Olivia Bulka's research while affiliated with University of Toronto and other places

Chloroform (CF) and dichloromethane (DCM) are groundwater contaminants of concern due to their high toxicity and inhibition of important biogeochemical processes such as methanogenesis. Anaerobic biotransformation of CF and DCM has been well documented but typically independently of one another. CF is the electron acceptor for certain organohalide-respiring bacteria that use reductive dehalogenases (RDases) to dechlorinate CF to DCM. In contrast, known DCM degraders use DCM as their electron donor, which is oxidized using a series of methyltransferases and associated proteins encoded by the mec cassette to facilitate the entry of DCM to the Wood–Ljungdahl pathway. The SC05 culture is an enrichment culture sold commercially for bioaugmentation, which transforms CF via DCM to CO2. This culture has the unique ability to dechlorinate CF to DCM using electron equivalents provided by the oxidation of DCM to CO2. Here, we use metagenomic and metaproteomic analyses to identify the functional genes involved in each of these transformations. Though 91 metagenome-assembled genomes were assembled, the genes for an RDase—named acdA—and a complete mec cassette were found to be encoded on a single contig belonging to Dehalobacter. AcdA and critical Mec proteins were also highly expressed by the culture. Heterologously expressed AcdA dechlorinated CF and other chloroalkanes but had 100-fold lower activity on DCM. Overall, the high expression of Mec proteins and the activity of AcdA suggest a Dehalobacter capable of dechlorination of CF to DCM and subsequent mineralization of DCM using the mec cassette. IMPORTANCE Chloroform (CF) and dichloromethane (DCM) are regulated groundwater contaminants. A cost-effective approach to remove these pollutants from contaminated groundwater is to employ microbes that transform CF and DCM as part of their metabolism, thus depleting the contamination as the microbes continue to grow. In this work, we investigate bioaugmentation culture SC05, a mixed microbial consortium that effectively and simultaneously degrades both CF and DCM coupled to the growth of Dehalobacter. We identified the functional genes responsible for the transformation of CF and DCM in SC05. These genetic biomarkers provide a means to monitor the remediation process in the field.

Chloroform (CF) and dichloromethane (DCM) are groundwater contaminants of concern due to their high toxicity and inhibition of important biogeochemical processes. Biotransformation of CF and DCM have been well documented but always independent of one another. CF is dechlorinated to DCM by organohalide-respiring bacteria using reductive dehalogenases (RDases), while known DCM-degraders either ferment or mineralize DCM, both of which use the mec cassette to facilitate the entry of DCM to the Wood-Ljungdahl pathway. The SC05 culture, used commercially for bioaugmentation, is the first and only known stable enrichment culture to transform CF and DCM simultaneously. Here we use metagenomic and metaproteomic analysis to identify the functional genes involved in each of these transformations. A single Dehalobacter metagenome assembled genome contains the genes for an RDase, named acdA , and a complete mec cassette encoded on a single contig. AcdA and several of the Mec proteins were also highly expressed. Furthermore, AcdA displayed high dechlorination activity on CF and other chloroalkanes but did not show significant dechlorination of DCM. Overall, the high expression of Mec proteins and the activity of AcdA suggest a Dehalobacter capable of dechlorination of CF to DCM, and subsequent mineralization of DCM using the mec cassette. Importance Chloroform (CF) and dichloromethane (DCM) are harmful groundwater contaminants. SC05 is the first anaerobic microbial culture that can effectively and simultaneously remove both CF and DCM. Here we identify the key CF and DCM-degrading pathways in the culture, which are both found in a Dehalobacter strain. This Dehalobacter contains and expresses all the genes necessary for both CF dechlorination and DCM mineralization, which has never been observed. Identifying these functional genes will improve our knowledge of how CF and DCM bioremediation progress and provide means to monitor the process when SC05 is deployed in the field.

Chloroform (CF) and dichloromethane (DCM) contaminate groundwater sites around the world, which can be remediated through bioaugmentation. Although several strains of Dehalobacter restrictus can reduce CF to DCM, and multiple Peptococcaceae can ferment DCM, these processes cannot happen simultaneously due to CF sensitivity in the known DCM-degraders or electron donor competition. Here we present a mixed microbial culture that can simultaneously metabolize CF and DCM to carbon dioxide and create an additional enrichment culture fed only DCM. Through species-specific qPCR, we find that a Dehalobacter strain grows both while CF alone and DCM alone are converted, indicating its involvement in both metabolic steps. Additionally, the culture was maintained for over 1400 days without addition of exogenous electron donor, and through electron balance calculations we show that DCM mineralization produces sufficient reducing equivalents (likely hydrogen) for CF respiration. Together, these results suggest intraspecies electron transfer could occur to continually reduce CF in the culture. Minimizing the addition of electron donor reduces the cost of bioremediation, and understanding this mechanism informs strategies for culture maintenance and scale-up, and benefits contaminated sites where the culture is employed for remediation worldwide. SYNOPSIS Dechlorination of chloroform to dichloromethane and dichloromethane mineralization are performed concurrently by a Dehalobacter -containing mixed microbial community without provision of exogenous electron donor. TOC ART

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