechlorination of other chlorinated ethenes. Carbon monoxide formed and accumulated as a metabolic by-product in dechlorinating cultures and impacted reductive dechlorination activity. The isolation of a new Dehalogenimonas species able to effectively dechlorinate toxic chlorinated ethenes to benign ethene expands our understanding of the reductive dechlorination process, with implications for bioremediation and environmental monitoring. IMPORTANCE Chlorinated ethenes are risk drivers at many contaminated sites, and current bioremediation efforts focus on organohalide-respiring Dehalococcoides mccartyi strains to achieve detoxification. We isolated and characterized the first non-Dehalococcoides bacterium, "Candidatus Dehalogenimonas etheniformans" strain GP, capable of metabolic reductive dechlorination of TCE, all DCE isomers, and VC to environmentally benign ethene. In addition to hydrogen, the new isolate utilizes formate as electron donor for reductive dechlorination, providing opportunities for more effective electron donor delivery to the contaminated subsurface. The discovery that a broader microbial diversity can achieve detoxification of toxic chlorinated ethenes in anoxic aquifers illustrates the potential of naturally occurring microbes for biotechnological applications.
... The optimum pH value for the growth of Dhc is around neutral (Chang et al., 2018). When groundwater pH was lower than 5.5, dechlorination of CEs by Dhc would be inhibited (Yang et al., 2017a(Yang et al., , 2017b. Thus, during the operational period, pH monitoring and adjustment would be a necessity to maintain an optimal CE dechlorinating efficiency. ...
In this study, the developed innovative immobilized Clostridium butyricum (ICB) (hydrogen-producing bacteria) column scheme was applied to cleanup chlorinated-ethene [mainly cis-1,2-dichloroethene (cis-DCE)] polluted groundwater in situ via the anaerobic reductive dechlorinating processes. The objectives were to assess the effectiveness of the field application of ICB scheme on the cleanup of cis-DCE polluted groundwater, and characterize changes of microbial communities after ICB application. Three remediation wells and two monitor wells were installed within the cis-DCE plume. In the remediation well, a 1.2-m PVC column (radius = 2.5 cm) (filled with ICB beads) and 20 L of slow polycolloid-releasing substrate (SPRS) were supplied for hydrogen production enhancement and primary carbon supply, respectively. Groundwater samples from remediation and monitor wells were analyzed periodically for cis-DCE and its degradation byproducts, microbial diversity, reductive dehalogenase, and geochemical indicators. Results reveal that cis-DCE was significantly decreased within the ICB and SPRS influence zone. In a remediation well with ICB injection, approximately 98.4% of cis-DCE removal (initial concentration = 1.46 mg/L) was observed with the production of ethene (end-product of cis-DCE dechlorination) after 56 days of system operation. Up to 0.72 mg/L of hydrogen was observed in remediation wells after 14 days of ICB and SPRS introduction, which corresponded with the increased population of Deha-lococcoides spp. (Dhc) (increased from 3.76 × 10 3 to 5.08 × 10 5 gene copies/L). Results of metagenomics analyses show that the SPRS and ICB introduction caused significant impacts on the bacterial communities, and increased Bacteroides, Citrobacter, and Desulfovibrio populations were observed, which had significant contributions to the reductive dechlorination of cis-DCE. Application of ICB could effectively result in increased populations of Dhc and RDase genes, which corresponded with improved dechlorination of cis-DCE and vinyl chloride. Introduction of ICB and SPRS could be applied as a potential in situ remedial option to enhance anaerobic dechlorination efficiencies of chlorinated ethenes.
... (3) The pH in the treated part of the aquifer is occasionally unsuitable for OHRB. Dehalococcoides degrade chlorinated ethenes within a narrow pH range of 6.5-8.0 (Löffler et al., 2013), and exposure to a pH of 5.5 for 40 days has been found to have a long-term inhibiting effect on the VC-to-ethene step (Yang et al., 2017b). Degradation of the higher chlorinated compounds appears less sensitive to acidic pH (Lacroix et al., 2014;Yang et al., 2017a). ...
Over the last decade, activated carbon amendments have successfully been applied to retain chlorinated ethene subsurface contamination. The concept of this remediation technology is that activated carbon and bioamendments are injected into aquifer systems to enhance biodegradation. While the scientific basis of the technology is established, there is a need for methods to characterise and quantify the biodegradation at field scale. In this study, an integrated approach was applied to assess in situ biodegradation after the establishment of a cross sectional treatment zone in a TCE plume. The amendments were liquid activated carbon, hydrogen release donors and a Dehalococcoides containing culture. The integrated approach included spatial and temporal evaluations on flow and transport, redox conditions, contaminant concentrations, biomarker abundance and compound-specific stable isotopes. This is the first study applying isotopic and microbial techniques to assess field scale biodegradation enhanced by liquid activated carbon and bioamendments. The injection enhanced biodegradation from TCE to primarily cis-DCE. The Dehalococcoides abundances facilitated characterisation of critical zones with insufficient degradation and possible explanations. A conceptual model of isotopic data together with distribution and transport information improved process understanding; the degradation of TCE was insufficient to counteract the contaminant input by inflow into the treatment zone and desorption from the sediment. The integrated approach could be used to document and characterise the in situ degradation, and the isotopic and microbial data provided process understanding that could not have been gathered from conventional monitoring tools. However, quantification of degradation through isotope data was restricted for TCE due to isotope masking effects. The combination of various monitoring tools, applied frequently at high-resolution, with system understanding, was essential for the assessment of biodegradation in the complex, non-stationary system. Furthermore, the investigations revealed prospects for future research, which should focus on monitoring contaminant fate and microbial distribution on the sediment and the activated carbon.
... B ioremediation of chlorinated contaminants in acidic groundwater is challenging because most organohalide-respiring bacteria cease growth under acidic conditions (1)(2)(3). Sulfurospirillum strains that grow via tetrachloroethene (PCE) reductive dechlorination under acidic conditions are candidates for bioremediation of chlorinated contaminants in low-pH groundwater (2,4). Sulfurospirillum sp. ...
Sulfurospirillum sp. strain ACS DCE couples growth with reductive dechlorination of tetrachloroethene to cis -1,2-dichloroethene at pH values as low as 5.5. The genome sequence of strain ACS DCE consists of a circular 2,737,849-bp chromosome and a 39,868-bp plasmid and carries 2,737 protein-coding sequences, including two reductive dehalogenase genes.
... However, carbon substrate addition would accelerate the anaerobic fermentation processes, which caused the production of fatty acids and decreased pH values in groundwater (Luo et al., 2019). The pH drop would also cause the negative impact on the efficiency of dechlorination (Yang et al., 2017). Results from other researches indicate the poly-γ-glutamic acid (γ-PGA) can be used as the carbon substrate to improve the anaerobic TCE dechlorination efficiency, and up to 99% of TCE could be removed after γ-PGA addition (Sheu et al., 2018). ...
Trichloroethylene (TCE) is a frequently found organic contaminant in polluted-groundwater. In this microcosm study, effects of hydrogen-producing bacteria [Clostridium butyricum (Clostridium sp.)] and inhibitor of sulfate-reducing bacteria (SRB) addition on the enhancement of TCE dechlorination were evaluated. Results indicate that Clostridium sp. supplement could effectively enhance TCE reductive dechlorination (97.4% of TCE removal) due to increased hydrogen concentration and Dehalococcoides (DHC) populations (increased to 1×10⁴ gene copies/L). However, addition of Clostridium sp. also caused the increase in dsrA (dissimilatory sulfide reductase subunit A) (increased to 2×10⁸ gene copies/L), and thus, part of the hydrogen was consumed by SRB, which would limit the effective application of hydrogen by DHC. Control of Clostridium sp. addition is a necessity to minimize the adverse impact of Clostridium sp. on DHC growth. Ferric citrate caused the slight raise of the oxidation-reduction state, which resulted in growth inhibition of SRB. Molybdate addition inhibited the growth of SRB, and thus, the dsrA concentrations (dropped from 4×10⁷ to 9×10⁵ gene copies/L) and sulfate reduction efficiency were decreased. Increased DHC populations (increased from 8×10³ to 1×10⁵ gene copies/L) were due to increased available hydrogen (increased from 0 to 2 mg/L), which enhanced TCE dechlorination (99.3% TCE removal). Metagenomic analyses show that a significant microbial diversity was detected in microcosms with different treatments. Clostridium sp., ferric citrate, and molybdate addition caused a decreased SRB communities and increased fatty acid production microbial communities (increased from 4.9% to 20.2%), which would be beneficial to the hydrogen production and TCE dechlorination processes.
