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Methane is a potent greenhouse gas that enters the marine system in large quantities at seafloor methane seeps. At a newly discovered seep site off the coast of Point Dume, CA, ~ meter-scale carbonate chimneys host microbial communities that exhibit the highest methane-oxidizing potential recorded to date. Here, we provide a detailed assessment of chimney geobiology through correlative mineralogical, geochemical, and microbiological studies of seven chimney samples in order to clarify the longevity and heterogeneity of these highly productive systems. U-Th dating indicated that a methane-driven carbonate precipitating system at Point Dume has existed for ~20 Kyr, while millimeter-scale variations in carbon and calcium isotopic values, elemental abundances, and carbonate polymorphs revealed changes in carbon source, precipitation rates, and diagenetic processes throughout the chimneys' lifespan. Microbial community analyses revealed diverse modern communities with prominent anaerobic methanotrophs, sulfate-reducing bacteria, and Anaerolineaceae; communities were more similar within a given chimney wall transect than in similar horizons of distinct structures. The chimneys represent long-lived repositories of methane-oxidizing communities and provide a window into how carbon can be transformed, sequestered, and altered over millennia at the Point Dume methane seep., (© 2024 John Wiley & Sons Ltd.)

The Astrobiology Primer 3.0 (ABP3.0) is a concise introduction to the field of astrobiology for students and others who are new to the field of astrobiology. It provides an entry into the broader materials in this supplementary issue of Astrobiology and an overview of the investigations and driving hypotheses that make up this interdisciplinary field. The content of this chapter was adapted from the other 10 articles in this supplementary issue and thus represents the contribution of all the authors who worked on these introductory articles. The content of this chapter is not exhaustive and represents the topics that the authors found to be the most important and compelling in a dynamic and changing field.

Scientific ideas about the potential existence of life elsewhere in the universe are predominantly informed by knowledge about life on Earth. Over the past ∼4 billion years, life on Earth has evolved into millions of unique species. Life now inhabits nearly every environmental niche on Earth that has been explored. Despite the wide variety of species and diverse biochemistry of modern life, many features, such as energy production mechanisms and nutrient requirements, are conserved across the Tree of Life. Such conserved features help define the operational parameters required by life and therefore help direct the exploration and evaluation of habitability in extraterrestrial environments. As new diversity in the Tree of Life continues to expand, so do the known limits of life on Earth and the range of environments considered habitable elsewhere. The metabolic processes used by organisms living on the edge of habitability provide insights into the types of environments that would be most suitable to hosting extraterrestrial life, crucial for planning and developing future astrobiology missions. This chapter will introduce readers to the breadth and limits of life on Earth and show how the study of life at the extremes can inform the broader field of astrobiology.

Salt marshes are dynamic, highly productive ecosystems positioned at the interface between terrestrial and marine systems. They are exposed to large quantities of both natural and anthropogenic carbon input, and their diverse sediment-hosted microbial communities play key roles in carbon cycling and remineralization. To better understand the effects of natural and anthropogenic carbon on sediment microbial ecology, several sediment cores were collected from Little Sippewissett Salt Marsh (LSSM) on Cape Cod, MA, USA and incubated with either Spartina alterniflora cordgrass or diesel fuel. Resulting shifts in microbial diversity and activity were assessed via bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Both Spartina and diesel amendments resulted in initial decreases of microbial diversity as well as clear, community-wide shifts in metabolic activity. Multi-stage degradative frameworks shaped by fermentation were inferred based on anabolically active lineages. In particular, the metabolically versatile Marinifilaceae were prominent under both treatments, as were the sulfate-reducing Desulfovibrionaceae , which may be attributable to their ability to utilize diverse forms of carbon under nutrient limited conditions. By identifying lineages most directly involved in the early stages of carbon processing, we offer potential targets for indicator species to assess ecosystem health and highlight key players for selective promotion of bioremediation or carbon sequestration pathways., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Frates, Spietz, Silverstein, Girguis, Hatzenpichler and Marlow.)

