Microbial-mediated hydrocarbon transformation plays a vital role in the attenuation of natural and anthropogenic-sourced petroleum contamination in the environment, particularly in marine systems. Indigenous microbial communities in marine habitats are resilient to influxes of petroleum, and it is well documented that many taxa are capable of responding and utilizing these compounds. Coastal ecosystems are often either subjected to or at risk for oil contamination and are of particular concern due to their significant environmental and economic value. The research projects presented here focused on coastal ecosystems and investigated microbial community compositions via next-generation sequencing of 16S rRNA genes, the genetic potential for anaerobic hydrocarbon biodegradation within these communities via molecular surveys of marker genes, and the response of anaerobic populations to exposure of a hydrocarbon via microcosm studies or to products of hydrocarbon transformation processes (i.e. photolysis) via sulfate reduction assays (SRAs).

Chesapeake Bay is the largest estuary in the United States, and experiences high nutrient loading and water column hypoxia due to watershed runoff, as well as petroleum contamination from urban runoff, atmospheric deposition, and spills directly into the water column. Past studies have demonstrated that aerobic hydrocarbon-degrading bacteria can be enriched from the water column and from the sediment. However, evidence for anaerobic biodegradation of hydrocarbons had not been demonstrated at the time of our study. Given the recurring seasonal water column hypoxia and the transient exposure to hydrocarbons, we hypothesized that the potential for degradation under anaerobic conditions may exist in Chesapeake Bay sediments. Here, molecular surveys and microcosms were utilized to investigate microbial community composition and the potential for anaerobic hydrocarbon degradation among sites along a transect of the Bay. Sampling locations were chosen both within and outside areas of recurring hypoxia. Distinct geochemical gradients along the transect were revealed. Low oxygen, low sulfate, and high methane concentrations were observed in the upper Bay, as were significantly higher levels of taxa associated with anaerobic processes (e.g., sulfate reducers and methanogens). In contrast, higher oxygen, higher sulfate, and very low methane were measured in the lower Bay. Sulfate-reducers and methanogens decreased in abundance in lower Bay sediments as well. Similarly, molecular surveys showed more frequent detection of marker genes associated with the anaerobic activation of hydrocarbons via the ‘fumarate addition’ pathway (e.g., assA, bssA) in the upper Bay, and microcosms established under sulfate-reducing and/or methanogenic conditions suggested that the model hydrocarbon, hexadecane, was being converted to methane by indigenous sediment communities obtained from the upper Bay sites. These findings illustrate the variability of microbial communities between different locations in Chesapeake Bay as well as differences in their response to a hydrocarbon. Together, the data highlighted the significance that anaerobic processes could potentially play in the event of an oil spill in Chesapeake Bay.

The Gulf of Mexico (GoM) is one of the most environmentally and economically important coastal regions in the United States. The Deepwater Horizon (DWH) spill in the GoM was the largest accidental release of crude oil into U.S. waters. Extensive research was carried out on the response of microbial communities to the discharged oil and gas. Collectively, studies emphasized the importance of both aerobic and anaerobic hydrocarbon transformation processes and concluded that native microbial populations responded quickly to the petroleum, promoting contaminant removal from the environment. Two of the research projects presented herein aimed to (1) further study the impact that released oil, once weathered, can have on indigenous anaerobic microbial communities, and to (2) characterize microbial populations associated with weathered oil residues (i.e., sand patties) that have remained in the environment years after the spill and to determine the role these populations have in the attenuation of residual contamination.

Once introduced into the environment, oil is subjected to a number of weathering processes, including evaporation, emulsification, and photooxidation. Photooxidation of oil can lead to the incorporation of oxygen molecules into hydrocarbon constituents, which can subsequently result in enhanced bioavailability and/or increased toxicity to certain organisms. Microbial toxicity studies are typically conducted using individual aerobic taxa, as opposed to indigenous communities or anaerobic microorganisms, and little is known with regard to how photolyzed oil affects anaerobes. Experiments presented here assessed the impact that photooxidized hydrocarbons can have on sulfate-reducing communities in coastal sediments. We hypothesized that photolyzed oil or photolyzed oil components would inhibit the sulfate-reducing communities. Three distinct GoM coastal locations were chosen for study. Sediment microbial communities were characterized via 16S rRNA gene sequencing, and the impact of irradiated crude oil or irradiated PAHs (i.e., pyrene, phenanthrene, and a phenanthrene/anthracene mixture) was tested via sulfate reduction assays (SRAs). Sulfate-reducing taxa varied in both abundance and composition across sampling sites. Overall, no impact on sulfate reduction rates was observed for any of the photolyzed compounds at any of the coastal locations investigated. Data suggested that water-soluble photogenerated products did not negatively impact sulfate-reducing communities and that these compounds could potentially be utilized by sulfate-reducing microorganisms. These findings highlight the resilience of native microbial communities in response to an influx of weathered hydrocarbons, as well as the potential of these populations to further mediate hydrocarbon transformation processes.

Weathering of oil released during the DWH spill also led to the formation of water-in-oil emulsions. Many of these emulsions washed ashore early after the onset of the spill, whereas an unknown quantity sank in nearshore environments, resulting in the formation of submerged oil mats (SOMs). Fragments of these buried mats continued to wash ashore coastal beaches and marshes years after the spill in the form of oil:sand aggregates (e.g., tar balls, sand patties, etc.). The third research project presented here aimed to use next-generation sequencing of 16S rRNA genes to characterize microbial communities associated with individual oil:sand aggregates collected from different GoM beaches, to use metagenomic sequencing to survey for marker genes associated with hydrocarbon transformation pathways to determine the genetic capacity for biodegradation within the microbial populations, and to conduct targeted metabolomics via mass spectrometry to assess whether these communities mediate transformation of hydrocarbons in situ (i.e., once aggregates are deposited on the beach). Given the presumed differences in residence times and exposure to different environmental conditions, we hypothesized that sand patty microbial communities would be different between sites. Together, molecular surveys demonstrated that individual aggregates had either an anaerobic, facultative anaerobic, or aerobic signature with regard to both the taxonomic composition of communities and the metabolic potential associated with hydrocarbon degradation pathways. Several taxa with known or suspected hydrocarbon-degrading ability were detected (e.g., Marinobacter, Alcanivorax, Mycobacterium), and specific taxa varied among samples. Additionally, profiles of functional genes involved in aerobic and anaerobic hydrocarbon transformation pathways (e.g., assA, alkB) also varied among samples and corresponded with 16S rRNA gene profiles. Results from beach sand and seawater samples confirmed that microbial populations were distinct from those obtained from sand patties. Taxonomic profiles of core communities (i.e., taxa comprising ≥1% of libraries) identified ten shared operational taxonomic units (OTUs) between aggregates and beach sand and seven shared OTUs between aggregates and seawater. Targeted mass spectrometry putatively identified metabolites indicative of aerobic and/or anaerobic hydrocarbon transformation processes (e.g., toluic acid, hydroxybenzoic acid, phenylpropionic acid), and showed that these compounds were not detected in beach sand. These findings provide evidence that aggregate-associated microbes are capable of hydrocarbon degradation and also highlight the potential role that microorganisms likely play in the long-term attenuation of remnant oil present in the environment years after the DWH spill.