... Furthermore, unsuitable environmental parameters, such as a low pH, dissolved O 2 or high redox values can negatively affect Dehalococcoides spp. [16][17][18][19]. ...
... pH changes, sulfide accumulation) often results in the accumulation of the more toxic intermediates, DCE and VC. [24][25][26] In this study, we investigated the effects of arsenic contamination on the dechlorination activity and gene expression in an axenic D. mccartyi strain 195 culture (Dhc195) in laboratory microcosms. To achieve this objective, we integrated results from physiological analyses, cell growth quantification, transcriptomic studies, and extracellular metabolomic analyses after perturbation by As(III) or As(V). ...
Arsenic and trichloroethene (TCE) are among the most prevalent groundwater contaminants in the United States. Co-contamination of these two compounds has been detected at 63% of current TCE-contaminated National Priorities List sites. When in situ TCE reductive dechlorination is stimulated by the addition of fermentable substrates to generate a reducing environment, the presence of arsenic can be problematic because of the potential for increased mobilization and toxicity caused by the reduction of arsenate [As(V)] to arsenite [As(III)]. This study assesses the effects of arsenic exposure on the TCE-dechlorinating activities of Dehalococcoides mccartyi strain 195. Our results indicate that 9.1 μM As(III) caused a 50% decrease in D. mccartyi cell growth. While As(V) concentrations up to 200 μM did not initially impact TCE dechlorination, inhibition was observed in cultures amended with 200 μM As(V) and 100 μM As(V) in 12 and 17 days, respectively, corresponding with the accumulation of As(III). Transcriptomic and metabolomic analyses were performed to evaluate cellular responses to both As(V) and As(III) stress. Amendment of amino acids enhanced arsenic tolerance of D. mccartyi. Results from this study improve our understanding of potential inhibitions of D. mccartyi metabolism caused by arsenic and can inform the design of bioremediation strategies at co-contaminated sites.
... Furthermore, the activity of Dhc is affected by the pH, with pH 6.8e7.8 being optimum (Middeldorp et al., 1999). Dhc appear to be inhibited for pH below 5 and above 10, with recovered activity when favorable pH conditions are reestablished (Rowlands, 2004), however, the duration of low pH exposure affects the ability of Dhc to recover activity at circumneutral pH (Yang et al., 2017). ...
Electrokinetics is being applied in combination with common insituremediation technologies, e.g. permeable reactive barriers, bioremediation and in-situ chemical oxidation, to overcome experienced limitations in remediation of chlorinated ethenes in low-permeable subsurface soils. The purpose of this review is to evaluate state-of-theart for identification of major knowledge gaps to obtain robust and successful field-implementations. Some of the major knowledge gaps include the behavior and influence
of induced transient changes in soil systems, transport velocities of chlorinated ethenes, and significance of site-specific parameters on transport velocities, e.g. heterogeneous soils and hydrogeochemistry. Furthermore, the various ways of reporting voltage distribution and transport rates complicate the comparison of transport velocities across studies. It was found, that for the combined EK-techniques, it is important to control the pH and redox changes caused by electrolysis for steady transport, uniform distribution of the electric field etc. Specifically for electrokinetically enhanced bioremediation, delivery
of lactate and biodegrading bacteria is of the same order of magnitude. This review shows that enhancement of remediation technologies can be achieved by electrokinetics, but major knowledge gaps must be examined to mature EK as robust methods for successful remediation of chlorinated ethene contaminated sites.
... The negative impact of low pH on reductive dechlorination has been well documented. [22][23][24][25][26]50 Although members of the genus Sulf urospirillum have been shown to reductively dechlorinate PCE and TCE to cis-DCE at low pH (pH 5.5), 26 this is the first time that growth of Dehalococcoides populations has been demonstrated at pH 5.5 after a rapid decrease from pH 7.0 to 5.5 (Table 2) and also after progressive decreases and long-term cultivation at pH 5.5 ( Figure 2). Thus, Dehalococcoides is likely a key player in the complete chlorinated ethene detoxification at field sites even when pH is below 6.0. ...
... No significant pH drop was observed in LC microcosms without SPRS addition. Yang et al. (2017) demonstrated that the activity of DHC could not be expected at low pH (<5.5). The observed pH values in microcosms were higher than 6 during the first half of the operation period, which were appropriate for the growth of Dehalococcoides spp. ...
Deteriorated environmental conditions during the bioremediation of trichloroethene (TCE)-polluted groundwater cause decreased treatment efficiencies. This study assessed the effect of applying immobilized Clostridium butyricum (a hydrogen-producing bacterium) in silica gel on enhancing the reductive dechlorination efficiency of TCE with the slow polycolloid-releasing substrate (SPRS) supplement in groundwater. The responses of microbial communities with the immobilized system (immobilized Clostridium butyricum and SPRS amendments) were also characterized by the metagenomics assay. A complete TCE removal in microcosms was obtained within 30 days with the application of this immobilized system via reductive dechlorination processes. An increase in the population of Dehalococcoides spp. was observed using the quantitative polymerase chain reaction (qPCR) analysis. Results of metagenomics assay reveal that the microbial communities in the immobilized system were distinct from those in systems with SPRS only. Bacterial communities associated with TCE biodegradation also increased in microcosms treated with the immobilized system. The immobilized system shows a great potential to promote the TCE dechlorination efficiency, and the metagenomics-based approach provides detailed insights into dechlorinating microbial community dynamics. The results would be helpful in designing an in situ immobilized system to enhance the bioremediation efficiency of TCE-contaminated groundwater.
... While Zhao, Ding and He (2016) describe the genome and an extrachromosomal element of the chloroethene dechlorinating strain 11a5, Dam et al. (2017) reconstructed the genomes of novel strains from 1,2,3,4-tetrachlorodibenzo-pdioxin dechlorinating enrichments. Yang et al. (2017) showed that Dehaloccoides mccartyi can survive low pH and that dechlorination is at least partially recovered at circum-neutral pH. The underlying reaction during dehalogenation of 1,2-dichloroethene by D. mccartyi strains 195 and BTF08 was shown to be a direct dichloroelemination to ethane by Franke et al. (2017), via a triple-element compoundspecific stable isotope analysis approach. ...
Many groundwater aquifers around the world are contaminated with trichloroethene (TCE), which can be harmful to human and ecosystem health. Permeable Reactive Barriers (PRB) are commonly used to remediate TCE-contaminated groundwaters especially when a point source is ill defined. Using biosolids from wastewater treatment plants as a PRB filling material can provide a source of carbon and nutrients for dechlorinating bacterial activity. However, under the anaerobic conditions of the PRB, methanogenesis can also occur which can adversely affect reductive dechlorination. We conducted bench scale experiments to evaluate the effect of biosolids on TCE reductive dechlorination and found that methanogenesis was significantly higher in the reactors amended with biosolids, but that reductive dechlorination did not decrease. Furthermore, the microbial communities in the biosolid-enhanced reactors were more abundant with obligate dechlorinators, such as Dehalobacter and Dehalogenimonas, than the reactors amended only with the dechlorinating culture. The biosolids enhanced the presence and abundance of methanogens and acetogens, which had a positive effect on maintaining an efficient dechlorinating microbial community and provided the necessary enzymes, cofactors, and electron donors. These results indicate that waste materials such as biosolids can be turned into a valuable resource for bioremediation of TCE and likely other contaminants.
