Metabolite-profiling to assess <italic>in<italic><italic> situ<italic> anaerobic microbial hydrocarbon degradation in diverse environments

Abstract

It is evident that complete mineralization of hydrocarbons and related compounds can occur under anaerobic conditions. A large variety of hydrocarbon classes have been shown to be amenable to decay by enriched microbial consortia and to a lesser extent, pure cultures. With the exception of anaerobic methane oxidation, significant advances have been made towards understanding the mechanistic nature of anaerobic hydrocarbon activation and mineralization over the last 20 years. The ability to detect metabolic intermediates formed by biodegradation of petroleum-related compounds (alkylbenzene, polycyclic aromatic, n-alkane and alicyclic) during cultivation of single isolates, enrichments, and most importantly from in situ field experiments has become a valuable tool for garnering real-time data about the microbial processes occurring in hydrocarbon laden environments, and assessing the loss of specific contaminants in complex mixtures. For this thesis, field and laboratory approaches were used to evaluate metabolic processes that contribute to the loss of hydrocarbons in diverse environments. The petroleum constituents were either concentrated via anthropogenic activities or naturally occurring (e.g. reservoirs, methane hydrates), and were analyzed to assay for signature metabolites that represent evidence of fundamental pathways for anaerobic hydrocarbon metabolism.

At a former refinery site in Casper, WY, a combination of field and laboratory approaches were used to determine the feasibility of natural attenuation as a remediation strategy for the surrounding hydrocarbon-contaminated aquifer and sediments. The application of field metabolomics revealed several metabolic intermediates, from a variety of hydrocarbon compound classes, within the petroleum-laden aquifer and substantiated the hypothesis that biodegradation is occurring within the environment. However, laboratory incubations using hydrocarbon-saturated sediments demonstrated a relative recalcitrance of contaminants of regulatory interest (benzene, ethylbenzene, 1,2,4- and 1,3,5-trimethylbenzene and 2-methylnaphthalene) that have been previously shown to be susceptible to anaerobic biodegradation. These findings were not due to the toxicity of the hydrocarbons (as validated by PLFA analysis), nor a limitation imposed by the terminal electron-accepting conditions (as indicated by continual sulfate-reduction). In addition, it was suggested that the persistence of benzene was due to the lack of endogenous microorganisms capable of anaerobic biodegradation of the aromatic compound. Thus, a precedent in the literature for the biodegradation of individual hydrocarbons does not necessarily deem these processes viable in all environmental systems. It is not only important to understand the activity of the microbial populations in the aquifer, but also to understand the abiotic processes that may limit or obscure the detection of biological processes in heterogeneous hydrocarbon mixtures.

Biocorrosion is a process that is potentially fueled by the turnover of hydrocarbons into organic acids and carbon dioxide under anaerobic conditions. Samples were collected from the Alaskan North Slope (ANS) oil fields in 2006 and 2008. Two sampling campaigns in Field B revealed alkysuccinic acids in the range of C1-C4, suggesting the addition of methane, ethane, propane and butane to fumarate, an analogous reaction that also occurs with higher molecular weight n-alkanes. Hyperthermophilic communities that may be involved in the production of low molecular weight alkylsuccinates were comprised of several types of Bacteria (syntrophs, fermentative, sulfate/sulfur reducing, iron-reducing) and methanogenic and sulfate-reducing Archaea. In 2008, an analysis of more than 50 samples from the A and B oil fields revealed akylbenzylsuccinates, benzoate associated intermediates, monoaromatic metabolites, naphthoic acids and ring-reduced naphthoates, alkylsuccinates and alkanoic acids. There were differences in Field A and B, specifically in Field B there were a greater number of metabolites, and similar metabolites from sample to sample. While fumarate addition has been widely accepted as the predominant mechanism for anaerobic hydrocarbon decay, the detection of metabolites related to carboxylation and hydroxylation reactions suggest that these processes should be further investigated, and that their metabolic importance in the functioning of microbial communities in petroleum reservoirs should not be discounted.

Methane is the shortest n-alkane, and is the most abundant hydrocarbon in the Earth\'s atmosphere. Though reverse methanogenesis is the current dogma for the anaerobic oxidation of methane (AOM), I proposed that methane is activated by addition to fumarate in a manner similar to that of ethane, propane, butane and longer n-alkanes. Previous investigations in petroleum-laden environments revealed the presence of methylsuccinate, particularly in oil field reservoirs and hydrocarbon-contaminated sediments. Laboratory incubations with sediments known to oxidize methane under anaerobic conditions revealed a suite of 13C -labeled downstream metabolic intermediates, suggesting the addition of methane to fumarate. The evidence for downstream metabolites does not refute reverse methanogenesis as the mechanism for AOM, nor does it provide direct evidence for the addition of methane to fumarate. However, this finding suggests that multiple pathways for the cycling of methane in anaerobic environments may exist, and that additional research should be conducted to characterize the biochemistry of this process