Coastal salt marshes are key sites of biogeochemical cycling and ideal systems in which to investigate the community structure of complex microbial communities. Here, we clarify structural-functional relationships among microorganisms and their mineralogical environment, revealing previously undescribed metabolic activity patterns and precise spatial arrangements within salt marsh sediment. Following 3.7-day in situ incubations with a non-canonical amino acid that was incorporated into new biomass, samples were resin-embedded and analysed by correlative fluorescence and electron microscopy to map the microscale arrangements of anabolically active and inactive organisms alongside mineral grains. Parallel sediment samples were examined by fluorescence-activated cell sorting and 16S rRNA gene sequencing to link anabolic activity to taxonomic identity. Both approaches demonstrated a rapid decline in the proportion of anabolically active cells with depth into salt marsh sediment, from ~60% in the top centimetre to 9.4%-22.4% between 2 and 10 cm. From the top to the bottom, the most prominent active community members shifted from sulfur cycling phototrophic consortia, to putative sulfate-reducing bacteria likely oxidizing organic compounds, to fermentative lineages. Correlative microscopy revealed more abundant (and more anabolically active) organisms around non-quartz minerals including rutile, orthoclase and plagioclase. Microbe-mineral relationships appear to be dynamic and context-dependent arbiters of biogeochemical cycling., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

At marine methane seeps, vast quantities of methane move through the shallow subseafloor, where it is largely consumed by microbial communities. This process plays an important role in global methane dynamics, but we have yet to identify all of the methane sinks in the deep sea. Here, we conducted a continental-scale survey of seven geologically diverse seafloor seeps and found that carbonate rocks from all sites host methane-oxidizing microbial communities with substantial methanotrophic potential. In laboratory-based mesocosm incubations, chimney-like carbonates from the newly described Point Dume seep off the coast of Southern California exhibited the highest rates of anaerobic methane oxidation measured to date. After a thorough analysis of physicochemical, electrical, and biological factors, we attribute this substantial metabolic activity largely to higher cell density, mineral composition, kinetic parameters including an elevated V max , and the presence of specific microbial lineages. Our data also suggest that other features, such as electrical conductance, rock particle size, and microbial community alpha diversity, may influence a sample's methanotrophic potential, but these factors did not demonstrate clear patterns with respect to methane oxidation rates. Based on the apparent pervasiveness within seep carbonates of microbial communities capable of performing anaerobic oxidation of methane, as well as the frequent occurrence of carbonates at seeps, we suggest that rock-hosted methanotrophy may be an important contributor to marine methane consumption., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

It is now routinely possible to sequence and recover microbial genomes from environmental samples. To the degree it is feasible to assign transcriptional and translational functions to these genomes, it should be possible, in principle, to largely understand the complete molecular inputs and outputs of a microbial community. However, gene-based tools alone are presently insufficient to describe the full suite of chemical reactions and small molecules that compose a living cell. Metabolomic tools have developed quickly and now enable rapid detection and identification of small molecules within biological and environmental samples. The convergence of these technologies will soon facilitate the detection of novel enzymatic activities, novel organisms, and potentially extraterrestrial life-forms on solar system bodies. This review explores the methodological problems and scientific opportunities facing researchers who hope to apply metabolomic methods in astrobiology-related fields, and how present challenges might be overcome.

Interactions among microorganisms and their mineralogical substrates govern the structure, function and emergent properties of microbial communities. These interactions are predicated on spatial relationships, which dictate metabolite exchange and access to key substrates. To quantitatively assess links between spatial relationships and metabolic activity, this study presents a novel approach to map all organisms, the metabolically active subset and associated mineral grains, all while maintaining spatial integrity of an environmental microbiome. We applied this method at an outgassing fumarole of Vanuatu's Marum Crater, one of the largest point sources of several environmentally relevant gaseous compounds, including H2O, CO2 and SO2. With increasing distance from the sediment-air surface and from mineral grain outer boundaries, organism abundance decreased but the proportion of metabolically active organisms often increased. These protected niches may provide more stable conditions that promote consistent metabolic activity of a streamlined community. Conversely, exterior surfaces accumulate more organisms that may cover a wider range of preferred conditions, implying that only a subset of the community will be active under any particular environmental regime. More broadly, the approach presented here allows investigators to see microbial communities 'as they really are' and explore determinants of metabolic activity across a range of microbiomes., (© FEMS 2020.)

