Environmental contamination remediation and management, Nov 22, 2023
Molecular biological tools (MBTs) are used to assess and characterize the microbiology and associ... more Molecular biological tools (MBTs) are used to assess and characterize the microbiology and associated biological processes at contaminated sites, including ecological roles, phylogenetic diversity, and metabolic and co-metabolic capabilities related to contaminant biodegradation and biotransformation. MBTs have changed our approach to the assessment and remediation of petroleum hydrocarbons in the environment. In contaminated-site management, several MBTs and stable isotope analysis methods can be used to measure the presence, abundance, and activity of petroleum hydrocarbon-degrading microorganisms and transformation pathways: (1) quantitative polymerase chain reaction (qPCR) to quantify microorganisms and genes encoding enzymes for biodegradation or biotransformation, (2) DNA sequencing to comprehensively identify the microorganisms presence and microbial community structure, (3) stable isotope probing (SIP) provides conclusive evidence of biodegradation through the use of 13 C-labeled surrogate compounds of petroleum hydrocarbons that microbes use as carbon and/or energy sources, and (4) compoundspecific isotope analysis (CSIA) measures the ratio of naturally occurring stable isotopes within a contaminant to indicate (bio)degradation. This chapter discusses the use, benefits, and limitations of MBTs. Several real-world case studies are provided to demonstrate how to investigate the biodegradation of petroleum hydrocarbon constituents under varying field conditions to better characterize governing biogeochemical processes and to better inform remedial decisions.
Passive multilevel samplers (MLS) containing a solid matrix for microbial colonization were used ... more Passive multilevel samplers (MLS) containing a solid matrix for microbial colonization were used in conjunction with a push-pull biostimulation experiment designed to promote biological U(VI) and Tc(VII) reduction. MLS were deployed at 24 elevations in the injection well and two down gradient wells to investigate the spatial variability in microbial community composition and growth prior to and following biostimulation. The
Journal Of Environmental Science And Health, Part A, May 20, 2014
Multiple lines of evidence were used to document the natural attenuation of perchlorate in a shal... more Multiple lines of evidence were used to document the natural attenuation of perchlorate in a shallow alluvial aquifer. In the upgradient, aerobic portion of the aquifer, perchlorate did not biodegrade. However, natural flushing by groundwater flow is reducing perchlorate concentrations in the aquifer over time. Perchlorate concentrations in the source area are expected to meet cleanup criteria in 11 to 27 years without active remedial measures. At the distal end of the plume, perchlorate is rapidly degraded as it migrates upward through organic rich littoral zone sediments. Apparent first-order degradation rates in groundwater were about 0.20 d(-1) and are consistent with laboratory macrocosm rates (0.12 d(-1)). qPCR results show a distinct region of the littoral zone where perchlorate degraders are elevated. The Eh within this zone varies from +0.1 to +0.3 V indicating perchlorate degraders can thrive in moderately oxidizing conditions. The study has shown that (i) there was no apparent perchlorate biodegradation in aerobic aquifer; (ii) perchlorate declines over time in aerobic aquifer due to flushing; (iii) there was a rapid perchlorate attenuation in organic rich littoral zone; and, (iv) qPCR results show large increases in perchlorate degraders in the littoral zone.