Leveraging the capabilities of microorganisms to reduce (degrade or transform) concentrations of pollutants in soil and groundwater can be a cost-effective, natural remedial approach to manage contaminated sites. Traditional design and implementation of bioremediation strategies consist of lab-scale biodegradation studies or collection of field-scale geochemical data to infer associated biological processes. While both lab-scale biodegradation studies and field-scale geochemical data are useful for remedial decision-making, additional insights can be gained through the application of Molecular Biological Tools (MBTs) to directly measure contaminant-degrading microorganisms and associated bioremediation processes. Field-scale application of a standardized framework pairing MBTs with traditional contaminant and geochemical analyses was successfully performed at two contaminated sites. At a site with trichloroethene (TCE) impacted groundwater, framework application informed design of an enhanced bioremediation approach. Baseline abundances of 16S rRNA genes for a genus of obligate organohalide-respiring bacteria (i.e., Dehalococcoides) were measured at low abundances (101–102 cells/mL) within the TCE source and plume areas. In combination with geochemical analyses, these data suggested that intrinsic biodegradation (i.e., reductive dechlorination) may be occurring, but activities were limited by electron donor availability. The framework was utilized to support development of a full-scale enhanced bioremediation design (i.e., electron donor addition) and to monitor remedial performance. Additionally, the framework was applied at a second site with residual petroleum hydrocarbon (PHC) impacted soils and groundwater. MBTs, specifically qPCR and 16S gene amplicon rRNA sequencing, were used to characterize intrinsic bioremediation mechanisms. Functional genes associated with anaerobic biodegradation of diesel components (e.g., naphthyl-2-methyl-succinate synthase, naphthalene carboxylase, alkylsuccinate synthase, and benzoyl coenzyme A reductase) were measured to be 2–3 orders of magnitude greater than unimpacted, background samples. Intrinsic bioremediation mechanisms were determined to be sufficient to achieve groundwater remediation objectives. Nonetheless, the framework was further utilized to assess that an enhanced bioremediation could be a successful remedial alternative or complement to source area treatment. While bioremediation of chlorinated solvents, PHCs, and other contaminants has been demonstrated to successfully reduce environmental risk and reach site goals, the application of field-scale MBT data in combination with contaminant and geochemical data analyses to design, implement, and monitor a site-specific bioremediation approach can result in more consistent remedy effectiveness.
Trichloroethylene (TCE) was once a widely applied industrial solvent, but is now an infamous contaminant in groundwater. Although anaerobic reductive dechlorination is considered a greener remediation approach, the accumulation of toxic intermediates, such as vinyl chloride (VC), and a longer remediation period are highly concerning. Biostimulation and bioaugmentation have been developed to solve these problems. The former method may not be effective, and the latter may introduce foreign genes. Here, we propose a new approach by applying environmental stresses to reshape the indigenous microbiome. In this study, by using the Taguchi method, the effects of heating, pH, salinity, and desiccation were systematically examined. The optimum conditions were defined as 50 °C, pH 9, 3.50% salinity (w/v), and 21% volumetric water content (θW). The top performing group, G7, can complete the conversion of 11.81 mg/L TCE into ethene in 3.0 days with a 1.23% abundance of Dehalococcoides mccartyi 195 (Dhc 195). Redundancy analysis confirmed that temperature and salinity were the predominant factors in reorganizing the microbiomes. The microbiome structure and its effectiveness can last for at least 90 d. The repetitive selection conditions and sustainable degradation capability strongly supported that microbiome reengineering is feasible for the rapid bioremediation of TCE-contaminated environmental matrices.
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. 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).
Chloroethenes are common soil and groundwater pollutants. Their dechlorination is impacted by environmental factors, such as the presence of metal ions. We here investigated the effect of ferrous iron on bacterial reductive dechlorination of chloroethenes and on methanogen community. Reductive dechlorination of tetrachloroethene was assayed with a groundwater sample originally containing 6.3 × 103 copies mL–1 of Dehalococcoides 16S rRNA gene and 2 mg L–1 of iron. Supplementation with 28 mg L–1 of ferrous iron enhanced the reductive dechlorination of cis-dichloroethene (cis-DCE) and vinyl chloride in the presence of methanogens. The supplementation shortened the time required for complete dechlorination of 1 mg L–1 of tetrachloroethene to ethene and ethane from 84 to 49 d. Methanogens, such as Candidatus ‘Methanogranum’, Methanomethylovorans and Methanocorpusculum, were significantly more abundant in iron-supplemented cultures than in non-supplemented cultures (P<<0.01i>). Upon methanogen growth inhibition by 2-bromoethanesulfonate and in the absence of iron supplementation, cis-DCE was not dechlorinated. Further, iron supplementation induced 71.3% dechlorination of cis-DCE accompanied by an increase in Dehalococcoides 16S rRNA and dehalogenase vcrA gene copies but not dehalogenase tceA gene copies. These observations highlight the cooperative effect of iron and methanogens on the reductive dechlorination of chloroethenes by Dehalococcoides spp.
In this study, the modified γ-poly-glutamic acid (m-PGA) was developed for continuous substrate supplement and pH control to bioremediate trichloroethene (TCE)-contaminated groundwater. The m-PGA was prepared by mixing γ-PGA and a stabilizer (a more sticky carbon source) [carboxymethyl cellulose, gelatin, starch, or emulsified colloidal substrate (ES)] together, which was used as a source of slow-release carbon substrate. Microcosm experiments were conducted to determine the feasibility of m-PGA for a long-term substrate releasing to enhance anaerobic TCE dechlorination. The globule diameter, viscosity, stability, adsorption and retardation effect, and zeta potential of γ-PGA mixtures were assessed. The mixture of γ-PGA and ES (volume ratio of 1:0.25) (m-PGA) had the most stable, biodegradable, long-lasting, TCE adsorption, and dispersive characteristics with a negative zeta potential (−132 mv) and uniform droplet. Up to 99% of TCE removal was achieved in microcosms with m-PGA supplement. M-PGA addition could result in an immediate TCE removal via physical adsorption and subsequent dechlorination mechanisms. In the pilot-scale study, 20 L of m-PGA was injected into an injection well to enhance TCE dechlorination. After 55 days of m-PGA injection, about 96% of TCE could be dechlorinated without byproduct accumulation. Increased ammonia concentrations were observed in microcosm and pilot-scale studies via the hydrolysis of amine from m-PGA, which could neutralize acidified groundwater. Results from the next generation sequencing indicate that strains with functions of reductive dechlorination, carbon biodegradation, B12 synthesis, and hydrogen production were detected after m-PGA injection.
Diffusion, sorption-desorption, and biodegradation influence chlorinated solvent storage in, and release (mass flux) from, low-permeability media. Although bioenhanced dissolution of non-aqueous phase liquids has been well-documented, less attention has been directed towards biologically-mediated enhanced diffusion from low-permeability media. This latter process was investigated using a heterogeneous aquifer cell, packed with 20–30 mesh Ottawa sand and lenses of varying permeability (1.0 × 10⁻¹²-1.2 × 10⁻¹¹ m²) and organic carbon (OC) content (<0.1–2%), underlain by trichloroethene (TCE)-saturated clay. Initial contaminant loading was attained by flushing with 0.5 mM TCE. Total chlorinated ethene removal by hydraulic flushing was then compared for abiotic and bioaugmented systems (KB-1® SIREM; Guelph, ON). A numerical model incorporating coupled diffusion and (de)sorption, facilitated quantification of bio-enhanced TCE release from low-permeability lenses, which ranged from 6 to 53%. Although Dehalococcoides mccartyi (Dhc) 16S rRNA genes were uniformly distributed throughout the porous media, strain-specific distribution, as indicated by the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA, was influenced by physical and chemical heterogeneity. Cells harboring bvcA comprised 44% of the total RDase genes in the lower clay layer and media surrounding high OC lenses, but only 2% of RDase genes at other locations. Conversely, cells harboring the vcrA gene comprised 50% of RDase genes in low-permeability media compared with 85% at other locations. These results demonstrate the influence of microbial processes on back diffusion, which was most evident in regions with pronounced contrasts in permeability and OC content. Bioenhanced mass transfer and changes in the relative abundance of Dhc strains likely impact bioremediation performance in heterogeneous systems.
