Final Report Summary - ANDEMIC (Unraveling key abiotic and biotic interactions for sustainable anaerobic biotransformation of chlorinated solvents in groundwater)
Chlorinated solvents such as tetrachloroethene/perchloroethene (PCE) and trichloroethene (TCE) and their daughter products cis-dichloroethene (cDCE) and vinyl chloride (VC) are potential carcinogens frequently found in groundwater and are classified as priority pollutants. Owing to their toxicity, even small spills render groundwater unsuitable for use, and cleanup is typically a costly and long-term undertaking. Development of effective, economically-feasible and environmentally-friendly technologies is therefore of great interest for the clean-up and remediation of groundwater and sites contaminated with these compounds.
Biological transformation of chlorinated solvents to non-toxic products is possible. The main biotransformation mechanism in nature is anaerobic reductive dechlorination, a process where chlorine atoms are sequentially removed and replaced by H atoms resulting in non-toxic ethene as ultimate product: PCE>TCE>cDCE>VC>ethene. Dehalococcoides (Phylum Chloroflexi) is the only organism shown so far of dechlorinating beyond cDCE and VC. Remarkably, Dehalococcoides obtains energy from this process for growth and cell maintenance. Rather than acting alone Dehalococcoides lives in microbial communities or consortia relying on other organisms to provide essential nutrients such as vitamins, an organic carbon source such as acetate and molecular hydrogen as an electron donor.
Bioaugmentation – the addition of active microbial cultures to a contaminated site – is a low-cost and potentially highly effective remediation alternative, particularly for chlorinated solvents, where dechlorination is carried out by very specialized microbes not present at every site. However, there remain serious scientific gaps to more widespread adoption of bioremediation and bioaugmentation. Diagnostics and prognostic monitoring approaches are critically needed to improve our ability to sustain high rates of microbial activity and make bioremediation more reliable and more predictable.
To overcome such limitations this project focuses on various aspects critical to improving and expanding the use of chlorinated solvent bioremediation. A stable microbial consortium (KB-1) developed in the Edwards’ lab (Univ. of Toronto) and used commercially for bioaugmentation at field sites was used as a model for lab-based and field experiments.
To successfully expand the use of bioremediation to uncommon and challenging settings such as fracture bedrock it is important to understand the abiotic-biotic and biotic-biotic interactions that influence microbial activity in the subsurface. In collaboration with industrial partners (Geosyntec Consultants and SiREM Labs, Guelph, Canada) a bioremediation field demonstration in fractured bedrock was conducted for the first time in Canada. While bioremediation has been successfully used at many sites with porous granular media, there is scant information on the feasibility of bioremediation approaches for treatment of chlorinated compounds in fractured bedrock, which poses a remedial challenge to any type of technology due to unpredictable groundwater flow patterns within the fractures. Rapid and complete reductive dechlorination could be achieved in those wells best hydraulically-connected to the re-circulation system; the latter was implemented to deliver and mix the fermentable substrate acting as electron donor (ethanol) and the microorganisms involved in the biotransformation process. In addition we applied a combined approach for monitoring biogeochemical processes in groundwater including: i) a tracer test to understand well-connectivity ii) monitoring of contaminant concentrations and other physical (e.g. temperature) and chemical (e.g. pH, sulfate and sulfide concentrations) groundwater properties and iii) monitoring of dechlorinating and non-dechlorinating microorganisms. In this manner, an improved understanding of the relationship between groundwater geochemical properties and microbial activity was gained and potential reasons for treatment failure or success at specific locations identified. This pioneering field demonstration opens up new possibilities for expanding the use of bioremediation to fractured bedrock settings and sites with average cool groundwater temperatures characteristic of colder climates.
Monitoring of biodegradative processes in the field can be substantially improved through the utilization of biomarkers such as those derived from molecular screens and isotopic fractionation signatures. In this project we developed various molecular probes targeting microorganisms within the KB-1 culture which might be critical to the activity of Dehalococcoides and ultimately the success of bioremediation. In addition, molecular probes were designed targeting functional genes (reductive dehalogenase genes) which allow discriminating between Dehalococcoides used for bioaugmentation, that is, from KB-1 and Dehalococcoides native to the contaminated site. This is important to verify whether the introduced Dehalococcoides is responsible for the bioremediation or the Dehalococcoides intrinsic to the site is doing the job.
