Amy V. Callaghan

Metagenomic analysis and metabolite profiling of deep–sea sediments from the Gulf of Mexico following the Deepwater Horizon oil spill

Marine subsurface environments such as deep-sea sediments, house abundant and diverse microbial communities that are believed to influence large-scale geochemical processes. These processes include the biotransformation and mineralization of numerous petroleum constituents. Thus, microbial communities in the Gulf of Mexico are thought to be responsible for the intrinsic bioremediation of crude oil released by the Deepwater Horizon (DWH) oil spill. While hydrocarbon contamination is known to enrich for aerobic, oil-degrading bacteria in deep-seawater habitats, relatively little is known about the response of communities in deep-sea sediments, where low oxygen levels may hinder such a response. Here, we examined the hypothesis that increased hydrocarbon exposure results in an altered sediment microbial community structure that reflects the prospects for oil biodegradation under the prevailing conditions. We explore this hypothesis using metagenomic analysis and metabolite profiling of deep-sea sediment samples following the DWH oil spill. The presence of aerobic microbial communities and associated functional genes was consistent among all samples, whereas, a greater number of Deltaproteobacteria and anaerobic functional genes were found in sediments closest to the DWH blowout site. Metabolite profiling also revealed a greater number of putative metabolites in sediments surrounding the blowout zone relative to a background site located 127 km away. The mass spectral analysis of the putative metabolites revealed that alkylsuccinates remained below detection levels, but a homologous series of benzylsuccinates (with carbon chain lengths from 5 to 10) could be detected. Our findings suggest that increased exposure to hydrocarbons enriches for Deltaproteobacteria, which are known to be capable of anaerobic hydrocarbon metabolism. We also provide evidence for an active microbial community metabolizing aromatic hydrocarbons in deep-sea sediments of the Gulf of Mexico.

The genome sequence of Desulfatibacillum alkenivorans AK-01: a blueprint for anaerobic alkane oxidation

Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.

Field and laboratory studies on the bioconversion of coal to methane in the San Juan Basin

The bioconversion of coal to methane in the San Juan Basin, New Mexico, was investigated. Production waters were analyzed via enrichment studies, metabolite-profiling, and culture-independent methods. Analysis of 16S rRNA gene sequences indicated the presence of methanogens potentially capable of acetoclastic, hydrogenotrophic, and methylotrophic metabolisms, predominantly belonging to the Methanosarcinales and Methanomicrobiales. Incubations of produced water and coal readily produced methane, but there was no correlation between the thermal maturity and methanogenesis. Coal methanogenesis was greater when samples with a greater richness of Firmicutes were utilized. A greater archaeal diversity was observed in the presence of several aromatic and short-chain fatty acid metabolites. Incubations amended with lactate, hydrogen, formate, and short-chain alcohols produced methane above un-amended controls. Methanogenesis from acetate was not observed. Metabolite profiling showed the widespread occurrence of putative aromatic ring intermediates including benzoate, toluic acids, phthalic acids, and cresols. The detection of saturated and unsaturated alkylsuccinic acids indicated n-alkane and cyclic alkane/alkene metabolism. Microarray analysis complemented observations based on hybridization to functional genes related to the anaerobic metabolism of aromatic and aliphatic substrates. These data suggest that coal methanogenesis is unlikely to be limited by methanogen biomass, but rather the activation and degradation of coal constituents.

Microbial transformation of the Deepwater Horizon oil spill—past, present, and future perspectives

The Deepwater Horizon blowout, which occurred on April 20, 2010, resulted in an unprecedented oil spill. Despite a complex effort to cap the well, oil and gas spewed from the site until July 15, 2010. Although a large proportion of the hydrocarbons was depleted via natural processes and human intervention, a substantial portion of the oil remained unaccounted for and impacted multiple ecosystems throughout the Gulf of Mexico. The depth, duration and magnitude of this spill were unique, raising many questions and concerns regarding the fate of the hydrocarbons released. One major question was whether or not microbial communities would be capable of metabolizing the hydrocarbons, and if so, by what mechanisms and to what extent? In this review, we summarize the microbial response to the oil spill as described by studies performed during the past four years, providing an overview of the different responses associated with the water column, surface waters, deep-sea sediments, and coastal sands/sediments. Collectively, these studies provide evidence that the microbial response to the Deepwater Horizon oil spill was rapid and robust, displaying common attenuation mechanisms optimized for low molecular weight aliphatic and aromatic hydrocarbons. In contrast, the lack of evidence for the attenuation of more recalcitrant hydrocarbon components suggests that future work should focus on both the environmental impact and metabolic fate of recalcitrant compounds, such as oxygenated oil components.