Chlorofluorocarbons including 1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113) often occur in groundwater plumes co-mingled with chlorinated solvents such as trichloroethene (TCE). We show that CFC-113 inhibits reductive dechlorination by Dehalococcoides mccartyi (Dhc) in a concentration-dependent manner, causing cis-1,2-dichloroethene (cis-DCE) stalls. Following a 17-day exposure of Dhc-containing consortium SDC-9 to 76 µM CFC-113, cis-DCE dechlorination activity did not recover after CFC-113 removal. River sediment microcosms demonstrated that CFC-113 was subject to microbial degradation under anoxic conditions and chlorotrifluoroethene (CTFE) was observed as a transformation product. No degradation of CFC-113 was observed in killed controls and in incubations with reactive minerals including mackinawite, green rust, magnetite and manganese dioxide. In vitro experiments with reduced corrinoid (i.e., vitamin B12) mediated reductive dechlorination of CFC-113 to CTFE and trifluoroethene (TFE) followed by reductive defluorination of TFE to cis-1,2-difluoroethene (cis-DFE) as an end product. This biomimetic degradation of CFC-113 to cis-DFE was also demonstrated in vivo using the corrinoid-producing homoacetogen Sporomusa ovata, suggesting the co-metabolic microbial reductive dechlorination and reductive defluorination of CFC-113 to cis-DFE is feasible under anoxic in situ conditions. The CFC-113 degradation intermediates CTFE, TFE, and cis-DFE did not inhibit TCE dechlorination by Dhc, indicating that the initial reductive transformation step can overcome cis-DCE stalls.
The effectiveness of using gamma poly-glutamic acid (γ-PGA) as the primary carbon and nitrogen sources to bioremediate trichloroethene (TCE)-contaminated groundwater was studied in this pilot-scale study. γ-PGA (40 L) solution was injected into the aquifer via the injection well (IW) for substrate supplement. Groundwater samples were collected from monitor wells and IW and analyzed for TCE and its byproducts, geochemical indicators, dechlorinating bacteria, and microbial diversity periodically. Injected γ-PGA resulted in an increase in total organic carbon (TOC) (up to 9820 mg/L in IW), and the TOC biodegradation caused the formation of anaerobic conditions. Increased ammonia concentration (because of amine release from γ-PGA) resulted in the neutral condition in groundwater, which benefited the growth of Dehalococcoides. The negative zeta potential and micro-scale diameter of γ-PGA allowed its globule to distribute evenly within soil pores. Up to 93% of TCE removal was observed (TCE dropped from 0.14 to 0.01 mg/L) after 59 days of γ-PGA injection, and TCE dechlorination byproducts were also biodegraded subsequently. Next generation sequence (NGS) analyses were applied to determine the dominant bacterial communities. γ-PGA supplement developed reductive dechlorinating conditions and caused variations in microbial diversity and dominant bacterial species. The dominant four groups of bacterial communities including dechlorinating bacteria, vinyl chloride degrading bacteria, hydrogen producing bacteria, and carbon biodegrading bacteria.
Trichloroethene (TCE) is a ubiquitous groundwater pollutant. Successful TCE bioremediation has been demonstrated at field sites using specialized microbial consortia harboring TCE-respiring Dehaloccocoides whose growth is cobalamin (vitamin B12)-dependent. Bioaugmentation cultures grown ex situ with ample exogenous vitamins in the medium and at neutral pH may become vitamin-limited or inhibited by acidic pH once injected into field sites, resulting in incomplete TCE dechlorination and accumulation of more toxic vinyl chloride (VC). Here, we report growth of the Dehalococcoides -containing bioaugmentation culture KB-1 in a TCE-amended mineral medium devoid of vitamins and in a VC-amended mineral medium at low pH (6.0 and 5.5). In cultures grown without exogenous vitamins or cobalamin, Acetobacterium , which can synthesize 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, and Sporomusa are the dominant acetogens. At neutral pH, a growing Acetobacterium population supports complete TCE dechlorination by Dehalococcoides at millimolar levels with a substantial increase in the amount of measured cobalamin (̴ 20-fold). Sustainable dechlorination of VC to ethene was achieved at a pH as low as 5.5, yet at low pH Acetobacterium is less abundant, potentially affecting the production of DMB and/or cobalamin. However, dechlorination activity at very low pH (< 5.0) was not stimulated by DMB supplementation, but was restored by raising pH to neutral. Assays in cell extracts revealed that vinyl chloride reductase (VcrA) activity declines significantly below pH 6.0 and is undetectable below pH 5.0. This study highlights the roles of and interplay between vitamin-producing populations and pH in microbial dechlorinating communities, and their importance for successful chlorinated ethenes bioremediation at field sites.
The degradation mechanism of the pollutant hexachloroethane (HCA) by a suspension of Pd-doped zerovalent iron microparticles (Pd-mZVI) in dissolved lactic acid polymers and oligomers (referred to as PLA) was investigated using gas chromatography and the indirect monitoring of iron corrosion by continuous measurements of pH, oxidation-reduction potential (ORP), and conductivity. The first experiments took place in the absence of HCA, to understand the evolution of the Pd-mZVI/PLA/H2O system. This showed that the evolution of pH, ORP, and conductivity is related to changes in solution chemistry due to iron corrosion and that the system is initially cathodically controlled by H⁺ mass transport to Pd surfaces because of the presence of an extensive PLA layer. We then investigated the effects of Pd-mZVI particles, temperature, initial HCA concentration, and PLA content on the Pd-mZVI/PLA/HCA/H2O system, to obtain a better understanding of the degradation mechanism. In all cases, HCA dechlorination first requires the production of atomic hydrogen H*—involving the accumulation of tetrachloroethylene (PCE) as an intermediate—before its subsequent reduction to non-chlorinated C2 and C4 compounds. The ratio between Pd-mZVI dosage, initial HCA concentration, and PLA content affects the rate of H* generation as well as the rate-determining step of the process. A pseudo-first-order equation can be applied when Pd-mZVI dosage is much higher than the theoretical stoichiometry (600 mg for [HCA]0 = 5–20 mg L⁻¹). Our results indicate that the HCA degradation mechanism includes mass transfer, sorption, surface reaction with H*, and desorption of the product.
Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within
their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an
approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel
slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry
(LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1,
BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but
also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified
chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three
enzymes and provides a path forward to further explore the largely unknown RDase protein family.
Six obligate anaerobic bacterial isolates (195, CBDB1, BAV1, VS, FL2 and GT) with strictly organohalide-respiring metabolisms were isolated from chlorinated solvent-contaminated aquifers, contaminated and uncontaminated river sediments or anoxic digester sludge. Cells were non-motile with a disc-shaped morphology of 0.3 to 1 μm in diameter, a thickness of 0.1-0.2 μm, and characteristic indentations on opposite flat sides of the cell. Growth occurred in completely synthetic, reduced medium amended with a haloorganic electron acceptor (i.e., mostly chlorinated but also some brominated compounds), hydrogen as electron donor, acetate as carbon source, and vitamins. No other growth-supporting redox couples were identified. Aqueous hydrogen consumption threshold concentrations were <1 nM. Growth ceased when vitamin B12 was omitted from the medium. Addition of sterile cell-free supernatant of Dehalococcoides-containing enrichment cultures enhanced dechlorination and growth of strains 195 and FL2, suggesting the existence of so far unidentified stimulants. Dechlorination occurred between pH 6.5 and 8.0 and over a temperature range of 15 to 35°C, with an optimum growth temperature between 25 and 30°C. The major PLFA were 14:0 (15.7 mol%), br15:0 (6.2 mol%), 16:0 (22.7 mol%), 10Me16:0 (25.8 mol%), and 18:0 (16.6 mol%). Unusual furan fatty acids including 9-(5-pentyl-2-furyl)-nonanoate (Fu18:2ω6) and 8-(5-hexyl-2-furyl)-octanoate (Fu18:2ω6) were detected in strains FL2, BAV1 and GT, but not in strains 195 and CBDB1. The 16S rRNA gene sequences of the six isolates shared greater than 98% identity, and phylogenetic analysis revealed an affiliation with the Chloroflexi phylum and a greater than 10% sequence divergence from other described isolates. The genome sizes and G+C contents ranged from 1.34 to 1.47 Mbp and 47 to 48.9 mol% G+C, respectively. Based on 16S rRNA gene sequence comparisons, genome-wide average nucleotide identity (ANI) and phenotypic characteristics, the organohalide-respiring isolates represent a new genus, for which the name Dehalococcoides gen. nov. is proposed. Isolates BAV1 (ATCC BAA-2100 = JCM 16839 = KCTC 5957), FL2 (ATCC BAA-2098 = DSM 23585 = JCM 16840 = KCTC 5959), GT (ATCC BAA-2099 = JCM 16841 = KCTC 5958), CBDB1, 195 (ATCC BAA-2266 = KCTC 15142), and VS are considered strains of the novel species Dehalococcoides mccartyi gen. nov., sp. nov. Dehalococcoides mccartyi sp. nov. strain 195T is designated as the type species of the genus.