In collaboration with industrial and academic partners in Canada and the US a microbial internal standard (MIS) that works in a similar manner as chemical internal standards in analytical chemistry was developed and validated. This MIS can be of great use for normalizing data related to the quantification of Dehalococcoides from different samples or processed by different users and methodologies. The idea is to develop tools that allow the standardization of microbial analysis in groundwater.
At bioremediated contaminated sites poor mass balance is commonly observed, that is, the amount of ethene produced is usually lower than the amount of chlorinated solvent present. We modeled the changes in C stable isotope ratios associated with the biodegradation of ethene in lab microcosms. The isotopic signature obtained was then used at field sites to evaluate whether poor mass balance and ethene disappearance was due to biological transformations or loss through dissolution or dispersion of ethene. This new monitoring tool provides new insights into the progress of bioremediation and can help site managers demonstrate to stakeholders and regulators that the remediation technology is progressing successfully.
Rather than acting as individual cells Dehalococcoides lives in consortia with many different species working together akin to a multicellular life form. Novel approaches are required to improve our knowledge at the molecular level of the unusual metabolism of such consortia. For this purpose we developed a non-targeted approach for the study of small molecules or metabolites (molecular mass between 150 and 2000Da) using high resolution mass spectrometry and an in-house data analysis pipeline which includes multivariate data analysis and mass defect network annotation of mass signals. This approach was used to test the response of the model mixed dechlorinating culture KB-1 and other dechlorinating cultures in the presence of different electron acceptors and also over time, from slow-dechlorinating to fast-dechlorinating cultures. This methodology allowed the annotation-the assignment of molecular formula to mass signal- of thousands of signals from the mass spectrometry analysis as well as the identification of masses or metabolic pathways that are relevant to certain responses or growth conditions. This discovery driven-approach opens up new possibilities for unraveling the biology of microbial communities and design targeted studies.
The scientific information acquired and the tools developed in AnDeMic might be extended to other consortia as well as to microbial systems used in other applications. Ultimately the knowledge gained through this project will contribute to design better strategies for the bioremediation of chlorinated solvents in contaminated aquifers.
Biological transformation of chlorinated solvents to non-toxic products is possible. The main biotransformation mechanism in nature is anaerobic reductive dechlorination, a process where chlorine atoms are sequentially removed and replaced by H atoms resulting in non-toxic ethene as ultimate product: PCE>TCE>cDCE>VC>ethene. Dehalococcoides (Phylum Chloroflexi) is the only organism shown so far of dechlorinating beyond cDCE and VC. Remarkably, Dehalococcoides obtains energy from this process for growth and cell maintenance. Rather than acting alone Dehalococcoides lives in microbial communities or consortia relying on other organisms to provide essential nutrients such as vitamins, an organic carbon source such as acetate and molecular hydrogen as an electron donor.
Bioaugmentation – the addition of active microbial cultures to a contaminated site – is a low-cost and potentially highly effective remediation alternative, particularly for chlorinated solvents, where dechlorination is carried out by very specialized microbes not present at every site. However, there remain serious scientific gaps to more widespread adoption of bioremediation and bioaugmentation. Diagnostics and prognostic monitoring approaches are critically needed to improve our ability to sustain high rates of microbial activity and make bioremediation more reliable and more predictable.
To overcome such limitations this project focuses on various aspects critical to improving and expanding the use of chlorinated solvent bioremediation. A stable microbial consortium (KB-1) developed in the Edwards’ lab (Univ. of Toronto) and used commercially for bioaugmentation at field sites was used as a model for lab-based and field experiments.