Enzymes involved in the anaerobic oxidation of n-alkanes: from methane to long-chain paraffins

Anaerobic microorganisms play key roles in the biogeochemical cycling of methane and non-methane alkanes. To date, there appear to be at least three proposed mechanisms of anaerobic methane oxidation (AOM). The first pathway is mediated by consortia of archaeal anaerobic methane oxidizers and sulfate-reducing bacteria (SRB) via “reverse methanogenesis” and is catalyzed by a homolog of methyl-coenzyme M reductase. The second pathway is also mediated by anaerobic methane oxidizers and SRB, wherein the archaeal members catalyze both methane oxidation and sulfate reduction and zero-valent sulfur is a key intermediate. The third AOM mechanism is a nitrite-dependent, “intra-aerobic” pathway described for the denitrifying bacterium, ‘Candidatus Methylomirabilis oxyfera.’ It is hypothesized that AOM proceeds via reduction of nitrite to nitric oxide, followed by the conversion of two nitric oxide molecules to dinitrogen and molecular oxygen. The latter can be used to functionalize the methane via a particulate methane monooxygenase. With respect to non-methane alkanes, there also appear to be novel mechanisms of activation. The most well-described pathway is the addition of non-methane alkanes across the double bond of fumarate to form alkyl-substituted succinates via the putative glycyl radical enzyme, alkylsuccinate synthase (also known as methylalkylsuccinate synthase). Other proposed mechanisms include anaerobic hydroxylation via ethylbenzene dehydrogenase-like enzymes and an “intra-aerobic” denitrification pathway similar to that described for ‘Methylomirabilis oxyfera.’

Metabolomic investigations of anaerobic hydrocarbon-impacted environments.

Metabolomics is a powerful tool for the assessment of expressed (in vivo or in situ) biological processes. Metabolite profiling is often employed during field investigations of hydrocarbon-laden environments for the purpose(s) of determining the extent of intrinsic or enhanced natural attenuation of contaminants, developing remediation strategies, and/or gaining a better understanding of microbial processes. During the last twenty-five years, the elucidation of anaerobic biodegradation pathways has not only provided metabolic and molecular biomarkers for environmental assessments of anaerobic hydrocarbon metabolism, but also an avenue for integrative field studies. The combination of metabolomics with compound-specific isotope analysis, molecular surveys and/or microcosm studies has demonstrated the need for multiple assessment methods for better resolution of in situ microbial activity.

Biosphere frontiers of subsurface life in the sedimented hydrothermal system of Guaymas Basin.

Temperature is one of the key constraints on the spatial extent, physiological and phylogenetic diversity, and biogeochemical function of subsurface life. A model system to explore these interrelationships should offer a suitable range of geochemical regimes, carbon substrates and temperature gradients under which microbial life can generate energy and sustain itself. In this theory and hypothesis article, we make the case for the hydrothermally heated sediments of Guaymas Basin in the Gulf of California as a suitable model system where extensive temperature and geochemical gradients create distinct niches for active microbial populations in the hydrothermally influenced sedimentary subsurface that in turn intercept and process hydrothermally generated carbon sources. We synthesize the evidence for high-temperature microbial methane cycling and sulfate reduction at Guaymas Basin – with an eye on sulfate-dependent oxidation of abundant alkanes – and demonstrate the energetic feasibility of these latter types of deep subsurface life in previously drilled Guaymas Basin locations of Deep-Sea Drilling Project 64.

Interrogation of Chesapeake Bay sediment microbial communities for intrinsic alkane-utilizing potential under anaerobic conditions.

