Abstract
The widespread use of hydrocarbon-based fuels has led to the contamination of many natural environments due to accidental spills or leaks. While anaerobic microorganisms indigenous to many fuel-contaminated groundwater sites can play a role in site remediation (e.g., monitored natural attenuation, MNA) via hydrocarbon biodegradation, multiple lines of evidence in support of such bioremediation are required. In this study, we investigated two fuel-contaminated groundwater sites for their potential to be managed by MNA. Microbial community composition, biogeochemical indicators, fumarate addition metabolites, and genes diagnostic of both alkane and alkyl-monoaromatic hydrocarbon activation were assessed. Fumarate addition metabolites and catabolic genes were detected for both classes of hydrocarbon biodegradation at both sites, providing strong evidence for in situ anaerobic hydrocarbon biodegradation. However, relevant metabolites and genes did not consistently co-occur within all groundwater samples. Using newly designed mixtures of quantitative polymerase chain reaction (qPCR) primers to target diverse assA and bssA genes, we measured assA gene abundances ranging from 105–108 copies/L, and bssA gene abundances ranging from 105–1010 copies/L at the sites. Overall, this study demonstrates the value of investigating fuel-contaminated sites using both metabolites and genes diagnostic of anaerobic hydrocarbon biodegradation for different classes of hydrocarbons to help assess field sites for management by MNA.
Highlights
The use of petroleum-based fuels to serve our global energy needs has resulted in the contamination of many pristine environments with hydrocarbons due to failing infrastructure or accidental spills
Several sampled wells collected from both sites A (Figure 1) and B (Figure 2) were characterized by BTEX (benzene, toluene, ethylbenzene, and xylenes) and alkane concentrations above the Alberta guidelines for allowable concentrations in non-drinking groundwater (benzene = 0.005 ppm; toluene = 0.024 ppm; ethylbenzene = 0.0016 ppm; xylenes = 0.02 ppm; alkanes = 2.2 ppm; [45]; see Table S1 in Supplementary Materials for tabulated hydrocarbon concentrations measured at both sites)
While the biogeochemical measurements suggested that nitrate, sulfate, or iron reduction could be electron acceptors in different portions of the site, it was difficult to discern clear trends or patterns that relate hydrocarbon concentrations with anaerobic electron accepting processes across the site. This heterogeneity was further exemplified by the microbial community profiling results, where microbial community compositions were different in each well sampled (Figure 3)
Summary
The use of petroleum-based fuels to serve our global energy needs has resulted in the contamination of many pristine environments with hydrocarbons due to failing infrastructure or accidental spills. In the absence of oxygen that serves as both a terminal electron acceptor and a powerful oxidant, anaerobic microorganisms employ distinct mechanisms for overcoming the stability of hydrocarbons for use as carbon and energy sources [6]. For unsubstituted hydrocarbons, such as benzene, naphthalene, or phenanthrene, evidence is increasingly pointing to carboxylation as an important activation step [7,8,9,10], the mechanism is not fully understood. This mechanism, commonly referred to as ‘fumarate addition’, is the most well-studied mechanism of hydrocarbon activation under anoxic conditions, and will be the focus of the work described here
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