Genomic Insights into the Marine Microbial Response to Oil Spills: Biogeographic Priming, Cryptic Hydrocarbon Cycling, and Substrate Specialization

Our seas, oceans, and coastal zones are under great stress and pollution, particularly by crude oil, which fuels the global economy. Subsurface petroleum reservoirs originate from geo-thermo-chemical reactions on biological debris over millions of years, resulting in a complex heterogeneous mixture of hydrocarbons, with major components consisting of alkanes with different chain lengths and branch points, cycloalkanes, branched cycloalkanes, mono-aromatic, and polycyclic aromatic hydrocarbons. Populations of hydrocarbon-degrading bacteria, including many species that cannot utilize other carbon sources, are present in all marine systems and play an important role in turnover and fate of these compounds. In this dissertation, the microbial response to petroleum components is probed in multiple environments to understand the role different chemical fractions play in eliciting different niches of oil consumers, and to identify factors controlling basal seed populations of hydrocarbon degraders poised to bloom to petroleum disasters. Through study in the subtropical North Atlantic Ocean, a cryptic long-chain alkane cycle has been confirmed, originating from cyanobacteria, dwarfing the quantity of other petroleum inputs to the ocean. In Chapter 1, I demonstrate waters in the mesopelagic underlying the photic zone hosted n-alkane degrading bacteria that bloomed rapidly when fed pentadecane, exhibiting exponential oxygen loss due to respiration within a week. Parallel experiments performed with sinking particles collected in situ from beneath the deep chlorophyll maximum—representing an export flux of particulate-phase pentadecane and its microbial consumers from the euphotic zone—exhibited similarly rapid bloom timing with pentadecane, but with greater oxygen decline. Notably, bloom onset timing for other petroleum compounds with no biogenic origin in the mesopelagic is an order of magnitude slower compared to biogenic alkanes. Metagenomic analyses of pentadecane blooms exposes the metabolic pathways used for pentadecane consumption. Analysis of gene abundance in unaltered seawater from oligotrophic settings reveals long-chain alkane genes are prolific in this setting and highlights the much lower prevalence of genes related to aromatic and short-chain alkane consumption. This work emphasizes the impact of phytoplankton-derived alkanes on the widespread abundance of long-chain alkane degraders in the ocean. The lack of biological hydrocarbon accumulation in the ocean points to their efficient consumption by networks of microorganisms. From analysis of sinking particles out of the photic zone, we know biogenic alkane consumption largely occurs in the sunlit ocean. To gain an understanding of which microbes consume biogenic alkanes we analyze the Tara Oceans global dataset for the presence of the alkane-1-monooxygenase gene (alkB). Stations within the North Atlantic subtropical gyre reveal alkB-related genes are abundant in the surface ocean and deep chlorophyll maximum and these genes are phylogenetically distinct from the ancestrally related delta-9 fatty acid desaturase and xylene monooxygenase. Notably, a dominant clade of alkB-like monooxygenases belongs to the globally abundant Marine Group II (MGII) archaea and is consistently present in all surface and DCM stations. This highly successful group of surface-ocean-dwelling archaea is known for a chemoorganoheterotrophic lifestyle targeting lipids, proteins and amino acids and can utilize photoheterotrophy, but a key role in biohydrocarbon cycling was unexpected as MGII archaea are not among the ~300 genera of bacteria and archaea previously identified as hydrocarbon degraders. Our further analysis of MGII genomes show alkane-1-monooxygenase genes are present in every genus of the MGII taxonomic order, and that other genes required to shunt alkane-derived carboxylic acids into central carbon metabolism are common among MGII. In the Gulf of Mexico (GOM), the biodegradation of n-pentane in the deep ocean was investigated along a gradient of natural seepage influences, illuminating the regional influence natural seeps have on priming petroleum hydrocarbon consumption. This seawater-soluble, volatile, compound is known to partition to the ocean’s interior following release from the seafloor and is abundant in petroleum reservoirs and refined products. The predominant member of the microbial community in n-pentane blooms is Cycloclasticus, and interestingly one Cycloclasticus ecotype favors the seep-ridden region of the GOM, whereas the other favors the open ocean environment far from natural seepage. Metagenomic analysis of the contrasting Cycloclasticus variants indicate the open ocean adapted variant encodes more general pathways for alkane consumption including short-chain alkanes, aromatics, and long-chain alkanes and possess pathways for dissimilatory nitrate reduction and thiosulfate oxidation, whereas the near-seep variant specializes solely on short-chain alkanes and aerobic metabolism. Metagenomic reconstruction and phylogenetic analysis of Cycloclasticus from publicly available sequence data reveals distinct strategies in hydrocarbon generalization and specialization for each major clade within the Cycloclasticus genera. Cycloalkanes are a major component of petroleum and many refined petroleum products. Methylated cycloalkane incubations using methylcyclohexane and methylcyclopentane were conducted in stations spanning a transect across the Gulf of Mexico. In three out of four stations, a single strain belonging to a novel genus within the Poricccaceae family and class Gammaproteobacteria bloomed to consume the methyl-cycloalkane provided within 18-21 days. Metagenomic reconstruction and analysis of the central carbon metabolism reveal a distinct strategy to oxidize cycloalkanes. A larger phylogenomic analysis reveals this methyl-cycloalkane consumer belongs to a monophyletic clade of genomes belonging to the same genera which originate from other environments with petroleum influence at subseafloor aquifers, hydrothermal vents, and petroleum mesocosms from a variety of marine sources. A defining feature of this clade is the presence of a divergent particulate hydrocarbon monooxygenase, which in the phylogenetic tree of the CuMMO enzyme superfamily (of ammonia and hydrocarbon monooxygenases) forms a distinct novel monophyletic clade from all other ammonia, methane, ethane, and ethylene monooxygenases