Based on the transient exposure of Chesapeake Bay sediments to hydrocarbons and the metabolic versatility of known anaerobic alkane-degrading microorganisms, it was hypothesized that distinct Bay sediment communities, governed by geochemical gradients, would have intrinsic alkane-utilizing potential under sulfate-reducing and/or methanogenic conditions. Sediment cores were collected along a transect of the Bay. Community DNA was interrogated via pyrosequencing of 16S rRNA genes, PCR of anaerobic hydrocarbon activation genes, and qPCR of 16S rRNA genes and genes involved in sulfate reduction/methanogenesis. Site sediments were used to establish microcosms amended with n-hexadecane under sulfate-reducing and methanogenic conditions. Sequencing of 16S rRNA genes indicated that sediments associated with hypoxic water columns contained significantly greater proportions of Bacteria and Archaea consistent with syntrophic degradation of organic matter and methanogenesis compared to less reduced sediments. Microbial taxa frequently associated with hydrocarbon-degrading communities were found throughout the Bay, and the genetic potential for hydrocarbon metabolism was demonstrated via the detection of benzyl-(bssA) and alkylsuccinate synthase (assA) genes. Although microcosm studies did not indicate sulfidogenic alkane degradation, the data suggested that methanogenic conversion of alkanes was occurring. These findings highlight the potential role that anaerobic microorganisms could play in the bioremediation of hydrocarbons in the Bay.

Microbial enzymes that oxidize hydrocarbons

Hydrocarbons are ubiquitous compounds of both natural and anthropogenic origin. Natural sources are derived via biosynthesis reactions in bacteria, phytoplankton, plants and fungi (Ladygina et al., 2006), as well as diagenesis and catagenesis (Horsfield and Rullkotter, 1994). As a result, hydrocarbons are environmentally distributed in microbial mats (Green and Jahnke, 2010), deep subsurface oil reservoirs (Sephton and Hazen, 2013), coalbeds (Strąpoc et al., 2011), and natural oil seeps (Kvenvolden and Cooper, 2003). Hydrocarbons are energy-rich biological substrates. However, accessing this energy requires novel enzymes. The metabolic ability of microorganisms to transform hydrocarbons is not surprising given that microbes have been exposed to these compounds throughout the course of evolution. The coupling of microbial processes and hydrocarbon transformation have important implications with regard to our understanding of biogeochemical cycling, biotransformation of environmental pollutants, subsurface production and oxidation of methane, and host/pathogen relationships. This special issue highlights recent advances in our understanding of the enzymes that govern hydrocarbon transformation in both microbial and environmental systems. From a microbes metabolic perspective, the largest thermodynamic benefit of hydrocarbon metabolism is derived from the oxidation of hydrocarbons with molecular oxygen. Harnessing the free energy inherent in oxygen reactivity requires an organism to have evolved mechanisms of activating oxygen and processing the reactive intermediates in ways that lead to the selective oxidation of the desired substrate, without incurring indiscriminate reactions within the organism itself. To date, the mechanisms involved in aerobic hydrocarbon oxidation are well-described, and the most prevalent feature of these processes is the dual role of oxygen as a physiological requirement and as a reactant (Berthe-Corti and Fetzner, 2002; National Research Council, 2003). Several articles in this issue review these aerobic mechanisms and the requisite enzymes. Specifically, these articles address aerobic enzymes involved in alkane oxidation (Ji et al., 2013; Wang and Shao, 2013) and new insights to the characterization of the model methanotroph, Methylosinus trichosporium OB3b (Matsen et al., 2013; Yang et al., 2013). In the absence of oxygen, anaerobic and facultative microorganisms use alternative electrons acceptors, such as sulfate, nitrate, iron, manganese, and carbon dioxide to oxidize hydrocarbons. The last 30 years have yielded new insights into the novel biochemistry of anaerobic microorganisms with respect to hydrocarbon activation and degradation. Several pathways have emerged as common themes among physiologically diverse microorganisms utilizing a range of hydrocarbon substrates under a myriad of terminal electron accepting conditions (Heider and Schuhle, 2013). Among recent discoveries are new mechanisms and enzymes involved in anaerobic alkane oxidation. In this issue, we have included review articles by Callaghan (2013) and Agrawal and Gieg (2013) outlining the current state of knowledge regarding anaerobic methane and non-methane alkane oxidation and methods for the in situ detection of anaerobic alkane biodegradation. In addition to hydrocarbon metabolism, which is widespread among microbial taxa, some microorganisms synthesize hydrocarbons, and the purpose of these biosynthesized hydrocarbons is not well-elucidated. For example, the microalga Botryococcus braunii can synthesize up to 75% of its dry weight in alkanes (Banerjee et al., 2002). In some organisms, the alkanes constitute protective coatings, whereas in other microbes, they may serve as a strategy to store excess energy when other required nutrients are not available for metabolism and protein synthesis. Protective coatings, however, can serve as substrates for antagonistic microorganisms, and the strategies employed to oxidize hydrocarbons on the outside of cells appear to be different than those employed to utilize hydrocarbon stores within cells. The article by Pedrini et al. (2013) provides detailed evidence for microbial oxidation of alkanes in a host/pathogen relationship. Given the tragedy of the Deepwater Horizon oil spill, the role of microorganisms in mitigating natural and anthropogenic hydrocarbon inputs to marine and terrestrial ecosystems has been brought to the forefront of scientific inquiry. Three papers in this issue focus on microbial activity in oil-impacted environments. Porter and Young (2013) investigate the distribution and potential use of bamA as a biomarker for anaerobic aromatic hydrocarbon degradation in environmental samples. Kimes et al. (2013) evaluate the use of metabolite profiling coupled with metagenomics to investigate the response of sediment microbial communities to the Deepwater Horizon oil spill. Finally, Bertrand et al. (2013) elucidate the identity and mechanisms of alkane hydroxylases found in organisms isolated from deep-sea vents, an environment in which the natural release of chemosynthetic alkanes may serve as an important fuel source. While the flux of hydrocarbons through anthropogenic combustion is much larger than the flux via microbial processing and tends to dominate the publics attention due to concerns about global climate change, the selectivity and diversity of microbial transformations of hydrocarbons remains a powerful reminder of the unrivaled elegance of molecular evolution. The future, in which we continue to isolate novel organisms, harness significant advances in sequencing technology, and develop new environmental monitoring tools will undoubtedly unveil exciting and unexpected aspects of the fundamental roles that microbial metabolism plays in hydrocarbon transformation.