Tetrachloroethene (PCE)-and trichloroethene (TCE)-impacted sites pose significant challenges even... more Tetrachloroethene (PCE)-and trichloroethene (TCE)-impacted sites pose significant challenges even when site characterization activities indicate that biodegradation has occurred naturally. Although site-specific, regulatory, and economic factors play roles in the remedy-selection process, the application of molecular biological tools to the bioremediation field has streamlined the assessment of remedial alternatives and allowed for detailed evaluation of the chosen remedial technology. The case study described here was performed at a PCE-impacted site at which reductive dechlorination of PCE and TCE had led to accumulation of cis-dichlorethene (cis-DCE) with concentrations ranging from approximately 10 to 100 mg/L. Bio-Trap ® samplers and quantitative polymerase chain reaction (qPCR) enumeration of Dehalococcoides spp. were used to evaluate three remedial options: monitored natural attenuation, biostimulation with HRC ® , and biostimulation with HRC-S ®. Dehalococcoides populations in HRC-S-amended Bio-Traps deployed in impacted wells were on the order of 10 3 to 10 4 cells/bead but were below detection limits in most unamended and HRC-amended Bio-Traps. Thus the in situ Bio-Trap study identified biostimulation with HRC-S as the recommended approach, which was further evaluated with a pilot study. After the pilot HRC-S injection, Dehalococcoides populations increased to 10 6 to 10 7 cells/bead, and concentrations of cis-DCE and vinyl chloride decreased with concurrent ethene production. Based on these results, a full-scale HRC-S injection was designed and implemented at the site. As with the pilot study, full-scale HRC-S injection promoted growth of Dehalococcoides spp. and stimulated reductive dechlorination of the daughter products cis-DCE and vinyl chloride. O
Biostimulation to promote reductive dechlorination is widely practiced, but the value of adding a... more Biostimulation to promote reductive dechlorination is widely practiced, but the value of adding an exogenous nitrogen (N) source (e.g., NH4+) during treatment is unclear. This study investigates the effect of NH4+ availability on organohalide-respiring Dehalococcoides mccartyi (Dhc) growth and reductive dechlorination in enrichment cultures derived from groundwater (PW4) and river sediment (TC) impacted with chlorinated ethenes. In PW4 cultures, the addition of NH4+ increased cis-1,2-dichloroethene (cDCE)-to-ethene dechlorination rates about five-fold (20.6±1.6 versus 3.8±0.5 μM Cl- d-1), and the total number of Dhc 16S rRNA gene copies were about 43-fold higher in incubations with NH4+ (1.8 ± 0.9 x 108 mL-1) compared to incubations without NH4+ (4.1 ± 0.8 x 107 mL-1). In TC cultures, NH4+ also stimulated cDCE-to-ethene dechlorination and Dhc growth. qPCR revealed that Cornell-type Dhc capable of N2 fixation dominated PW4 cultures without NH4+, but their relative abundance decreased in cultures with NH4+ amendment (i.e., 99% versus 54% of total Dhc). Pinellas-type Dhc incapable of N2 fixation were responsible for cDCE dechlorination in TC cultures and diazotrophic community members met their fixed N requirement in medium without NH4+. Responses to NH4+ were apparent at the community level and N2-fixing bacterial populations increased in incubations without NH4+. Quantitative assessment of Dhc nitrogenase genes, transcripts, and proteomics data linked Cornell-type Dhc nifD and nifK expression with fixed N limitation. NH4+ additions also demonstrated positive effects on Dhc in situ dechlorination activity in the vicinity of well PW4. These findings demonstrate that biostimulation with NH4+ can enhance Dhc reductive dechlorination rates; however, a "do nothing" approach that relies on indigenous diazotrophs can achieve similar dechlorination end points and avoids the potential for stalled dechlorination due to inhibitory levels of NH4+ or transformation products (i.e., nitrous oxide).
Applied Microbiology and Biotechnology, Apr 9, 2019
Quantifying functional biomarker genes and their transcripts provides critical lines of evidence ... more Quantifying functional biomarker genes and their transcripts provides critical lines of evidence for contaminant biodegradation; however, accurate quantification depends on qPCR primers that contain no, or minimal, mismatches with the target gene. Developing accurate assays has been particularly challenging for genes encoding fumarate-adding enzymes (FAE) due to the high level of genetic diversity in this gene family. In this study, metagenomics applied to a field-derived, o-xylene-degrading methanogenic consortium revealed genes encoding FAE that would not be accurately quantifiable by any previously available PCR assays. Sequencing indicated that a gene similar to the napthylmethylsuccinate synthase gene (nmsA) was most abundant, although benzylsuccinate synthase genes (bssA) also were present along with genes encoding alkylsuccinate synthase (assA). Upregulation of the nmsA-like gene was observed during o-xylene degradation. Protein homology modeling indicated that mutations in the active site, relative to a BssA that acts on toluene, increase binding site volume and accessibility, potentially to accommodate the relatively larger o-xylene. The new nmsA-like gene was also detected at substantial concentrations at field sites with a history of xylene contamination.
h i g h l i g h t s • The biodegradation potential of 1,2-DCA and VC is proposed at three field s... more h i g h l i g h t s • The biodegradation potential of 1,2-DCA and VC is proposed at three field sites. • Site-Specific Limiting Factors are presented to Biodegradation of 1,2-DCA and VC. • Hydrogeological, geochemical and qPCR data are presented to explain biodegradation. • Dehalococcoides proved to be a key dechlorinator to VC and chlorinated ethanes. • Biodegradation is a viable strategy to chlorinated ethanes and VC.
