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Sediment and pore water samples from all drill sites of International Ocean Discovery Program (IODP) Expedition 385 were analyzed quantitatively for aliphatic hydrocarbons, petroleum (C9–C44) hydrocarbons, and aromatic and polyaromatic compounds. All hydrocarbon classes showed concentration peaks in deep, hot sediments just above and below deeply buried sills (Sites U1545 and U1546), indicating that they were formed by thermal maturation of buried organic matter in the thermal aureole of sill intrusion and have, to a large extent, remained in situ. Plotting hydrocarbon concentrations against in situ temperature shows a pronounced increase in concentration between 65° and 80°C, the thermal limit of hydrocarbon-degrading microbial populations. A smaller hydrocarbon maximum is associated with surficial sediments: within the upper 4 m of the sediment column, the concentrations of total saturated hydrocarbons and of total petroleum hydrocarbons were almost always higher compared to the next sediment samples in downhole sequence, compatible with biogenic hydrocarbon input that reaches all drill sites in Guaymas Basin. The U-shaped hydrocarbon profiles suggest a biological filter that degrades surficial hydrocarbon input and deeply sourced hydrocarbons as soon as the temperature regime in gradually cooling, slowly accumulating sediments permits microbial activity. 

We compiled DNA and RNA isolation protocols for sediment bulk extraction and their yields from Guaymas Basin subsurface sediments recovered during International Ocean Discovery Program Expedition 385 and evaluated their sensitivity for metagenomic and amplicon analyses of subsurface microbial communities. Guaymas Basin sediments present a challenge for DNA and RNA recovery due to high concentrations of hydrocarbons, steep thermal gradients, and rapidly declining cell numbers downhole. Metagenomic library construction and sequencing was possible from as little as 0.2 to 0.5 ng DNA/cm3 sediment; polymerase chain reaction (PCR) amplification of 16S rRNA genes required in most cases approximately 1–2 ng DNA/cm3 sediment. At in situ temperatures of 50° to 60°C, decreasing DNA recovery leads to increasingly uncertain hit or miss outcomes and failures for metagenomic and amplicon analyses. DNA concentration profiles show that, even before these hot temperatures are reached, relatively moderate temperatures (near 40°C) have a major effect on microbial abundance and DNA yield. Comparison with cell count profiles shows that hydrothermal influence reduces downhole cell densities by multiple orders of magnitude compared to nonhydrothermal sediments. This effect is also visible at relatively moderate temperatures. RNA recovery is highly sensitive to downhole increasing temperatures and decreasing cell numbers, and was most efficient for microbial communities in cool, relatively shallow subsurface sediments. 

The Guaymas Basin in the Gulf of California is characterized by active seafloor spreading, the rapid deposition of organic-rich sediments, steep geothermal gradients, and abundant methane of mixed thermogenic and microbial origin. Subsurface sediment samples from eight drilling sites with distinct geochemical and thermal profiles were selected for DNA extraction and PCR amplification to explore the diversity of methane-cycling archaea in the Guaymas Basin subsurface. We performed PCR amplifications with general (mcrIRD), and ANME-1 specific primers that target the alpha subunit of methyl coenzyme M reductase (mcrA). Diverse ANME-1 lineages associated with anaerobic methane oxidation were detected in seven out of the eight drilling sites, preferentially around the methane-sulfate interface, and in several cases, showed preferences for specific sampling sites. Phylogenetically, most ANME-1 sequences from the Guaymas Basin subsurface were related to marine mud volcanoes, seep sites, and the shallow marine subsurface. The most frequently recovered methanogenic phylotypes were closely affiliated with the hyperthermophilic Methanocaldococcaceae, and found at the hydrothermally influenced Ringvent site. The coolest drilling site, in the northern axial trough of Guaymas Basin, yielded the greatest diversity in methanogen lineages. Our survey indicates the potential for extensive microbial methane cycling within subsurface sediments of Guaymas Basin. 

Previous studies of microbial communities in subseafloor sediments reported that microbial abundance and diversity decrease with sediment depth and age, and microbes dominating at depth tend to be a subset of the local seafloor community. However, the existence of geographically widespread, subsurface-adapted specialists is also possible. Here, we usemetagenomic and metatranscriptomic analyses of the hydrothermally heated, sediment layers of Guaymas Basin (Gulf of California, Mexico) to examine the distribution and activity patterns of bacteria and archaea along thermal, geochemical and cell count gradients. We find that the composition and distribution of metagenome-assembled genomes (MAGs), dominated by numerous lineages of Chloroflexota and Thermoproteota, correlate with biogeochemical parameters as long as temperatures remain moderate, but downcore increasing temperatures beyond ca. 45 ºC override other factors. Consistently, MAG size and diversity decrease with increasing temperature, indicating a downcore winnowing of the subsurface biosphere. By contrast, specific archaeal MAGs within the Thermoproteota and Hadarchaeota increase in relative abundance and in recruitment of transcriptome reads towards deeper, hotter sediments, marking the transition towards a specialized deep, hot biosphere. 

Analyses of gene expression of subsurface bacteria and archaea provide insights into their physiological adaptations to in situ subsurface conditions. We examined patterns of expressed genes in hydrothermally heated subseafloor sediments with distinct geochemical and thermal regimes in Guaymas Basin, Gulf of California, Mexico. RNA recovery and cell counts declined with sediment depth, however, we obtained metatranscriptomes from eight sites at depths spanning between 0.8 and 101.9m below seafloor. We describe the metabolic potential of sediment microorganisms, and discuss expressed genes involved in tRNA, mRNA, and rRNA modifications that enable physiological flexibility of bacteria and archaea in the hydrothermal subsurface. Microbial taxa in hydrothermally influenced settings like Guaymas Basin may particularly depend on these catalytic RNA functions since they modulate the activity of cells under elevated temperatures and steep geochemical gradients. Expressed genes for DNA repair, protein maintenance and circadian rhythm were also identified. The concerted interaction of many of these genes may be crucial for microorganisms to survive and to thrive in the Guaymas Basin subsurface biosphere. 

Hydrocarbons are degraded by specialized types of bacteria, archaea, and fungi. Their occurrence in marine hydrocarbon seeps and sediments prompted a study of their role and their potential interactions, using the hydrocarbon-rich hydrothermal sediments of Guaymas Basin in the Gulf of California as a model system. This sedimented vent site is characterized by localized hydrothermal circulation that introduces seawater sulfate into methane- and hydrocarbon-rich sediments, and thus selects for diverse hydrocarbon-degrading communities of which methane, alkane- and aromatics-oxidizing sulfate-reducing bacteria and archaea have been especially well-studied. Current molecular and cultivation surveys are detecting diverse fungi in Guaymas Basin hydrothermal sediments, and draw attention to possible fungal-bacterial interactions. In this Hypothesis and Theory article, we report on background, recent results and outcomes, and underlying hypotheses that guide current experiments on this topic in the Edgcomb and Teske labs in 2021, and that we will revisit during our ongoing investigations of bacterial, archaeal, and fungal communities in the deep sedimentary subsurface of Guaymas Basin.