Permanently cold deep-sea sediments (2500-3500 m water depth) with and without indications of thermogenic hydrocarbon seepage were exposed to naphtha to examine the presence and potential of cold-adapted aerobic hydrocarbon-degrading microbial populations. Monitoring these microcosms for volatile hydrocarbons by GC-MS revealed sediments without in situ hydrocarbons responded more rapidly to naphtha amendment than hydrocarbon seep sediments overall, but seep sediments removed aromatic hydrocarbons benzene, toluene, ethylbenzene and xylene (BTEX) more readily. Naphtha-driven aerobic respiration was more evident in surface sediment (0-20 cmbsf) than deeper anoxic layers (>130 cmbsf) that responded less rapidly. In all cases, enrichment of Gammaproteobacteria included lineages of Oleispira, Pseudomonas, and Alteromonas known to be associated with marine oil spills. On the other hand, taxa known to be prevalent in situ and diagnostic for thermogenic hydrocarbon seepage in deep sea sediment, did not respond to naphtha amendment. This suggests a limited role for these prevalent seep-associated populations in the context of aerobic hydrocarbon biodegradation.

Endospore distributions in marine sediments are influenced by geological conduits providing routes for sub-surface to surface microbial dispersal. To examine this phenomenon in more detail, endospore abundance was determined by quantifying dipicolinic acid (DPA) in 16 deep sea sediment cores from hydrocarbon prospective areas in the NW Atlantic Ocean. DPA concentrations were compared with measurements of over 250 different gaseous and liquid hydrocarbon compounds used to assess for the presence of thermogenic hydrocarbons. This revealed significantly higher levels of endospores at hydrocarbon seep sites than at most hydrocarbon-negative sites. In one exceptional case, a hydrocarbon-negative core in close proximity to adjacent thermogenic seep sites exhibited high endospore abundance, indicating either deposition of endospores emitted from the subsurface via nearby seepage, or alternatively a historical thermogenic hydrocarbon seep where hydrocarbons can no longer be detected by standard geochemistry approaches. This work expands the application of DPA as a proxy for quantifying dormant endospore biogeography of both recent and paleoenvironmental hydrocarbon seep sites.

Microorganisms are the ocean's first responders to marine pollution events, yet baseline studies rarely focus on microbial communities. Temporal and spatial microbial biodiversity baselines were established using bacterial 16S rRNA gene amplicon sequencing of seafloor sediments in a deep-water oil prospective area along the Scotian Slope off Canada's east coast sampled during 2015-2018. Bacterial diversity was generally similar in space and time, with members of the family Woeseiaceae detected consistently in >1 % relative abundance, similar to seabed sediments in other parts of the world. Anomalous biodiversity results at one site featured lower Woe-seiaceae as well as higher levels of bacterial groups specifically associated with cold seeps such as Aminicenantes. This was unexpected given that site selection was based on sediment geochemistry not revealing any petroleum hydrocarbons in these locations. This finding highlights the sensitivity and specificity of microbial DNA sequencing in environmental monitoring. Microbiome assessments like this one represent an important strategy for incorporating microbial biodiversity as a new and useful metric for establishing robust environmental baselines that are necessary for understanding ecosystem responses to marine pollution.

Deep sea hydrocarbon seep detection relies predominantly on geochemical analyses of seabed marine sediment cores to identify the presence of gas or oil. The presence of seeping hydrocarbons in these locations alters local redox stratification and microbial community structure. Cultivation-independent biodiversity surveys of microbial communities can offer a proxy for seeping hydrocarbons, but this strategy has not been extensively investigated in deep water settings. In this study, 16S rRNA gene sequencing of bacterial communities was performed on sediment cores obtained in >2500 m water depth at 43 different locations in the Northwest Atlantic Ocean. Cores extending down to 10 m below seafloor (mbsf) were assessed for gas composition, gas isotopes and liquid hydrocarbons. Over 650 bacterial biodiversity libraries were constructed from different sediment depths at these locations. Sites with strong evidence for the presence of thermogenic or biogenic hydrocarbon seepage were reflected in bacterial population analyses that revealed significant differences between hydrocarbon seep and non-seep locations. Specific bacterial indicators were associated with different sediment depth intervals including Caldatribacteriota and Campilobacterota OTUs observed in high relative sequence abundance at 20-50 cmbsf in hydrocarbon seep sediments. These groups were differentially abundant between sites with thermogenic and biogenic hydrocarbon seepage.

ABSTRACT Deep sea hydrocarbon seep detection relies predominantly on geochemical analyses of seabed marine sediment cores to identify the presence of gas or oil. The presence of seeping hydrocarbons in these locations alters resident microbial community structure, leading to culture-based biodegradation assays as a complement to geochemical tools for seep detection. Biodiversity surveys of microbial communities can offer a similar proxy for seeping hydrocarbons, but this strategy has not been extensively investigated in deep water settings. In this study, 16S rRNA gene sequencing of bacterial communities was performed on sediment cores obtained in >2500 m water depth at 43 different locations in the NW Atlantic Ocean. Core samples from as deep as 10 metres below seafloor (mbsf) were assessed for gas composition, gas isotopes and liquid hydrocarbons. Over 650 bacterial 16S rRNA gene amplicon libraries were constructed from different sediment depths at these locations. Select sites showed strong evidence for the presence of thermogenic or biogenic hydrocarbons such that bacterial population analyses revealed significant differences between hydrocarbon seep and non-seep locations. Specific bacterial indicators were associated with different sediment depth intervals. Caldatribacteriota and Campilobacterota OTUs were observed in high relative sequence abundance in hydrocarbon seep sediments, particularly in the 20-50 cmbsf interval. Furthermore, these groups were differentially abundant between sites with thermogenic and biogenic hydrocarbons. The patterns revealed here suggest that microbial screening has the potential to play a key role in hydrocarbon seep detection and characterisation in remote deep-sea environments.