Organohalide-respiring bacteria have key roles in the natural chlorine cycle; however, most of th... more Organohalide-respiring bacteria have key roles in the natural chlorine cycle; however, most of the current knowledge is based on cultures from contaminated environments. We demonstrate that grape pomace compost without prior exposure to chlorinated solvents harbors a Dehalogenimonas (Dhgm) species capable of using chlorinated ethenes, including the human carcinogen and common groundwater pollutant vinyl chloride (VC) as electron acceptors. Grape pomace microcosms and derived solid-free enrichment cultures were able to dechlorinate trichloroethene (TCE) to less chlorinated daughter products including ethene. 16S rRNA gene amplicon and qPCR analyses revealed a predominance of Dhgm sequences, but Dehalococcoides mccartyi (Dhc) biomarker genes were not detected. The enumeration of Dhgm 16S rRNA genes demonstrated VCdependent growth, and 6.55 ± 0.64 × 10 8 cells were measured per μmol of chloride released. Metagenome sequencing enabled the assembly of a Dhgm draft genome, and 52 putative reductive dehalogenase (RDase) genes were identified. Proteomic workflows identified a putative VC RDase with 49 and 56.1% amino acid similarity to the known VC RDases VcrA and BvcA, respectively. A survey of 1,173 groundwater samples collected from 111 chlorinated solvent-contaminated sites in the United States and Australia revealed that Dhgm 16S rRNA genes were frequently detected and outnumbered Dhc in 65% of the samples. Dhgm are likely greater contributors to reductive dechlorination of chlorinated solvents in contaminated aquifers than is currently recognized, and non-polluted environments represent sources of organohalide-respiring bacteria with novel RDase genes.
Ground Water Monitoring and Remediation, Nov 13, 2013
ABSTRACT A microbial survey of hydrocarbon-impacted groundwater and vadose zone at a Midwestern r... more ABSTRACT A microbial survey of hydrocarbon-impacted groundwater and vadose zone at a Midwestern refinery employed molecular biological tools to elucidate the microbial processes involved in bioremediation occurring in the subsurface. qPCR analysis of bio-traps incubated in groundwater indicated that a large and diverse microbial community was present throughout the site and suggested that mechanisms of benzene, toluene, ethylbenzene, and xylene (BTEX) biodegradation included aerobic oxidation, sulfate reduction, methanogenesis, and possibly Fe+3 reduction. To assess the role of vadose zone microorganisms in hydrocarbon attenuation, RNA was extracted from soil core samples, and reverse transcriptase-qPCR (RT-qPCR) analysis indicated that microbial activity in the vadose zone generally increased with depth, likely supported by hydrocarbons and methane volatilizing from the groundwater. Stable isotope probing (SIP) with 13C6-benzene provided direct evidence of benzene biodegradation in six of the eight wells studied. The highest levels of 13C were detected in dissolved inorganic carbon (DIC) extracted from the two monitoring wells closest to the river. The influx of nutrients and oxygen coming from the river may help to maintain a robust population of hydrocarbon degraders in these wells. While qPCR analysis indicated that microorganisms with the genetic potential for hydrocarbon biodegradation were ubiquitous at the site, RT-qPCR and SIP results were used to refine the site conceptual model by identifying areas where that genetic potential was actively being expressed and locations where biodegradation was lagging.
Increasingly, molecular biological tools, most notably quantitative polymerase chain reaction (qP... more Increasingly, molecular biological tools, most notably quantitative polymerase chain reaction (qPCR), are being employed to provide a more comprehensive assessment of bioremediation of petroleum hydrocarbons and fuel oxygenates. While qPCR enumeration of key organisms or catabolic genes can aid in site management decisions, evaluation of site activities conducted to stimulate biodegradation would ideally include a direct measure of gene expression to infer activity. In the current study, reverse-transcriptase (RT) qPCR was used to monitor gene expression to evaluate the effectiveness of an oxygen infusion system to promote biodegradation of BTEX and MTBE. During system operation, dissolved oxygen (DO) levels at the infusion points were greater than 30 mg/L, contaminant concentrations decreased, and transcription of two aromatic oxygenase genes and Methylibium petroleiphilum PM1-like 16S rRNA copies increased by as many as 5 orders of magnitude. Moreover, aromatic oxygenase gene transcription and PM1 16s rRNA increased at downgradient locations despite low DO levels even during system operation. Conversely, target gene expression substantially decreased when the system was deactivated. RT-qPCR results also corresponded to increases in benzene and MTBE attenuation rates. Overall, monitoring gene expression complemented traditional groundwater analyses and conclusively demonstrated that the oxygen infusion system promoted BTEX and MTBE biodegradation.