This study sought to characterize bacterial and archaeal populations in a perchloroethene- and butyrate-fed enrichment culture containing hydrogen-consuming "Dehalococcoides ethenogenes" strain 195 and a Methanospirillum hungatei strain. Phylogenetic characterization of this microbial community was done via 16S rRNA gene clone library and gradient gel electrophoresis analyses. Fluorescence in situ hybridization was used to quantify populations of "Dehalococcoides" and Archaea and to examine the colocalization of these two groups within culture bioflocs. A technique for enrichment of planktonic and biofloc-associated biomass was developed and used to assess differences in population distribution and gene expression patterns following provision of substrate. On a per-milliliter-of-culture basis, most D. ethenogenes genes (the hydrogenase gene hupL; the highly expressed gene for an oxidoreductase of unknown function, fdhA; the RNA polymerase subunit gene rpoB; and the 16S rRNA gene) showed no statistical difference in expression between planktonic and biofloc enrichments at either time point studied (1 to 2 and 6 h postfeeding). Normalization of transcripts to ribosome (16S rRNA) levels supported that planktonic and biofloc-associated D. ethenogenes had similar gene expression profiles, with one notable exception; planktonic D. ethenogenes showed higher expression of tceA relative to biofloc-associated cells at 6 h postfeeding. These trends were compared to those for the hydrogen-consuming methanogen in the culture, M. hungatei. The vast majority of M. hungatei cells, ribosomes (16S rRNA), and transcripts of the hydrogenase gene mvrD and the housekeeping gene rpoE were observed in the biofloc enrichments. This suggests that, unlike the comparable activity of D. ethenogenes from both enrichments, planktonic M. hungatei is responsible for only a small fraction of the hydrogenotrophic methanogenesis in this culture.
Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated
ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring Dehalococcoides sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene
(TCE), at high rates. By using reversed genetics, the corresponding gene (vcrA) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family
of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The vcrA gene was found to be cotranscribed with vcrB, encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and vcrC, encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The vcrAB genes were subsequently found to be present and expressed in other cultures containing VC-respiring Dehalococcoides organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the
vcrA gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation.
Gastrointestinal pathogens are faced with an extremely acidic environment. Within moments, a pathogen such as Escherichia coli O157:H7 can move from the nurturing pH 7 environment of a hamburger to the harsh pH 2 milieu of the stomach. Surprisingly, certain microorganisms that grow at neutral pH have elegantly regulated systems that enable survival during excursions into acidic environments. The best-characterized acid-resistance system is found in E. coli.
The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different
Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and
bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20
and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples
from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less
than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.
Remediation of chlorinated solvent source zones is very difficult, at times controversial, and must be based on state-of-the-art knowledge of the behavior of nonaqueous phase liquids in the subsurface, as well as site specific geology, chemistry, biology and hydrogeology.
This volume begins with an overview of the current state-of-the-practice that serves as an introduction to the rest of the book. The second chapter summarizes the challenges involved in source zone remediation, which has been and remains contentious, expensive and difficult, for a variety of reasons. Following are chapters providing more focused discussions of specific aspects of this overall challenge:
· Two chapters on source zone characterization, the first summarizing the current issues and techniques, and the second focusing on several innovative diagnostic methods.
· Two chapters on modeling, the first focused on modeling source zone remediation itself, and the second focused on the responses of downgradient plumes to source remediation.
· A chapter on the use of mass flux and mass discharge information to improve source zone management and remediation.
· A series of chapters on specific source zone remediation methods, including hydraulic displacement and recovery, in situ chemical oxidation (ISCO), in situ chemical reduction (ISCR), enhanced flushing with cosolvents and surfactants, in situ bioremediation, and finally source zone monitored natural attenuation. · A chapter on combined remedies, discussing the fundamental issues involved in developing effective combined remedies.
· A chapter on the costs of source zone treatment, using several hypothetical site scenarios, comparing different technologies on a total and net present cost basis.
The last two chapters consider the future of source zone remediation, beginning with a discussion of alternate management strategies, followed by a summary of the research and development needed to improve the state of the practice.
Each chapter in this volume has been reviewed for technical content by two or more experts in each subject area covered. This volume will provide a useful reference for practitioners, researchers and students involved in remediation of chlorinated solvent source zones.
In Situ Remediation of Chlorinated Solvent Plumes
H.F. Stroo & C.H. Ward
Editors
This volume presents a critical analysis and timely synthesis of the past two decades of intensive research, development and demonstrations on the in situ remediation of chlorinated solvent plumes. The intended audiences include the decision makers, practicing engineers and hydrogeologists who will select, design and operate these remedial systems, as well as the researchers seeking to improve the current state of the science and technology. Our hope is that this volume will serve as a useful resource to assist remediation professionals in applying and developing these technologies as effectively as possible.
Topics addressed in this volume include:
• An overview of the current state of understanding of chlorinated solvents remediation.
• A summary of the chemistry of solvents, the physical, chemical and biological processes that underpin the most frequently employed remediation technologies, and the engineering and implementation issues that influence their efficacy.
• A discussion of site characterization, source zone and plume management, and modeling strategies and tools.
• An analysis of the advantages, performance and relative costs of a range of remedial technologies, including bioremediation technologies such as monitored natural attenuation, biostimulation and bioaugmentation for anaerobic degradation of chlorinated solvents; and physical-chemical technologies such as air sparging, chemical oxidation and reduction, barrier walls, and in-well treatment.
• A cost assessment of the most frequently used technologies, with case studies of several template sites and analyses of the capital costs, as well as costs for laboratory testing, pilot-scale demonstration, design, system operation, monitoring and maintenance during operations, and demolition and restoration after remediation. In addition, analogous cost data are presented for pump-and-treat systems for each template site to illustrate the potential cost savings associated with the use of alternative approaches.
• A summary of emerging technologies such as phytoremediation and electrolytic reactive barriers to illustrate their current stage of maturity and the potential applicability of these approaches for specific situations.
• An assessment of the research needed to more cost-effectively address what remains of a multi-billion dollar legacy environmental contamination problem.
Each chapter in this volume has been thoroughly reviewed for technical content by one or more experts in each of the subject areas covered. The editors and chapter authors have produced a well-written and up-to-date treatise that we hope will be a useful reference for decision-makers, practitioners and developers of advanced technologies for in situ remediation of chlorinated solvent plumes.
This volume reviews the past 10 to 15 years of intensive research and development that have led to bioaugmentation becoming an accepted technology. It includes background information on the basic microbial processes involved, as well as a thorough summary of the most important bioaugmentation strategies. It will serve as a valuable resource for environmental remediation professionals who seek to understand, evaluate and implement bioaugmentation. Topics include:
• A brief history and overview of bioaugmentation.
• A detailed review of the discovery of Dehalococcoides and the development of reductive dechlorination of chlorinated solvents as a remedial technology.
• The state-of-the-science for the production and handling of Dehalococcoides bioaugmentation cultures.
• A practical guide for deciding whether to bioaugment with Dehalococcoides.
• Design considerations for implementing bioaugmentation.
• A summary of the monitoring options during bioaugmentation with Dehalococcoides.
• Reviews of other bioaugmentation techniques, including aerobic cometabolism of chlorinated solvents, and treatment of carbon tetrachloride and methyl tert butyl ether.
• An analysis of the costs for bioaugmentation of chlorinated aliphatic compounds in groundwater.
• An assessment of and the uncertainties and opportunities for future bioaugmentation research and development.
Each chapter in this volume has been thoroughly reviewed for technical content by two or more experts in each subject area covered. This volume will provide a useful reference for both practitioners and researchers involved in groundwater remediation.