The deep biosphere is the largest microbial habitat on Earth and features abundant bacterial endospores. Whereas dormancy and survival at theoretical energy minima are hallmarks of microbial physiology in the subsurface, ecological processes such as dispersal and selection in the deep biosphere remain poorly understood. We investigated the biogeography of dispersing bacteria in the deep sea where upward hydrocarbon seepage was confirmed by acoustic imagery and geochemistry. Thermophilic endospores in the permanently cold seabed correlated with underlying seep conduits reveal geofluid-facilitated cell migration pathways originating in deep petroleum-bearing sediments. Endospore genomes highlight adaptations to life in anoxic petroleum systems and bear close resemblance to oil reservoir microbiomes globally. Upon transport out of the subsurface, viable thermophilic endospores reenter the geosphere by sediment burial, enabling germination and environmental selection at depth where new petroleum systems establish. This microbial dispersal loop circulates living biomass in and out of the deep biosphere.

Summary paragraph The deep biosphere is the largest microbial habitat on Earth and features abundant bacterial endospores 1,2 . Whereas dormancy and survival at theoretical energy minima are hallmarks of subsurface microbial populations 3 , the roles of fundamental ecological processes like dispersal and selection in these environments are poorly understood 4 . Here we combine geophysics, geochemistry, microbiology and genomics to investigate biogeography in the subsurface, focusing on bacterial endospores in a deep-sea setting characterized by thermogenic hydrocarbon seepage. Thermophilic endospores in permanently cold seabed sediments above petroleum seep conduits were correlated with the presence of hydrocarbons, revealing geofluid-facilitated cell migration pathways originating in deep oil reservoirs. Genomes of thermophilic bacteria highlight adaptations to life in anoxic petroleum systems and reveal that these dormant populations are closely related to oil reservoir microbiomes from around the world. After transport out of the subsurface and into the deep-sea, thermophilic endospores re-enter the geosphere by sedimentation. Viable thermophilic endospores spanning the top several metres of the seabed correspond with total endospore counts that are similar to or exceed the global average. Burial of dormant cells enables their environmental selection in sedimentary formations where new petroleum systems establish, completing a geological microbial loop that circulates living biomass in and out of the deep biosphere.

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At marine cold seeps, gaseous and liquid hydrocarbons migrate from deep subsurface origins to the sediment-water interface. Cold seep sediments are known to host taxonomically diverse microorganisms, but little is known about their metabolic potential and depth distribution in relation to hydrocarbon and electron acceptor availability. Here we combined geophysical, geochemical, metagenomic and metabolomic measurements to profile microbial activities at a newly discovered cold seep in the deep sea. Metagenomic profiling revealed compositional and functional differentiation between near-surface sediments and deeper subsurface layers. In both sulfate-rich and sulfate-depleted depths, various archaeal and bacterial community members are actively oxidizing thermogenic hydrocarbons anaerobically. Depth distributions of hydrocarbon-oxidizing archaea revealed that they are not necessarily associated with sulfate reduction, which is especially surprising for anaerobic ethane and butane oxidizers. Overall, these findings link subseafloor microbiomes to various biochemical mechanisms for the anaerobic degradation of deeply-sourced thermogenic hydrocarbons.

At marine cold seeps, gaseous and liquid hydrocarbons migrate from deep subsurface origins to the sediment-water interface. Cold seep sediments are known to host taxonomically diverse microorganisms, but little is known about their metabolic potential and depth distribution in relation to hydrocarbon and electron acceptor availability. In this work, we combined geochemical, metagenomic and metabolomic measurements in distinct sediment redox regimes to profile microbial activities within the uppermost 350 cm of a newly discovered cold seep in the NW Atlantic deep sea (2.3 km water depth). Depth-resolved metagenomic profiling revealed compositional and functional differentiation between near-surface sediments (dominated by Proteobacteria) and deeper subsurface layers (dominated by Atribacteria, Chloroflexi, Euryarchaeota and Lokiarchaeota). Metabolic capabilities of community members were inferred from 376 metagenome-assembled genomes spanning 46 phyla (including five novel candidate phyla). In deeper sulfate-reducing and methanogenic sediments, various community members are capable of anaerobically oxidizing short-chain alkanes (alkyl-CoM reductase pathway), longer-chain alkanes (fumarate addition pathway), and aromatic hydrocarbons (fumarate addition and subsequent benzoyl-CoA pathways). Geochemical profiling demonstrated that hydrocarbon substrates are abundant in this location, thermogenic in origin, and subject to biodegradation. The detection of alkyl-/arylalkylsuccinate metabolites, together with carbon isotopic signatures of ethane, propane and carbon dioxide, support that microorganisms are actively degrading hydrocarbons in these sediments. Hydrocarbon oxidation pathways operate alongside other deep seabed metabolisms such as sulfide oxidation, hydrogen oxidation, carbon fixation, fermentation and reductive dehalogenation. Upward migrated thermogenic hydrocarbons thus sustain diverse microbial communities with activities that affect subseafloor biogeochemical processes across the redox spectrum in deep sea cold seeps.