Harnessing the metabolic potential of uncultured microbial communities is a compelling opportunity for the biotechnology industry, an approach that would vastly expand the portfolio of usable feedstocks. Methane is particularly promising because it is abundant and energy-rich, yet the most efficient methane-activating metabolic pathways involve mixed communities of anaerobic methanotrophic archaea and sulfate reducing bacteria. These communities oxidize methane at high catabolic efficiency and produce chemically reduced by-products at a comparable rate and in near-stoichiometric proportion to methane consumption. These reduced compounds can be used for feedstock and downstream chemical production, and at the production rates observed in situ they are an appealing, cost-effective prospect. Notably, the microbial constituents responsible for this bioconversion are most prominent in select deep-sea sediments, and while they can be kept active at surface pressures, they have not yet been cultured in the lab. In an industrial capacity, deep-sea sediments could be periodically recovered and replenished, but the associated technical challenges and substantial costs make this an untenable approach for full-scale operations. In this study, we present a novel method for incorporating methanotrophic communities into bioindustrial processes through abstraction onto low mass, easily transportable carbon cloth artificial substrates. Using Gulf of Mexico methane seep sediment as inoculum, optimal physicochemical parameters were established for methane-oxidizing, sulfide-generating mesocosm incubations. Metabolic activity required >∼40% seawater salinity, peaking at 100% salinity and 35 °C. Microbial communities were successfully transferred to a carbon cloth substrate, and rates of methane-dependent sulfide production increased more than threefold per unit volume. Phylogenetic analyses indicated that carbon cloth-based communities were substantially streamlined and were dominated by Desulfotomaculum geothermicum. Fluorescence in situ hybridization microscopy with carbon cloth fibers revealed a novel spatial arrangement of anaerobic methanotrophs and sulfate reducing bacteria suggestive of an electronic coupling enabled by the artificial substrate. This system: 1) enables a more targeted manipulation of methane-activating microbial communities using a low-mass and sediment-free substrate; 2) holds promise for the simultaneous consumption of a strong greenhouse gas and the generation of usable downstream products; and 3) furthers the broader adoption of uncultured, mixed microbial communities for biotechnological use., (© 2018 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.)

Biological methane oxidation is a globally relevant process that mediates the flux of an important greenhouse gas through both aerobic and anaerobic metabolic pathways. However, measuring these metabolic rates presents many obstacles, from logistical barriers to regulatory hurdles and poor precision. Here we present a new approach for investigating microbial methane metabolism based on hydrogen atom dynamics, which is complementary to carbon-focused assessments of methanotrophy. The method uses monodeuterated methane (CH 3 D) as a metabolic substrate, quantifying the aqueous D/H ratio over time using off-axis integrated cavity output spectroscopy. This approach represents a nontoxic, comparatively rapid, and straightforward approach that supplements existing radiotopic and stable carbon isotopic methods; by probing hydrogen atoms, it offers an additional dimension for examining rates and pathways of methane metabolism. We provide direct comparisons between the CH 3 D procedure and the well-established 14 CH 4 radiotracer method for several methanotrophic systems, including type I and II aerobic methanotroph cultures and methane-seep sediment slurries and carbonate rocks under anoxic and oxic incubation conditions. In all applications tested, methane consumption values calculated via the CH 3 D method were directly and consistently proportional to 14 C radiolabel-derived methane oxidation rates. We also employed this method in a nontraditional experimental setup, using flexible, gas-impermeable bags to investigate the role of pressure on seep sediment methane oxidation rates. Results revealed an 80% increase over atmospheric pressure in methanotrophic rates the equivalent of ~900-m water depth, highlighting the importance of this parameter on methane metabolism and exhibiting the flexibility of the newly described method. IMPORTANCE Microbial methane consumption is a critical component of the global carbon cycle, with wide-ranging implications for climate regulation and hydrocarbon exploitation. Nonetheless, quantifying methane metabolism typically involves logistically challenging methods and/or specialized equipment; these impediments have limited our understanding of methane fluxes and reservoirs in natural systems, making effective management difficult. Here, we offer an easily implementable, precise method using monodeuterated methane (CH 3 D) that advances three specific aims. First, it allows users to directly compare methane consumption rates between different experimental treatments of the same inoculum. Second, by empirically linking the CH 3 D procedure with the well-established 14 C radiocarbon approach, we determine absolute scaling factors that facilitate rate measurements for several aerobic and anaerobic systems of interest. Third, CH 3 D represents a helpful tool in evaluating the relationship between methane activation and full oxidation in methanotrophic metabolisms. The procedural advantages, consistency, and novel research questions enabled by the CH 3 D method should prove useful in a wide range of culture-based and environmental microbial systems to further elucidate methane metabolism dynamics.

