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Methane is a major greenhouse gas and a key component of global biogeochemical cycles. Microbial methane often deviates from isotope and isotopolog equilibrium in surface environments but approaches equilibrium in deep subsurface sediments. The origin of this near-equilibrium isotopic signature in methane, whether directly produced by methanogens or achieved through anaerobic oxidation of methane (AOM), remains uncertain. Here, we show that, in the absence of AOM, microbial methane produced from deep-sea sediments exhibits isotopolog compositions approaching thermodynamic equilibrium due to energy limitation. In contrast, microbial methane from salt marsh and thermokarst lakes exhibits significant hydrogen and clumped isotopic disequilibrium due to high free-energy availability. We propose that clumped isotopologs of methane provide a proxy for characterizing the bioenergetics of environments for methane production. Together, these observations demonstrate methane clumped isotopes as a powerful tool to better understand the relation between methane metabolisms and the energy landscape in natural environments. 

Hydrothermal sediments host phylogenetically diverse and physiologically complex microbial communities. Previous studies of microbial community structure in hydrothermal sediments have typically used short-read sequencing approaches. To improve on these approaches, we use LoopSeq, a high-throughput synthetic long-read sequencing method that has yielded promising results in analyses of microbial ecosystems, such as the human gut microbiome. In this study, LoopSeq is used to obtain near-full length (approximately 1,400–1,500 nucleotides) bacterial 16S rRNA gene sequences from hydrothermal sediments in Guaymas Basin. Based on these sequences, high-quality alignments and phylogenetic analyses provided new insights into previously unrecognized taxonomic diversity of sulfur-cycling microorganisms and their distribution along a lateral hydrothermal gradient. Detailed phylogenies for free-living and syntrophic sulfur-cycling bacterial lineages identified well-supported monophyletic clusters that have implications for the taxonomic classification of these groups. Particularly, we identify clusters withinCandidatusDesulfofervidus that represent unexplored physiological and genomic diversity. In general, LoopSeq-derived 16S rRNA gene sequences aligned consistently with reference sequences in GenBank; however, chimeras were prevalent in sequences as affiliated with the thermophilicCandidatusDesulfofervidus andThermodesulfobacterium, and in smaller numbers within the sulfur-oxidizing familyBeggiatoaceae. Our analysis of sediments along a well-documented thermal and geochemical gradient show how lineages affiliated with different sulfur-cycling taxonomic groups persist throughout surficial hydrothermal sediments in the Guaymas Basin. 

Abstract Growing evidence suggests that interactions among heterotrophic microorganisms influence the efficiency and rate of organic matter turnover. These interactions are dynamic and shaped by the composition and availability of resources in their surrounding environment. Heterotrophic microorganisms inhabiting marine environments often encounter fluctuations in the quality and quantity of carbon inputs, ranging from simple sugars to large, complex compounds. Here, we experimentally tested how the chemical complexity of carbon substrates affects competition and growth dynamics between two heterotrophic marine isolates. We tracked cell density using species-specific polymerase chain reaction (PCR) assays and measured rates of microbial CO2 production along with associated isotopic signatures (13C and 14C) to quantify the impact of these interactions on organic matter remineralization. The observed cell densities revealed substrate-driven interactions: one species exhibited a competitive advantage and quickly outgrew the other when incubated with a labile compound whereas both species seemed to coexist harmoniously in the presence of more complex organic matter. Rates of CO2 respiration revealed that coincubation of these isolates enhanced organic matter turnover, sometimes by nearly 2-fold, compared to their incubation as mono-cultures. Isotopic signatures of respired CO2 indicated that coincubation resulted in a greater remineralization of macromolecular organic matter. These results demonstrate that simple substrates promote competition whereas high substrate complexity reduces competitiveness and promotes the partitioning of degradative activities into distinct niches, facilitating coordinated utilization of the carbon pool. Taken together, this study yields new insight into how the quality of organic matter plays a pivotal role in determining microbial interactions within marine environments. 