NABIR UMTRA Old Rifle Biostimulation Sediment Data Correlations between modifying electron donor ... more NABIR UMTRA Old Rifle Biostimulation Sediment Data Correlations between modifying electron donor and acceptor accessibility, the in-situ microbial community, and bioreduction of Uranium at the FRC and UMTRA research sites indicated that significant modifications in the rate, amount and by inference the potential stability of immobilized Uranium are feasible in these environments. The in-situ microbial community at these sites was assessed with a combination of lipid and real-time molecular techniques providing quantitative insights of effects of electron donor and manipulations.
Bioremediation can be an effective treatment strategy at contaminated sites when applied appropri... more Bioremediation can be an effective treatment strategy at contaminated sites when applied appropriately and implemented properly. Thus it is important to have methods that can be used to reliably assess biodegradation potential and ultimately the feasibility and performance of bioremediation as a treatment approach. While an important component of site monitoring, decreasing trends in contaminant concentrations can result from physical processes, do not document that loss is due to biodegradation, and can be difficult to discern in practice. Therefore direct measures of microbes responsible for biodegradation of compounds have been developed. These methods fall into two categories: direct measure of biodegradation functional genes using methods such as quantitative PCR and microarrays and indirect measures in changes in microbial community structure using methods such as PCR-DGGE and T-RFLP when target genes are unknown. This chapter describes the various molecular methods being used and examples where they have been applied for the assessment of environmental samples.
Ground Water Monitoring and Remediation, Mar 19, 2019
This column reviews the general features of PHT3D Version 2, a reactive multicomponent transport ... more This column reviews the general features of PHT3D Version 2, a reactive multicomponent transport model that couples the geochemical modeling software PHREEQC-2 (Parkhurst and Appelo 1999) with three-dimensional groundwater flow and transport simulators MODFLOW-2000 and MT3DMS (Zheng and Wang 1999). The original version of PHT3D was developed by Henning Prommer and Version 2 by Henning Prommer and Vincent Post (Prommer and Post 2010). More detailed information about PHT3D is available at the website http://www.pht3d.org. The review was conducted separately by two reviewers. This column is presented in two parts.
Bio-Trap ®-based in situ microcosm studies were conducted to evaluate EHC-M ® stimulated degradat... more Bio-Trap ®-based in situ microcosm studies were conducted to evaluate EHC-M ® stimulated degradation of mono-, di-, and trichlorobenzenes in anaerobic groundwater at a site in Michigan. The data show that the EHC-M ® amendment stimulated an overall increase in microbial activity and a shift in the microbial community structure, indicating more reduced conditions. Stable isotope probing with 13 C 6-chlorobenzene demonstrated attenuation of chlorobenzene and subsequent separation and characterization of the 12 C-and 13 C-deoxyribonucleic acid (DNA) fractions were used to identify the attenuating microbes. These data clearly show the participation of an obligate aerobe in the chlorobenzene biodegradation process. Decreases in concentrations of trichlorobenzenes were also observed in comparison to a control. Due to the thermodynamically favorable reducing conditions stimulated by EHC-M ® , the mechanism of degradation of the trichlorobenzenes is presumed to be reductive dehalogenation. However, on the strength of the DNA-based analysis of microbial community structure, concurrent microaerophilic degradation of chlorobenzene or its metabolites was definitively demonstrated and cannot be ruled out for the other chlorobenzenes. O
The objective of this work was to field‐demonstrate the Min‐Trap® sampler technology, a new in‐si... more The objective of this work was to field‐demonstrate the Min‐Trap® sampler technology, a new in‐situ passive monitoring tool that offers distinct advantages for collecting mineralogical data. The Min‐Trap consists of a solid porous medium (e.g., silica sand) contained within a water‐permeable mesh and support housing that is deployed inside a monitoring well. The porous medium serves as a solid substrate upon which target minerals passively form. Analysis of the sample medium using chemical, microscopic, spectroscopic, or other techniques can provide direct evidence of geochemical conditions and the formation of target minerals in‐situ. The abiotic degradation of chlorinated volatile organic compounds via the reducing power stored in reactive minerals (e.g., iron sulfides) is a recognized important process, and Min‐Traps are well suited to document the formation of these minerals during remediation. Min‐Traps were tested in multiple wells at two field sites undergoing active remediat...