Competing Interests: The authors declare no conflict of interest.

Marine methane seep habitats represent an important control on the global flux of methane. Nucleotide-based meta-omics studies outline community-wide metabolic potential, but expression patterns of environmentally relevant proteins are poorly characterized. Proteomic stable isotope probing (proteomic SIP) provides additional information by characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities, offering enhanced detection through system-wide product integration. Here we applied proteomic SIP to (15)[Formula: see text] and CH4 amended seep sediment microcosms in an attempt to track protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 unique proteins were identified, 11% of which were (15)N-labeled. Consistent with the dominant anaerobic oxidation of methane (AOM) activity commonly observed in anoxic seep sediments, proteins associated with sulfate reduction and reverse methanogenesis-including the ANME-2 associated methylenetetrahydromethanopterin reductase (Mer)-were all observed to be actively synthesized ((15)N-enriched). Conversely, proteins affiliated with putative aerobic sulfur-oxidizing epsilon- and gammaproteobacteria showed a marked decrease over time in our anoxic sediment incubations. The abundance and phylogenetic range of (15)N-enriched methyl-coenzyme M reductase (Mcr) orthologs, many of which exhibited novel post-translational modifications, suggests that seep sediments provide niches for multiple organisms performing analogous metabolisms. In addition, 26 proteins of unknown function were consistently detected and actively expressed under conditions supporting AOM, suggesting that they play important roles in methane seep ecosystems. Stable isotope probing in environmental proteomics experiments provides a mechanism to determine protein durability and evaluate lineage-specific responses in complex microbial communities placed under environmentally relevant conditions. Our work here demonstrates the active synthesis of a metabolically specific minority of enzymes, revealing the surprising longevity of most proteins over the course of an extended incubation experiment in an established, slow-growing, methane-impacted environmental system.