To successfully expand the use of bioremediation to uncommon and challenging settings such as fracture bedrock it is important to understand the abiotic-biotic and biotic-biotic interactions that influence microbial activity in the subsurface. In collaboration with industrial partners (Geosyntec Consultants and SiREM Labs, Guelph, Canada) a bioremediation field demonstration in fractured bedrock was conducted for the first time in Canada. While bioremediation has been successfully used at many sites with porous granular media, there is scant information on the feasibility of bioremediation approaches for treatment of chlorinated compounds in fractured bedrock, which poses a remedial challenge to any type of technology due to unpredictable groundwater flow patterns within the fractures. Rapid and complete reductive dechlorination could be achieved in those wells best hydraulically-connected to the re-circulation system; the latter was implemented to deliver and mix the fermentable substrate acting as electron donor (ethanol) and the microorganisms involved in the biotransformation process. In addition we applied a combined approach for monitoring biogeochemical processes in groundwater including: i) a tracer test to understand well-connectivity ii) monitoring of contaminant concentrations and other physical (e.g. temperature) and chemical (e.g. pH, sulfate and sulfide concentrations) groundwater properties and iii) monitoring of dechlorinating and non-dechlorinating microorganisms. In this manner, an improved understanding of the relationship between groundwater geochemical properties and microbial activity was gained and potential reasons for treatment failure or success at specific locations identified. This pioneering field demonstration opens up new possibilities for expanding the use of bioremediation to fractured bedrock settings and sites with average cool groundwater temperatures characteristic of colder climates.
Monitoring of biodegradative processes in the field can be substantially improved through the utilization of biomarkers such as those derived from molecular screens and isotopic fractionation signatures. In this project we developed various molecular probes targeting microorganisms within the KB-1 culture which might be critical to the activity of Dehalococcoides and ultimately the success of bioremediation. In addition, molecular probes were designed targeting functional genes (reductive dehalogenase genes) which allow discriminating between Dehalococcoides used for bioaugmentation, that is, from KB-1 and Dehalococcoides native to the contaminated site. This is important to verify whether the introduced Dehalococcoides is responsible for the bioremediation or the Dehalococcoides intrinsic to the site is doing the job.
In collaboration with industrial and academic partners in Canada and the US a microbial internal standard (MIS) that works in a similar manner as chemical internal standards in analytical chemistry was developed and validated. This MIS can be of great use for normalizing data related to the quantification of Dehalococcoides from different samples or processed by different users and methodologies. The idea is to develop tools that allow the standardization of microbial analysis in groundwater.
At bioremediated contaminated sites poor mass balance is commonly observed, that is, the amount of ethene produced is usually lower than the amount of chlorinated solvent present. We modeled the changes in C stable isotope ratios associated with the biodegradation of ethene in lab microcosms. The isotopic signature obtained was then used at field sites to evaluate whether poor mass balance and ethene disappearance was due to biological transformations or loss through dissolution or dispersion of ethene. This new monitoring tool provides new insights into the progress of bioremediation and can help site managers demonstrate to stakeholders and regulators that the remediation technology is progressing successfully.
Rather than acting as individual cells Dehalococcoides lives in consortia with many different species working together akin to a multicellular life form. Novel approaches are required to improve our knowledge at the molecular level of the unusual metabolism of such consortia. For this purpose we developed a non-targeted approach for the study of small molecules or metabolites (molecular mass between 150 and 2000Da) using high resolution mass spectrometry and an in-house data analysis pipeline which includes multivariate data analysis and mass defect network annotation of mass signals. This approach was used to test the response of the model mixed dechlorinating culture KB-1 and other dechlorinating cultures in the presence of different electron acceptors and also over time, from slow-dechlorinating to fast-dechlorinating cultures. This methodology allowed the annotation-the assignment of molecular formula to mass signal- of thousands of signals from the mass spectrometry analysis as well as the identification of masses or metabolic pathways that are relevant to certain responses or growth conditions. This discovery driven-approach opens up new possibilities for unraveling the biology of microbial communities and design targeted studies.
The scientific information acquired and the tools developed in AnDeMic might be extended to other consortia as well as to microbial systems used in other applications. Ultimately the knowledge gained through this project will contribute to design better strategies for the bioremediation of chlorinated solvents in contaminated aquifers.