Environmental contamination remediation and management, Nov 22, 2023
Molecular biological tools (MBTs) are used to assess and characterize the microbiology and associ... more Molecular biological tools (MBTs) are used to assess and characterize the microbiology and associated biological processes at contaminated sites, including ecological roles, phylogenetic diversity, and metabolic and co-metabolic capabilities related to contaminant biodegradation and biotransformation. MBTs have changed our approach to the assessment and remediation of petroleum hydrocarbons in the environment. In contaminated-site management, several MBTs and stable isotope analysis methods can be used to measure the presence, abundance, and activity of petroleum hydrocarbon-degrading microorganisms and transformation pathways: (1) quantitative polymerase chain reaction (qPCR) to quantify microorganisms and genes encoding enzymes for biodegradation or biotransformation, (2) DNA sequencing to comprehensively identify the microorganisms presence and microbial community structure, (3) stable isotope probing (SIP) provides conclusive evidence of biodegradation through the use of 13 C-labeled surrogate compounds of petroleum hydrocarbons that microbes use as carbon and/or energy sources, and (4) compoundspecific isotope analysis (CSIA) measures the ratio of naturally occurring stable isotopes within a contaminant to indicate (bio)degradation. This chapter discusses the use, benefits, and limitations of MBTs. Several real-world case studies are provided to demonstrate how to investigate the biodegradation of petroleum hydrocarbon constituents under varying field conditions to better characterize governing biogeochemical processes and to better inform remedial decisions.
Passive multilevel samplers (MLS) containing a solid matrix for microbial colonization were used ... more Passive multilevel samplers (MLS) containing a solid matrix for microbial colonization were used in conjunction with a push-pull biostimulation experiment designed to promote biological U(VI) and Tc(VII) reduction. MLS were deployed at 24 elevations in the injection well and two down gradient wells to investigate the spatial variability in microbial community composition and growth prior to and following biostimulation. The
Journal Of Environmental Science And Health, Part A, May 20, 2014
Multiple lines of evidence were used to document the natural attenuation of perchlorate in a shal... more Multiple lines of evidence were used to document the natural attenuation of perchlorate in a shallow alluvial aquifer. In the upgradient, aerobic portion of the aquifer, perchlorate did not biodegrade. However, natural flushing by groundwater flow is reducing perchlorate concentrations in the aquifer over time. Perchlorate concentrations in the source area are expected to meet cleanup criteria in 11 to 27 years without active remedial measures. At the distal end of the plume, perchlorate is rapidly degraded as it migrates upward through organic rich littoral zone sediments. Apparent first-order degradation rates in groundwater were about 0.20 d(-1) and are consistent with laboratory macrocosm rates (0.12 d(-1)). qPCR results show a distinct region of the littoral zone where perchlorate degraders are elevated. The Eh within this zone varies from +0.1 to +0.3 V indicating perchlorate degraders can thrive in moderately oxidizing conditions. The study has shown that (i) there was no apparent perchlorate biodegradation in aerobic aquifer; (ii) perchlorate declines over time in aerobic aquifer due to flushing; (iii) there was a rapid perchlorate attenuation in organic rich littoral zone; and, (iv) qPCR results show large increases in perchlorate degraders in the littoral zone.