This book summarizes the current state of knowledge concerning bacteria that use halogenated organic compounds as respiratory electron acceptors. The discovery of organohalide-respiring bacteria has expanded the range of electron acceptors used for energy conservation, and serves as a prime example of how scientific discoveries are enabling innovative engineering solutions that have transformed remediation practice. Individual chapters provide in-depth background information on the discovery, isolation, phylogeny, biochemistry, genomic features, and ecology of individual organohalide-respiring genera, including Dehalococcoides, Dehalogenimonas, Dehalobacter, Desulfitobacterium and Sulfurospirillum, as well as organohalide-respiring members of the Deltaproteobacteria. The book introduces readers to the fascinating biology of organohalide-respiring bacteria, offering a valuable resource for students, engineers and practitioners alike.
Chlorinated solvents have been a primary focus of the remediation industry since the 1980s, and many remedial technologies have been developed, tested, and applied to remove these constituents from contaminated aquifers. The relative ease of stimulating organohalide-respiring bacteria in situ and the availability of low cost electron donor substrates and effective bioaugmentation cultures have allowed in situ bioremediation technologies to be applied successfully at thousands of sites around the world. Typically, the success of the remediation is dependent more on the site characteristics (e.g., geochemistry, geology, hydrology, contaminant concentration, etc.) than the fidelity of the microbes. As we begin to address the most challenging contaminated sites that remain to be remediated, including those with free product contamination, complicated geologies (e.g., low permeability soils or fractured rock), or complex contaminant mixtures, in situ bioremediation may not be the sole technology applied at these sites but it will likely be an important component of many remedies. Therefore, fundamental understandings of microbiology and the development of novel application approaches remain essential to ensure continued success in remediation of the most difficult chlorinated solvent-contaminated sites.
To date, organohalide respiration (OHR) has been restricted to the bacterial domain of life. Known organohalide-respiring bacteria
(OHRB) are spread among several phyla comprising both Gram-positive and Gram-negative bacteria. As a unique trait, OHRB benefit from reductive dehalogenase enzymes enabling them to use different organohalides as terminal electron acceptors and occupy a wide range of terrestrial and aquatic environments. This chapter comprises three sections: First, we give an overview of phylogeny of known OHRB and briefly discuss physiological and genetic characteristics of each group. Second, the environmental distribution of OHRB is presented. Owing to the application of molecular diagnostic approaches, OHRB are being increasingly detected not only from organohalide-contaminated groundwaters and sediments but also from pristine environments, including deep oceanic sediments and soils that are ample sources of naturally occurring organohalides. Finally, we highlight important factors that impact the ecology of OHRB and their interaction with other microbial guilds.
Despite advances in physicochemical remediation technologies, in situ bioremediation treatment based on Dehalococcoides mccartyi (Dhc) reductive dechlorination activity remains a cornerstone approach to remedy sites impacted with chlorinated ethenes. Selecting the best remedial strategy is challenging due to uncertainties and complexity associated with biological and geochemical factors influencing Dhc activity. Guidelines based on measurable biogeochemical parameters have been proposed, but contemporary efforts fall short of meaningfully integrating the available information. Extensive groundwater monitoring datasets have been collected for decades, but have not been systematically analyzed and used for developing tools to guide decision-making. In the present study, geochemical and microbial datasets collected from 35 wells at five contaminated sites were used to demonstrate that a data mining prediction model using the classification and regression tree (CART) algorithm can provide improved predictive understanding of a site's reductive dechlorination potential. The CART model successfully predicted the 3-month-ahead reductive dechlorination potential with 75.8% and 69.5% true positive rate (i.e., sensitivity) for the training set and the test set, respectively. The machine learning algorithm ranked parameters by relative importance for assessing in situ reductive dechlorination potential. The abundance of Dhc 16S rRNA genes, CH4, Fe(2+), NO3(-), NO2(-), and SO4(2-) concentrations, total organic carbon (TOC) amounts, and oxidation-reduction potential (ORP) displayed significant correlations (p < 0.01) with dechlorination potential, with NO3(-), NO2(-), and Fe(2+) concentrations exhibiting precedence over other parameters. Contrary to prior efforts, the power of data mining approaches lies in the ability to discern synergetic effects between multiple parameters that affect reductive dechlorination activity. Overall, these findings demonstrate that data mining techniques (e.g., machine learning algorithms) effectively utilize groundwater monitoring data to derive predictive understanding of contaminant degradation, and thus have great potential for improving decision-making tools. A major need for realizing the predictive capabilities of data mining approaches is a curated, open-access, up-to-date and comprehensive collection of biogeochemical groundwater monitoring data.
As part of their lifecycle neutralophilic bacteria are often exposed to varying environmental stresses, amongst which fluctuations in pH are the most frequent. In particular, acid environments can be encountered in many situations from fermented food to the gastric compartment of the animal host. Herein we review the current knowledge of the molecular mechanisms adopted by a range of Gram-positive and Gram-negative bacteria, mostly those affecting human health, for coping with acid stress. Because organic and inorganic acids have deleterious effects on the activity of the biological macromolecules to the point of significantly reducing growth and even threatening their viability, it is not unexpected that neutralophilic bacteria have evolved a number of different protective mechanisms, that provide them with an advantage in otherwise life-threatening conditions. The overall logic of these is to protect the cell from the deleterious effects of a harmful level of protons. Among the most favoured mechanisms are the pumping out of protons, production of ammonia, and proton-consuming decarboxylation reactions, as well as modifications of the lipid content in the membrane. Several examples are provided to describe mechanisms adopted to sense the external acidic pH. Particular attention is paid to Escherichia coli extreme acid resistance mechanisms, the activity of which ensure survival and may be directly linked to virulence.This article is protected by copyright. All rights reserved.
In chloroethene-contaminated sites undergoing in situ bioremediation, groundwater acidification is a frequent problem in the source zone, and buffering strategies have to be implemented to maintain the pH in the neutral range. An alternative to conventional soluble buffers is silicate mineral particles as a long-term source of alkalinity. In previous studies, the buffering potential of these minerals has been evaluated based on abiotic dissolution tests and geochemical modeling. In the present study, the buffering potential of four silicate minerals (andradite, diopside, fayalite and forsterite) was tested in batch cultures amended with tetrachloroethene (PCE), and inoculated with different organohalide-respiring consortia. Another objective of this study was to determine the influence of pH on the different steps of PCE dechlorination. The consortia showed significant differences in sensitivities towards acidic pH for the different dechlorination steps. Molecular analysis indicated that Dehalococcoides spp. that were present in all consortia, were the most pH sensitive organohalide-respiring guild members compared to Sulfurospirillum spp. and Dehalobacter spp. In batch cultures with silicate mineral particles as pH buffering agents, all four minerals tested were able to maintain the pH in the appropriate range for reductive dechlorination of chloroethenes. However, complete dechlorination to ethene was only observed with forsterite, diopside, and fayalite. Dissolution of andradite increased the redox potential and did not allow dechlorination. With forsterite, diopside and fayalite, dechlorination to ethene was observed but at much lower rates for the last two dechlorination steps compared with the positive control. This indicated an inhibition effect of silicate minerals and/or its dissolution products on reductive dechlorination of cis-DCE and VC. Hence, despite the proven pH buffering potential of silicate minerals, compatibility with the bacterial community involved in in situ bioremediation has to be carefully evaluated prior to their use for pH control at a specific site.
The long-term buffering potential of three silicate minerals (diopside, fayalite and forsterite) present as fine particles in porous quartz sand medium was evaluated in flow-through column experiments over a period of 6.5 months. The columns were operated with PCE concentrations close to saturation and inoculated with the organohalide-respiring consortium SDC-9™, which is able to completely dechlorinate PCE to ethene at high concentrations. In the absence of pH buffering agents, fermentation and organohalide respiration drove the pH close to 6.1, leading to severe inhibition of PCE dechlorination. Forsterite and fayalite were able to maintain the pH close to 7.5 and 6.5, respectively, and to sustain the production of VC and ethene. Diopside gradually lost its buffering capacity during the first 84 days due to the formation of a low reactive leached layer but dechlorination to cis-DCE was still achieved. Among the three minerals tested, forsterite was identified as the best buffering agent. Its presence led to the best PCE removal performance and the highest relative abundance of Dehalococcoides. This study showed that forsterite and fayalite are promising sources of long-term pH buffering for in situ bioremediation of source-zone PCE.