Unlabelled: Marine methane seeps are globally distributed geologic features in which reduced fluids, including methane, are advected upward from the subsurface. As a result of alkalinity generation during sulfate-coupled methane oxidation, authigenic carbonates form slabs, nodules, and extensive pavements. These carbonates shape the landscape within methane seeps, persist long after methane flux is diminished, and in some cases are incorporated into the geologic record. In this study, microbial assemblages from 134 native and experimental samples across 5,500 km, representing a range of habitat substrates (carbonate nodules and slabs, sediment, bottom water, and wood) and seepage conditions (active and low activity), were analyzed to address two fundamental questions of seep microbial ecology: (i) whether carbonates host distinct microbial assemblages and (ii) how sensitive microbial assemblages are to habitat substrate type and temporal shifts in methane seepage flux. Through massively parallel 16S rRNA gene sequencing and statistical analysis, native carbonates are shown to be reservoirs of distinct and highly diverse seep microbial assemblages. Unique coupled transplantation and colonization experiments on the seafloor demonstrated that carbonate-associated microbial assemblages are resilient to seep quiescence and reactive to seep activation over 13 months. Various rates of response to simulated seep quiescence and activation are observed among similar phylogenies (e.g., Chloroflexi operational taxonomic units) and similar metabolisms (e.g., putative S oxidizers), demonstrating the wide range of microbial sensitivity to changes in seepage flux. These results imply that carbonates do not passively record a time-integrated history of seep microorganisms but rather host distinct, diverse, and dynamic microbial assemblages., Importance: Since their discovery in 1984, the global distribution and importance of marine methane seeps have become increasingly clear. Much of our understanding of methane seep microorganisms-from metabolisms to community ecology-has stemmed from detailed studies of seep sediments. However, it has become apparent that carbonates represent a volumetrically significant habitat substrate at methane seeps. Through combined in situ characterization and incubation experiments, this study demonstrates that carbonates host microbial assemblages distinct from and more diverse than those of other seep habitats. This emphasizes the importance of seep carbonates as biodiversity locales. Furthermore, we demonstrate that carbonate-associated microbial assemblages are well adapted to withstand fluctuations in methane seepage, and we gain novel insight into particular taxa that are responsive (or recalcitrant) to changes in seep conditions., (Copyright © 2015 Case et al.)

The atmospheric flux of methane from the oceans is largely mitigated through microbially mediated sulphate-coupled methane oxidation, resulting in the precipitation of authigenic carbonates. Deep-sea carbonates are common around active and palaeo-methane seepage, and have primarily been viewed as passive recorders of methane oxidation; their role as active and unique microbial habitats capable of continued methane consumption has not been examined. Here we show that seep-associated carbonates harbour active microbial communities, serving as dynamic methane sinks. Microbial aggregate abundance within the carbonate interior exceeds that of seep sediments, and molecular diversity surveys reveal methanotrophic communities within protolithic nodules and well-lithified carbonate pavements. Aggregations of microbial cells within the carbonate matrix actively oxidize methane as indicated by stable isotope FISH-nanoSIMS experiments and (14)CH4 radiotracer rate measurements. Carbonate-hosted methanotrophy extends the known ecological niche of these important methane consumers and represents a previously unrecognized methane sink that warrants consideration in global methane budgets.

Seep sediments are dominated by intensive microbial sulfate reduction coupled to the anaerobic oxidation of methane (AOM). Through geochemical measurements of incubation experiments with methane seep sediments collected from Hydrate Ridge, we provide insight into the role of iron oxides in sulfate-driven AOM. Seep sediments incubated with (13)C-labeled methane showed co-occurring sulfate reduction, AOM, and methanogenesis. The isotope fractionation factors for sulfur and oxygen isotopes in sulfate were about 40‰ and 22‰, respectively, reinforcing the difference between microbial sulfate reduction in methane seeps versus other sedimentary environments (for example, sulfur isotope fractionation above 60‰ in sulfate reduction coupled to organic carbon oxidation or in diffusive sedimentary sulfate-methane transition zone). The addition of hematite to these microcosm experiments resulted in significant microbial iron reduction as well as enhancing sulfate-driven AOM. The magnitude of the isotope fractionation of sulfur and oxygen isotopes in sulfate from these incubations was lowered by about 50%, indicating the involvement of iron oxides during sulfate reduction in methane seeps. The similar relative change between the oxygen versus sulfur isotopes of sulfate in all experiments (with and without hematite addition) suggests that oxidized forms of iron, naturally present in the sediment incubations, were involved in sulfate reduction, with hematite addition increasing the sulfate recycling or the activity of sulfur-cycling microorganisms by about 40%. These results highlight a role for natural iron oxides during bacterial sulfate reduction in methane seeps not only as nutrient but also as stimulator of sulfur recycling.