Tetrachloroethene (PCE)-and trichloroethene (TCE)-impacted sites pose significant challenges even... more Tetrachloroethene (PCE)-and trichloroethene (TCE)-impacted sites pose significant challenges even when site characterization activities indicate that biodegradation has occurred naturally. Although site-specific, regulatory, and economic factors play roles in the remedy-selection process, the application of molecular biological tools to the bioremediation field has streamlined the assessment of remedial alternatives and allowed for detailed evaluation of the chosen remedial technology. The case study described here was performed at a PCE-impacted site at which reductive dechlorination of PCE and TCE had led to accumulation of cis-dichlorethene (cis-DCE) with concentrations ranging from approximately 10 to 100 mg/L. Bio-Trap ® samplers and quantitative polymerase chain reaction (qPCR) enumeration of Dehalococcoides spp. were used to evaluate three remedial options: monitored natural attenuation, biostimulation with HRC ® , and biostimulation with HRC-S ®. Dehalococcoides populations in HRC-S-amended Bio-Traps deployed in impacted wells were on the order of 10 3 to 10 4 cells/bead but were below detection limits in most unamended and HRC-amended Bio-Traps. Thus the in situ Bio-Trap study identified biostimulation with HRC-S as the recommended approach, which was further evaluated with a pilot study. After the pilot HRC-S injection, Dehalococcoides populations increased to 10 6 to 10 7 cells/bead, and concentrations of cis-DCE and vinyl chloride decreased with concurrent ethene production. Based on these results, a full-scale HRC-S injection was designed and implemented at the site. As with the pilot study, full-scale HRC-S injection promoted growth of Dehalococcoides spp. and stimulated reductive dechlorination of the daughter products cis-DCE and vinyl chloride. O
Biostimulation to promote reductive dechlorination is widely practiced, but the value of adding a... more Biostimulation to promote reductive dechlorination is widely practiced, but the value of adding an exogenous nitrogen (N) source (e.g., NH4+) during treatment is unclear. This study investigates the effect of NH4+ availability on organohalide-respiring Dehalococcoides mccartyi (Dhc) growth and reductive dechlorination in enrichment cultures derived from groundwater (PW4) and river sediment (TC) impacted with chlorinated ethenes. In PW4 cultures, the addition of NH4+ increased cis-1,2-dichloroethene (cDCE)-to-ethene dechlorination rates about five-fold (20.6±1.6 versus 3.8±0.5 μM Cl- d-1), and the total number of Dhc 16S rRNA gene copies were about 43-fold higher in incubations with NH4+ (1.8 ± 0.9 x 108 mL-1) compared to incubations without NH4+ (4.1 ± 0.8 x 107 mL-1). In TC cultures, NH4+ also stimulated cDCE-to-ethene dechlorination and Dhc growth. qPCR revealed that Cornell-type Dhc capable of N2 fixation dominated PW4 cultures without NH4+, but their relative abundance decreased in cultures with NH4+ amendment (i.e., 99% versus 54% of total Dhc). Pinellas-type Dhc incapable of N2 fixation were responsible for cDCE dechlorination in TC cultures and diazotrophic community members met their fixed N requirement in medium without NH4+. Responses to NH4+ were apparent at the community level and N2-fixing bacterial populations increased in incubations without NH4+. Quantitative assessment of Dhc nitrogenase genes, transcripts, and proteomics data linked Cornell-type Dhc nifD and nifK expression with fixed N limitation. NH4+ additions also demonstrated positive effects on Dhc in situ dechlorination activity in the vicinity of well PW4. These findings demonstrate that biostimulation with NH4+ can enhance Dhc reductive dechlorination rates; however, a "do nothing" approach that relies on indigenous diazotrophs can achieve similar dechlorination end points and avoids the potential for stalled dechlorination due to inhibitory levels of NH4+ or transformation products (i.e., nitrous oxide).
Applied Microbiology and Biotechnology, Apr 9, 2019
Quantifying functional biomarker genes and their transcripts provides critical lines of evidence ... more Quantifying functional biomarker genes and their transcripts provides critical lines of evidence for contaminant biodegradation; however, accurate quantification depends on qPCR primers that contain no, or minimal, mismatches with the target gene. Developing accurate assays has been particularly challenging for genes encoding fumarate-adding enzymes (FAE) due to the high level of genetic diversity in this gene family. In this study, metagenomics applied to a field-derived, o-xylene-degrading methanogenic consortium revealed genes encoding FAE that would not be accurately quantifiable by any previously available PCR assays. Sequencing indicated that a gene similar to the napthylmethylsuccinate synthase gene (nmsA) was most abundant, although benzylsuccinate synthase genes (bssA) also were present along with genes encoding alkylsuccinate synthase (assA). Upregulation of the nmsA-like gene was observed during o-xylene degradation. Protein homology modeling indicated that mutations in the active site, relative to a BssA that acts on toluene, increase binding site volume and accessibility, potentially to accommodate the relatively larger o-xylene. The new nmsA-like gene was also detected at substantial concentrations at field sites with a history of xylene contamination.
h i g h l i g h t s • The biodegradation potential of 1,2-DCA and VC is proposed at three field s... more h i g h l i g h t s • The biodegradation potential of 1,2-DCA and VC is proposed at three field sites. • Site-Specific Limiting Factors are presented to Biodegradation of 1,2-DCA and VC. • Hydrogeological, geochemical and qPCR data are presented to explain biodegradation. • Dehalococcoides proved to be a key dechlorinator to VC and chlorinated ethanes. • Biodegradation is a viable strategy to chlorinated ethanes and VC.