Accurate control of groundwater pH is of critical importance for in situ biological treatment of chlorinated solvents. The use of ground silicate minerals mixed with groundwater is an appealing buffering strategy as silicate minerals may act as long-term sources of alkalinity. In a previous study, we developed a geochemical model for evaluation of the pH buffering capacity of such minerals. The model included the main microbial processes driving groundwater acidification as well as mineral dissolution. In the present study, abiotic mineral dissolution experiments were conducted with five silicate minerals (andradite, diopside, fayalite, forsterite, nepheline). The goal of the study was to validate the model and to test the buffering capacity of the candidate minerals identified previously. These five minerals increased the pH from acidic to neutral and slightly basic values. The model was revised and improved to represent better the experimental observations. In particular, the experiments revealed the importance of secondary mineral precipitation on the buffering potential of silicates, a process not included in the original formulation. The main secondary phases likely to precipitate were identified through model calibration, as well as the degree of saturation at which they formed. The predictions of the revised geochemical model were in good agreement with the observations, with a correlation coefficient higher than 0.9 in most cases. This study confirmed the potential of silicates to act as pH control agents and showed the reliability of the geochemical model, which can be used as a design tool for field applications.
Acidification due to microbial dechlorination of trichloroethene (TCE) can limit the bio-enhanced dissolution of TCE dense non-aqueous phase liquid (DNAPL). This study related the dissolution enhancement of a TCE DNAPL to the pH buffer capacity of the medium and the type of electron donor used. In batch systems, dechlorination was optimal at pH7.1-7.5, but was completely inhibited below pH6.2. In addition, dechlorination in batch systems led to a smaller pH decrease at an increasing pH buffer capacity or with the use of formate instead of lactate as electron donor. Subsequently, bio-enhanced TCE DNAPL dissolution was quantified in diffusion-cells with a 5.5cm central sand layer, separating a TCE DNAPL layer from an aqueous top layer. Three different pH buffer capacities (2.9mM-17.9mM MOPS) and lactate or formate as electron donor were applied. In the lactate fed diffusion-cells, the DNAPL dissolution enhancement factor increased from 1.5 to 2.2 with an increase of the pH buffer capacity. In contrast, in the formate fed diffusion-cells, the DNAPL dissolution enhancement factor (2.4±0.3) was unaffected by the pH buffer capacity. Measurement of the pore water pH confirmed that the pH decreased less with an increased pH buffer capacity or with formate instead of lactate as electron donor. These results suggest that the significant impact of acidification on bio-enhanced DNAPL dissolution can be overcome by the amendment of a pH buffer or by applying a non acidifying electron donor like formate.
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 × 1011 DHC, the second loop bioaugmented with 3.9 × 1012 DHC, and the third loop bioaugmented with 3.9 × 1013 DHC. Groundwater monitoring was performed to evaluate DHC growth and migration, dechlorination rates, and aquifer geochemistry. The loop inoculated with 3.9 × 1012 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 × 1011 and 3.9 × 1013 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.
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.
Coupling thermal treatment with microbial reductive dechlorination is a promising remedy for tetrachloroethene (PCE) and trichloroethene (TCE) contaminated source zones. Laboratory experiments evaluated Dehalococcoides (Dhc) dechlorination performance, viability, and biomarker gene (DNA) and transcript (mRNA) abundances during exposure to elevated temperatures. The PCE-dechlorinating consortia BDI and OW produced ethene when incubated at temperatures of 30 °C, but vinyl chloride (VC) accumulated when cultures were incubated at 35 or 40 °C. Cultures incubated at 40 °C for less than 49 days resumed VC dechlorination following cooling; however, incubation at 45 °C resulted in complete loss of dechlorination activity. Dhc 16S rRNA, bvcA, and vcrA gene abundances in cultures showing complete dechlorination to ethene at 30 °C exceeded those measured in cultures incubated at higher temperatures, consistent with observed dechlorination activities. Conversely, biomarker gene transcript abundances per cell in cultures incubated at 35 and 40 °C were generally at least one order-of-magnitude greater than those measured in ethene-producing cultures incubated at 30 °C. Even in cultures accumulating VC, transcription of the vcrA gene, which is implicated in VC-to-ethene dechlorination, was up-regulated. Temperature stress caused the up-regulation of Dhc reductive dehalogenase gene expression indicating that Dhc gene expression measurements should be interpreted cautiously as Dhc biomarker gene transcript abundances may not correlate with dechlorination activity.
Chlorinated solvents such as perchloroethylene (PCE) and trichloroethylene (TCE) continue to be significant groundwater contaminants throughout the USA. In many cases efficient bioremediation of aquifers contaminated with these chemicals requires the addition of exogenous microorganisms, specifically members of the genus Dehalococcoides (DHC). This process is referred to as bioaugmentation. In this study a fed-batch fermentation process was developed for producing large volumes (to 3,200 L) of DHC-containing consortia suitable for treating contaminated aquifers. Three consortia enriched from three different sites were grown anaerobically with sodium lactate as an electron donor and PCE or TCE as an electron acceptor. DHC titers in excess of 10(11) DHC/L could be reproducibly obtained at all scales tested and with all three of the enrichment cultures. The mean specific DHC growth rate for culture SDC-9 was 0.036 +/- 0.005 (standard error, SE)/h with a calculated mean doubling time of 19.3 +/- 2.7 (SE) h. Finished cultures could be concentrated approximately tenfold by membrane filtration and stored refrigerated (4 degrees C) for more that 40 days without measurable loss of activity. Dehalogenation of PCE by the fermented cultures was affected by pH with no measurable activity at pH <5.0.
Soils harbor enormously diverse bacterial populations, and soil bacterial communities can vary greatly in composition across
space. However, our understanding of the specific changes in soil bacterial community structure that occur across larger spatial
scales is limited because most previous work has focused on either surveying a relatively small number of soils in detail
or analyzing a larger number of soils with techniques that provide little detail about the phylogenetic structure of the bacterial
communities. Here we used a bar-coded pyrosequencing technique to characterize bacterial communities in 88 soils from across
North and South America, obtaining an average of 1,501 sequences per soil. We found that overall bacterial community composition,
as measured by pairwise UniFrac distances, was significantly correlated with differences in soil pH (r = 0.79), largely driven by changes in the relative abundances of Acidobacteria, Actinobacteria, and Bacteroidetes across the range of soil pHs. In addition, soil pH explains a significant portion of the variability associated with observed
changes in the phylogenetic structure within each dominant lineage. The overall phylogenetic diversity of the bacterial communities
was also correlated with soil pH (R2 = 0.50), with peak diversity in soils with near-neutral pHs. Together, these results suggest that the structure of soil bacterial
communities is predictable, to some degree, across larger spatial scales, and the effect of soil pH on bacterial community
composition is evident at even relatively coarse levels of taxonomic resolution.
Enhanced reductive dehalogenation is an attractive treatment technology for in situ remediation of chlorinated solvent DNAPL source areas. Reductive dehalogenation is an acid-forming process with hydrochloric acid and also organic acids from fermentation of the electron donors typically building up in the source zone during remediation. This can lead to groundwater acidification thereby inhibiting the activity of dehalogenating microorganisms. Where the soils' natural buffering capacity is likely to be exceeded, the addition of an external source of alkalinity is needed to ensure sustained dehalogenation. To assist in the design of bioremediation systems, an abiotic geochemical model was developed to provide insight into the processes influencing the groundwater acidity as dehalogenation proceeds, and to predict the amount of bicarbonate required to maintain the pH at a suitable level for dehalogenating bacteria (i.e., >6.5). The model accounts for the amount of chlorinated solvent degraded, site water chemistry, electron donor, alternative terminal electron-accepting processes, gas release and soil mineralogy. While calcite and iron oxides were shown to be the key minerals influencing the soil's buffering capacity, for the extensive dehalogenation likely to occur in a DNAPL source zone, significant bicarbonate addition may be necessary even in soils that are naturally well buffered. Results indicated that the bicarbonate requirement strongly depends on the electron donor used and availability of competing electron acceptors (e.g., sulfate, iron (III)). Based on understanding gained from this model, a simplified model was developed for calculating a preliminary design estimate of the bicarbonate addition required to control the pH for user-specified operating conditions.