This study examines the potential for the biologically mediated anaerobic oxidation of methane (AOM) coupled to sulfate reduction on ancient Mars. Seven distinct fluids representative of putative martian groundwater were used to calculate Gibbs energy values in the presence of dissolved methane under a range of atmospheric CO2 partial pressures. In all scenarios, AOM is exergonic, ranging from -31 to -135 kJ/mol CH4. A reaction transport model was constructed to examine how environmentally relevant parameters such as advection velocity, reactant concentrations, and biomass production rate affect the spatial and temporal dependences of AOM reaction rates. Two geologically supported models for ancient martian AOM are presented: a sulfate-rich groundwater with methane produced from serpentinization by-products, and acid-sulfate fluids with methane from basalt alteration. The simulations presented in this study indicate that AOM could have been a feasible metabolism on ancient Mars, and fossil or isotopic evidence of this metabolic pathway may persist beneath the surface and in surface exposures of eroded ancient terrains.

Although the importance of trophic linkages, including 'top-down forcing', on energy flow and ecosystem productivity is recognized, the influence of metazoan grazing on Archaea and the biogeochemical processes that they mediate is unknown. Here, we test if: (1) Archaea provide a food source sufficient to allow metazoan fauna to complete their life cycle; (2) neutral lipid biomarkers (including crocetane) can be used to identify Archaea consumers; and (3) archaeal aggregates are a dietary source for methane seep metazoans. In the laboratory, we demonstrated that a dorvilleid polychaete, Ophryotrocha labronica, can complete its life cycle on two strains of Euryarchaeota with the same growth rate as when fed bacterial and eukaryotic food. Archaea were therefore confirmed as a digestible and nutritious food source sufficient to sustain metazoan populations. Both strains of Euryarchaeota used as food sources had unique lipids that were not incorporated into O. labronica tissues. At methane seeps, sulfate-reducing bacteria that form aggregations and live syntrophically with anaerobic-methane oxidizing Archaea contain isotopically and structurally unique fatty acids (FAs). These biomarkers were incorporated into tissues of an endolithofaunal dorvilleid polychaete species from Costa Rica (mean bulk δ(13)C=-92±4‰; polar lipids -116‰) documenting consumption of archaeal-bacterial aggregates. FA composition of additional soft-sediment methane seep species from Oregon and California provided evidence that consumption of archaeal-bacterial aggregates is widespread at methane seeps. This work is the first to show that Archaea are consumed by heterotrophic metazoans, a trophic process we coin as 'archivory'.

Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium best known as the predominant opportunistic pathogen infecting the lungs of cystic fibrosis patients. In this context, it is thought to form biofilms, within which locally reducing and acidic conditions can develop that favor the stability of ferrous iron [Fe(II)]. Because iron is a signal that stimulates biofilm formation, we performed a microarray study to determine whether P. aeruginosa strain PA14 exhibits a specific transcriptional response to extracellular Fe(II). Among the genes that were most upregulated in response to Fe(II) were those encoding the two-component system BqsR/BqsS, previously identified for its role in P. aeruginosa strain PAO1 biofilm decay (13); here, we demonstrate its role in extracellular Fe(II) sensing. bqsS and bqsR form an operon together with two small upstream genes, bqsP and bqsQ, and one downstream gene, bqsT. BqsR/BqsS sense extracellular Fe(II) at physiologically relevant concentrations (>10 μM) and elicit a specific transcriptional response, including its autoregulation. The sensor distinguishes between Fe(II), Fe(III), and other dipositive cations [Ca(II), Cu(II), Mg(II), Mn(II), Zn(II)] under aerobic or anaerobic conditions. The gene that is most upregulated by BqsR/BqsS, as measured by quantitative reverse transcription-PCR (qRT-PCR), is PA14_04180, which is predicted to encode a periplasmic oligonucleotide/oligosaccharide-binding domain (OB-fold) protein. Coincident with phenazine production during batch culture growth, Fe(II) becomes the majority of the total iron pool and bqsS is upregulated. The existence of a two-component system that senses Fe(II) indicates that extracellular Fe(II) is an important environmental signal for P. aeruginosa.