Organohalide-respiring bacteria have key roles in the natural chlorine cycle; however, most of th... more Organohalide-respiring bacteria have key roles in the natural chlorine cycle; however, most of the current knowledge is based on cultures from contaminated environments. We demonstrate that grape pomace compost without prior exposure to chlorinated solvents harbors a Dehalogenimonas (Dhgm) species capable of using chlorinated ethenes, including the human carcinogen and common groundwater pollutant vinyl chloride (VC) as electron acceptors. Grape pomace microcosms and derived solid-free enrichment cultures were able to dechlorinate trichloroethene (TCE) to less chlorinated daughter products including ethene. 16S rRNA gene amplicon and qPCR analyses revealed a predominance of Dhgm sequences, but Dehalococcoides mccartyi (Dhc) biomarker genes were not detected. The enumeration of Dhgm 16S rRNA genes demonstrated VCdependent growth, and 6.55 ± 0.64 × 10 8 cells were measured per μmol of chloride released. Metagenome sequencing enabled the assembly of a Dhgm draft genome, and 52 putative reductive dehalogenase (RDase) genes were identified. Proteomic workflows identified a putative VC RDase with 49 and 56.1% amino acid similarity to the known VC RDases VcrA and BvcA, respectively. A survey of 1,173 groundwater samples collected from 111 chlorinated solvent-contaminated sites in the United States and Australia revealed that Dhgm 16S rRNA genes were frequently detected and outnumbered Dhc in 65% of the samples. Dhgm are likely greater contributors to reductive dechlorination of chlorinated solvents in contaminated aquifers than is currently recognized, and non-polluted environments represent sources of organohalide-respiring bacteria with novel RDase genes.
Ground Water Monitoring and Remediation, Nov 13, 2013
ABSTRACT A microbial survey of hydrocarbon-impacted groundwater and vadose zone at a Midwestern r... more ABSTRACT A microbial survey of hydrocarbon-impacted groundwater and vadose zone at a Midwestern refinery employed molecular biological tools to elucidate the microbial processes involved in bioremediation occurring in the subsurface. qPCR analysis of bio-traps incubated in groundwater indicated that a large and diverse microbial community was present throughout the site and suggested that mechanisms of benzene, toluene, ethylbenzene, and xylene (BTEX) biodegradation included aerobic oxidation, sulfate reduction, methanogenesis, and possibly Fe+3 reduction. To assess the role of vadose zone microorganisms in hydrocarbon attenuation, RNA was extracted from soil core samples, and reverse transcriptase-qPCR (RT-qPCR) analysis indicated that microbial activity in the vadose zone generally increased with depth, likely supported by hydrocarbons and methane volatilizing from the groundwater. Stable isotope probing (SIP) with 13C6-benzene provided direct evidence of benzene biodegradation in six of the eight wells studied. The highest levels of 13C were detected in dissolved inorganic carbon (DIC) extracted from the two monitoring wells closest to the river. The influx of nutrients and oxygen coming from the river may help to maintain a robust population of hydrocarbon degraders in these wells. While qPCR analysis indicated that microorganisms with the genetic potential for hydrocarbon biodegradation were ubiquitous at the site, RT-qPCR and SIP results were used to refine the site conceptual model by identifying areas where that genetic potential was actively being expressed and locations where biodegradation was lagging.
Increasingly, molecular biological tools, most notably quantitative polymerase chain reaction (qP... more Increasingly, molecular biological tools, most notably quantitative polymerase chain reaction (qPCR), are being employed to provide a more comprehensive assessment of bioremediation of petroleum hydrocarbons and fuel oxygenates. While qPCR enumeration of key organisms or catabolic genes can aid in site management decisions, evaluation of site activities conducted to stimulate biodegradation would ideally include a direct measure of gene expression to infer activity. In the current study, reverse-transcriptase (RT) qPCR was used to monitor gene expression to evaluate the effectiveness of an oxygen infusion system to promote biodegradation of BTEX and MTBE. During system operation, dissolved oxygen (DO) levels at the infusion points were greater than 30 mg/L, contaminant concentrations decreased, and transcription of two aromatic oxygenase genes and Methylibium petroleiphilum PM1-like 16S rRNA copies increased by as many as 5 orders of magnitude. Moreover, aromatic oxygenase gene transcription and PM1 16s rRNA increased at downgradient locations despite low DO levels even during system operation. Conversely, target gene expression substantially decreased when the system was deactivated. RT-qPCR results also corresponded to increases in benzene and MTBE attenuation rates. Overall, monitoring gene expression complemented traditional groundwater analyses and conclusively demonstrated that the oxygen infusion system promoted BTEX and MTBE biodegradation.