Oxygen-sensitive Dehalococcoides bacteria play crucial roles in detoxification of chlorinated contaminants (e.g., chlorinated ethenes), and bioremediation monitoring relies on quantification of Dehalococcoides DNA and RNA biomarkers. To explore the effects of oxygen on Dehalococcoides activity, viability, and biomarker quantification, batch experiments with a tetrachloroethene-to-ethene dechlorinating consortium (Bio-Dechlor INOCULUM [BDI]) harboring multiple Dehalococcoides strains were performed to quantify the effects of < or = 4 mg/L dissolved oxygen. Oxygen inhibited reductive dechlorination, and only incomplete dechlorination to vinyl chloride (VC) occurred following oxygen consumption and extended incubation periods (89 days). Following 30 days of oxygen exposure and subsequent oxygen removal (i.e., reversibility experiments), all trichloroethene- (TCE-) fed cultures dechlorinated TCE to VC, but VC dechlorination to ethene occurred in only one out of fourteen replicates. These results suggest that Dehalococcoides strains respond differently to oxygen exposure, and strains catalyzing the VC-to-ethene dechlorination step are more susceptible to oxygen inhibition. Quantitative real-time PCR (qPCR) analysis detected a 1-1.5 order-of-magnitude decrease in the number of Dehalococcoides biomarker genes (i.e., 16S rRNA gene and the reductive dehalogenase [RDase] genes tceA, vcrA, bvcA) in the oxygen-amended cultures, but qPCR analysis failed to distinguish viable, dechlorinating from irreversibly inhibited (nonviable) Dehalococcoides cells. Reverse transcriptase qPCR (RT-qPCR) detected Dehalococcoides gene transcripts in the oxygen-amended, non-dechlorinating cultures, and biomarker transcription did not always correlate with dechlorination (in)activity. Enhanced molecular tools that complement existing protocols and provide quantitative information on the viability and activity of the Dehalococcoides population are desirable.
An anaerobic microbial consortium (referred to as ANAS) that reductively dechlorinates trichloroethene (TCE) completely to ethene with the transient production of cisdichloroethene (cDCE) and vinyl chloride was enriched from contaminated soil obtained from Alameda Naval Air Station. ANAS uses lactate as its electron donor and has been functionally stable for over 2 years. Following a brief exposure to oxygen, a subculture (designated VCC) derived from ANAS could dechlorinate TCE only to vinyl chloride with lactate as its electron donor. Three molecular methods were used concurrently to characterize the community structure of ANAS and VCC: clone library construction/clone sequencing, terminal restriction fragment length polymorphism (T-RFLP) analysis, and fluorescent in situ hybridization (FISH) with rRNA probes. The community structure of ANAS did not change significantly over the course of a single feeding/dechlorination cycle, and only minor fluctuations occurred over many feeding cycles spanning the course of 1 year. Clone libraries and T-RFLP analyses suggested that ANAS was dominated by populations belonging to three phylogenetic groups: Dehalococcoides species, Desulfovibrio species, and members of the Clostridiaceae (within the low G + C Gram-positives). FISH results suggest that members of the Cytophaga/Flavobacterium/Bacteroides (CFB) cluster and high G + C Gram-positives (HGCs) were numerically important in ANAS despite their under-representation in the clone libraries. Parallel analyses of VCC samples suggested that Dehalococcoides species and Clostridiaceae were only minor populations in this community. Instead, VCC had increased populations of organisms in the beta and gamma subclasses of the Proteobacteria as well as significant populations of organisms in the CFB cluster. It is possible that symbiotic interactions are occurring between some of ANAS's phylogenetic groups under the enrichment conditions, including interspecies hydrogen transfer from Desulfovibrio species to Dehalococcoides species. However, the nucleic acid-based analyses performed here would need to be supplemented with chemical species data in order to test any hypotheses about functional roles of various community members. Additionally, these results suggest that an organism outside the Dehalococcoides genus may be capable of dechlorinating cDCE to 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.
The ability to inoculate a PCE-NAPL source zone with no prior dechlorinating activity was examined using a near field-scale simulated aquifer. A known mass of PCE was added to establish a source zone, and the groundwater was depleted of oxygen using acetate and lactate prior to culture addition. An active and stable dechlorinating culture was used as an inoculum, and dechlorination activity was observed within 2 weeks following culture transfer. PCE reduction to TCE and cis-DCE was observed initially, and the formation of these compounds was accelerated by the addition of a long-term source of hydrogen (Hydrogen Releasing Compound). cis-DCE was the predominant chlorinated ethene present in the effluent after 225 days of operation, and production of VC and ethene lagged the formation of TCE and cis-OCE. However, dechlorination extent continued to improve over time, and VC eventually became a major product, suggesting that reinoculation was unnecessary. The detection of Dehalococcoides species in the source culture and in the simulated aquifer postinoculation indicated that the metabolic capability to dechlorinate beyond cis-DCE (t = 86 days and t = 245 days) was present. Elevated levels of TCE and cis-DCE were present in the source zone, but neither VC nor ethene were detected in the vicinity of NAPL. The results of this research indicated that adding dechlorinating cultures may be useful in the application of source zone bioremediation but that dechlorination beyond cis-DCE may be limited to regions downgradient of the source zone.
Two biokinetic models employing the Michaelis-Menten equation for anaerobic reductive dechlorination of tetrachloroethylene (PCE) and trichloroethylene (TCE) were developed. The models were compared with results from batch kinetic tests conducted over a wide range of PCE and TCE concentrations with two different dechlorinating cultures. One model applies Michaelis-Menten kinetics with competitive inhibition among chlorinated aliphatic hydrocarbons (CAHs), while the other model includes both competitive inhibition and Haldane inhibition at high CAH concentrations. Model simulations with competitive inhibition simulated the experimental results well for PCE concentrations lower than 300 microM. However, simulations deviated from the experimental observations for PCE or TCE concentrations greater than 300-400 microM. The kinetic model that incorporated both competitive and Haldane inhibitions better simulated experimental data for PCE concentrations near the solubility limit (1000 microM), and TCE concentrations at half its solubility limit (4000 microM). Based on the modeling analysis of the experimental results, the PM culture (Point Mugu, CA) had very high Haldane inhibition constants for cis-1,2-dichlororethylene (c-DCE) and vinyl chloride (VC) (6000 and 7000 microM, respectively), indicating very weak Haldane inhibition, while the EV culture (the Evanite site in Corvallis, OR) had lower Haldane inhibition constants for TCE, c-DCE, and VC of 900, 750, and 750 microM, respectively. The BM culture (a binary mixed culture of the PM and EV cultures) had transformation abilities that represented the mixture of the EV and PM cultures. Model simulations of the BM culture transformation abilities were well represented by separate rate equations and model parameters for the two independent cultures that were simultaneously solved. Modeling results indicated that a combination of competitive and Haldane inhibition kinetics is required to simulate dechlorination over a broad range of concentrations up to the solubility limit of PCE and half the solubility limit of TCE.
Strategies and procedures for enriching, isolating, and cultivating reductively dechlorinating bacteria that use chloroorganic compounds as metabolic electron acceptors from environmental samples are described. Further, nucleic acid-based approaches used to detect and quantify dechlorinator (i.e., Dehalococcoides)-specific genes are presented.
Human activities have released large amounts of toxic organic and inorganic chemicals into the environment. Toxic waste streams threaten dwindling drinking water supplies and impact terrestrial, estuarine and marine ecosystems. Cleanup is technically challenging and the costs based on traditional technologies are exceeding the economic capabilities of even the richest countries. Recent advances in our understanding of the microbiology contributing to contaminant transformation and detoxification has led to successful field demonstrations. Hence, harnessing the activity of naturally occurring bacteria, particularly the power of anaerobic reductive processes, is a promising approach to restore contaminated subsurface environments, protect drinking water reservoirs and to safeguard ecosystem health.
Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains
- KM Ritalahti
- BK Amos
- Y Sung