Metabolomic and Metagenomic Analysis of Two Crude Oil Production Pipelines Experiencing Differential Rates of Corrosion

Corrosion processes in two North Sea oil production pipelines were studied by analyzing pig envelope samples via metagenomic and metabolomic techniques. Both production systems have similar physico-chemical properties and injection waters are treated with nitrate, but one pipeline experiences severe corrosion and the other does not. Early and late pigging material was collected to gain insight into the potential causes for differential corrosion rates. Metabolites were extracted and analyzed via ultra-high performance liquid chromatography/high-resolution mass spectrometry with electrospray ionization (ESI) in both positive and negative ion modes. Metabolites were analyzed by comparison with standards indicative of aerobic and anaerobic hydrocarbon metabolism and by comparison to predicted masses for KEGG metabolites. Microbial community structure was analyzed via 16S rRNA gene qPCR, sequencing of 16S PCR products, and MySeq Illumina shotgun sequencing of community DNA. Metagenomic data were used to reconstruct the full length 16S rRNA genes and genomes of dominant microorganisms. Sequence data were also interrogated via KEGG annotation and for the presence of genes related to terminal electron accepting (TEA) processes as well as aerobic and anaerobic hydrocarbon degradation. Significant and distinct differences were observed when comparing the ‘high corrosion’ (HC) and the ‘low corrosion’ (LC) pipeline systems, especially with respect to the TEA utilization potential. The HC samples were dominated by sulfate-reducing bacteria (SRB) and archaea known for their ability to utilize simple carbon substrates, whereas LC samples were dominated by pseudomonads with the genetic potential for denitrification and aerobic hydrocarbon degradation. The frequency of aerobic hydrocarbon degradation genes was low in the HC system, and anaerobic hydrocarbon degradation genes were not detected in either pipeline. This is in contrast with metabolite analysis, which demonstrated the presence of several succinic acids in HC samples that are diagnostic of anaerobic hydrocarbon metabolism. Identifiable aerobic metabolites were confined to the LC samples, consistent with the metagenomic data. Overall, these data suggest that corrosion management might benefit from a more refined understanding of microbial community resilience in the face of disturbances such as nitrate treatment or pigging, which frequently prove insufficient to alter community structure toward a stable, less-corrosive assemblage.