NABIR UMTRA Old Rifle Biostimulation Sediment Data Correlations between modifying electron donor ... more NABIR UMTRA Old Rifle Biostimulation Sediment Data Correlations between modifying electron donor and acceptor accessibility, the in-situ microbial community, and bioreduction of Uranium at the FRC and UMTRA research sites indicated that significant modifications in the rate, amount and by inference the potential stability of immobilized Uranium are feasible in these environments. The in-situ microbial community at these sites was assessed with a combination of lipid and real-time molecular techniques providing quantitative insights of effects of electron donor and manipulations.
Bioremediation can be an effective treatment strategy at contaminated sites when applied appropri... more Bioremediation can be an effective treatment strategy at contaminated sites when applied appropriately and implemented properly. Thus it is important to have methods that can be used to reliably assess biodegradation potential and ultimately the feasibility and performance of bioremediation as a treatment approach. While an important component of site monitoring, decreasing trends in contaminant concentrations can result from physical processes, do not document that loss is due to biodegradation, and can be difficult to discern in practice. Therefore direct measures of microbes responsible for biodegradation of compounds have been developed. These methods fall into two categories: direct measure of biodegradation functional genes using methods such as quantitative PCR and microarrays and indirect measures in changes in microbial community structure using methods such as PCR-DGGE and T-RFLP when target genes are unknown. This chapter describes the various molecular methods being used and examples where they have been applied for the assessment of environmental samples.
Ground Water Monitoring and Remediation, Mar 19, 2019
This column reviews the general features of PHT3D Version 2, a reactive multicomponent transport ... more This column reviews the general features of PHT3D Version 2, a reactive multicomponent transport model that couples the geochemical modeling software PHREEQC-2 (Parkhurst and Appelo 1999) with three-dimensional groundwater flow and transport simulators MODFLOW-2000 and MT3DMS (Zheng and Wang 1999). The original version of PHT3D was developed by Henning Prommer and Version 2 by Henning Prommer and Vincent Post (Prommer and Post 2010). More detailed information about PHT3D is available at the website http://www.pht3d.org. The review was conducted separately by two reviewers. This column is presented in two parts.
Bio-Trap ®-based in situ microcosm studies were conducted to evaluate EHC-M ® stimulated degradat... more Bio-Trap ®-based in situ microcosm studies were conducted to evaluate EHC-M ® stimulated degradation of mono-, di-, and trichlorobenzenes in anaerobic groundwater at a site in Michigan. The data show that the EHC-M ® amendment stimulated an overall increase in microbial activity and a shift in the microbial community structure, indicating more reduced conditions. Stable isotope probing with 13 C 6-chlorobenzene demonstrated attenuation of chlorobenzene and subsequent separation and characterization of the 12 C-and 13 C-deoxyribonucleic acid (DNA) fractions were used to identify the attenuating microbes. These data clearly show the participation of an obligate aerobe in the chlorobenzene biodegradation process. Decreases in concentrations of trichlorobenzenes were also observed in comparison to a control. Due to the thermodynamically favorable reducing conditions stimulated by EHC-M ® , the mechanism of degradation of the trichlorobenzenes is presumed to be reductive dehalogenation. However, on the strength of the DNA-based analysis of microbial community structure, concurrent microaerophilic degradation of chlorobenzene or its metabolites was definitively demonstrated and cannot be ruled out for the other chlorobenzenes. O
The objective of this work was to field‐demonstrate the Min‐Trap® sampler technology, a new in‐si... more The objective of this work was to field‐demonstrate the Min‐Trap® sampler technology, a new in‐situ passive monitoring tool that offers distinct advantages for collecting mineralogical data. The Min‐Trap consists of a solid porous medium (e.g., silica sand) contained within a water‐permeable mesh and support housing that is deployed inside a monitoring well. The porous medium serves as a solid substrate upon which target minerals passively form. Analysis of the sample medium using chemical, microscopic, spectroscopic, or other techniques can provide direct evidence of geochemical conditions and the formation of target minerals in‐situ. The abiotic degradation of chlorinated volatile organic compounds via the reducing power stored in reactive minerals (e.g., iron sulfides) is a recognized important process, and Min‐Traps are well suited to document the formation of these minerals during remediation. Min‐Traps were tested in multiple wells at two field sites undergoing active remediat...
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