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 (α) 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. 

Abstract 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 use metagenomic 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. 

Abstract 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 101.9 m 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. 

Abstract The intrusion of igneous sills into organic-rich sediments accompanies the emplacement of igneous provinces, continental rifting, and sedimented seafloor spreading. Heat from intruding sills in these settings alters sedimentary organic carbon, releasing methane and other gasses. Recent studies hypothesize that carbon released by this mechanism impacts global climate, particularly during large igneous province emplacements. However, the direct impacts of sill intrusion, including carbon release, remain insufficiently quantified. Here, we present results from International Ocean Discovery Program (IODP) Expedition 385 comparing drill-core and wireline measurements from correlative sedimentary strata at adjacent sites cored in Guaymas Basin, Gulf of California, one altered by a recently intruded sill and one unaffected. We estimate 3.30 Mt of carbon were released due to this sill intrusion, representing an order of magnitude less carbon than inferences from outcrops and modeling would predict. This attenuated carbon release can be attributed to shallow intrusion and the high heat capacity of young, high-porosity sediments. Shallow intrusion also impacts sub-seafloor carbon cycling by disrupting advective fluxes, and it compacts underlying sediments, increasing potential carbon release in response to subsequent intrusions. 

Abstract Deep marine sediments (>1mbsf) harbor ~26% of microbial biomass and are the largest reservoir of methane on Earth. Yet, the deep subsurface biosphere and controls on its contribution to methane production remain underexplored. Here, we use a multidisciplinary approach to examine methanogenesis in sediments (down to 295 mbsf) from sites with varying degrees of thermal alteration (none, past, current) at Guaymas Basin (Gulf of California) for the first time. Traditional (13C/12C and D/H) and multiply substituted (13CH3D and 12CH2D2) methane isotope measurements reveal significant proportions of microbial methane at all sites, with the largest signal at the site with past alteration. With depth, relative microbial methane decreases at differing rates between sites. Gibbs energy calculations confirm methanogenesis is exergonic in Guaymas sediments, with methylotrophic pathways consistently yielding more energy than the canonical hydrogenotrophic and acetoclastic pathways. Yet, metagenomic sequencing and cultivation attempts indicate that methanogens are present in low abundance. We find only one methyl-coenzyme M (mcrA) sequence within the entire sequencing dataset. Also, we identify a wide diversity of methyltransferases (mtaB, mttB), but only a few sequences phylogenetically cluster with methylotrophic methanogens. Our results suggest that the microbial methane in the Guaymas subsurface was produced over geologic time by relatively small methanogen populations, which have been variably influenced by thermal sediment alteration. Higher resolution metagenomic sampling may clarify the modern methanogen community. This study highlights the importance of using a multidisciplinary approach to capture microbial influences in dynamic, deep subsurface settings like Guaymas Basin. 

Abstract In Guaymas Basin, organic-rich hydrothermal sediments produce complex hydrocarbon mixtures including saturated, aromatic and alkylated aromatic compounds. We examined sediments from push cores from Guyamas sites with distinct temperature and geochemistry profiles to gain a better understanding on abiotic and biological hydrocarbon alteration. Here we provide evidence for biodegradation of hopanoids, producing saturated hydrocarbons like drimane and homodrimane as intermediate products. These sesquiterpene by-products are present throughout cooler sediments, but their relative abundance is drastically reduced within hotter hydrothermal sediments, likely due to hydrothermal mobilization. Within the sterane pool we detect a trend toward aromatization of steroidal compounds within hotter sediments. The changes in hopane and sterane biomarker composition at different sites reflect temperature-related differences in geochemical and microbial hydrocarbon alterations. In contrast to traditionally observed microbial biodegradation patterns that may extend over hundreds of meters in subsurface oil reservoirs, Guaymas Basin shows highly compressed changes in surficial sediments.