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Methylotrophic methanogenesis in the sulfate-rich zone of coastal and marine sediments couples with anaerobic oxidation of methane (AOM), forming the cryptic methane cycle. This study provides evidence of cryptic methane cycling in the sulfate-rich zone across a land–ocean transect of four stations–two brackish, one marine, and one hypersaline–within the Carpinteria Salt Marsh Reserve (CSMR), southern California, USA. Samples from the top 20 cm of sediment from the transect were analyzed through geochemical and molecular (16S rRNA) techniques, in-vitro methanogenesis incubations, and radiotracer incubations utilizing 35S-SO4, 14C-mono-methylamine, and 14C-CH4. Sediment methane concentrations were consistently low (3 to 28 µM) at all stations, except for the marine station, where methane increased with depth reaching 665 µM. Methanogenesis from mono-methylamine was detected throughout the sediment at all stations with estimated CH4 production rates in the sub-nanomolar to nanomolar range per cm3 sediment and day. 16S rRNA analysis identified methanogenic archaea (Methanosarcinaceae, Methanomassiliicoccales, and Methanonatronarchaeacea) capable of producing methane from methylamines in sediment where methylotrophic methanogenesis was found to be active. Metabolomic analysis of porewater showed mono-methylamine was mostly undetectable (<3 µM) or present in trace amounts (<10 µM) suggesting rapid metabolic turnover. In-vitro methanogenesis incubations of natural sediment showed no linear methane buildup, suggesting a process limiting methane emissions. AOM activity, measured with 14C-CH4, overlapped with methanogenesis from mono-methylamine activity at all stations, with rates ranging from 0.03 to 19.4 nmol cm− 3 d− 1. Geochemical porewater analysis showed the CSMR sediments are rich in sulfate and iron. Porewater sulfate concentrations (9–91 mM) were non-limiting across the transect, supporting sulfate reduction activity (1.5–2,506 nmol cm− 3 d− 1). Porewater sulfide and iron (II) profiles indicated that the sediment transitioned from a predominantly iron-reducing environment at the two brackish stations to a predominantly sulfate-reducing environment at the marine and hypersaline stations, which coincided with the presence of phyla (Desulfobacterota) involved in these processes. AOM activity overlapped with sulfate reduction and porewater iron (II) concentrations suggesting that AOM is likely coupled to sulfate and possibly iron reduction at all stations. However, 16S rRNA analysis identified anaerobic methanotrophs (ANME-2) only at the marine and hypersaline stations while putative methanogens were found in sediment across all stations. In one sediment horizon at the marine station, methanogen families (Methanosarcinaceae, Methanosaetaceae, Methanomassiliicoccales, and Methanoregulaceae) and ANME 2a,2b, and 2c groups were found together. Collectively, our data suggest that at the brackish stations methanogens alone may be involved in cryptic methane cycling, while at the marine and hypersaline stations both groups may be involved in the process. Differences in rate constants from incubations with 14C-labeled methane and mono-methylamine suggest a non-methanogenic process oxidizing mono-methylamine to inorganic carbon, likely mediated by sulfate-reducing bacteria. Understanding the potential competition of sulfate reducers with methanogens for mono-methylamine needs further investigation as it might be another important process responsible for low methane emissions in salt marshes. 

Methane seeps harbor uncharacterized animal–microbe symbioses with unique nutritional strategies. Three undescribed sea spider species (family Ammotheidae; genusSericosura) endemic to methane seeps were found along the eastern Pacific margin, from California to Alaska, hosting diverse methane- and methanol-oxidizing bacteria on their exoskeleton. δ¹³C tissue isotope values of in situ specimens corroborated methane assimilation (−45‰, on average). Live animal incubations with¹³C-labeled methane and methanol, followed by nanoscale secondary ion mass spectrometry, confirmed that carbon derived from both compounds was actively incorporated into the tissues within five days. Methano- and methylotrophs of the bacterial families Methylomonadaceae, Methylophagaceae and Methylophilaceae were abundant, based on environmental metagenomics and 16S rRNA sequencing, and fluorescence and electron microscopy confirmed dense epibiont aggregations on the sea spider exoskeleton. Egg sacs carried by the males hosted identical microbes suggesting vertical transmission. We propose that these sea spiders farm and feed on methanotrophic and methylotrophic bacteria, expanding the realm of animals known to harness C1 compounds as a carbon source. These findings advance our understanding of the biology of an understudied animal lineage, unlocking some of the unique nutritional links between the microbial and faunal food webs in the oceans. 

ABSTRACT The microbial recycling of organic matter in marine sediments depends upon electron acceptors that are utilized based on availability and energetic yield. Since sulfate is the most abundant oxidant once oxygen has been depleted, the sulfide produced after sulfate reduction becomes an important electron donor for autotrophic microbes. The ability of sulfide to be re‐oxidized through multiple metabolic pathways and intermediates with variable oxidation states prompts investigation into which species are preferentially utilized and what are the factors that determine the fate of reduced sulfur species. Quantifying these sulfur intermediates in porewaters is a critical first step towards achieving a more complete understanding of the oxidative sulfur cycle, yet this has been accomplished in very few studies, none of which include oligotrophic sedimentary environments in the open ocean. Here we present profiles of porewater sulfur intermediates from sediments underlying oligotrophic regions of the ocean, which encompass about 75% of the ocean's surface and are characterized by low nutrient levels and productivity. Aiming at addressing uncertainties about if and how sulfide produced by the degradation of scarce sedimentary organic matter plays a role in carbon fixation in the sediment, we determine depth profiles of redox‐sensitive metals and sulfate isotope compositions and integrate these datasets with 16S rRNA microbial community composition data and solid‐phase sulfur concentrations. We did not find significant correlations between sulfur species or trace metals and specific sulfur cycling taxa, which suggests that microorganisms in pelagic and oxic sediments may be generalists utilizing flexible metabolisms to oxidize organic matter through different electron acceptors. 

Discovering new deep hydrothermal vent systems is one of the biggest challenges in ocean exploration. They are a unique window to elucidate the physical, geochemical, and biological processes that occur on the seafloor and are involved in the evolution of life on Earth. In this study, we present a molecular analysis of the microbial composition within the newly discovered hydrothermal vent field,JaichMaa ‘ja ‘ag, situated in the Southern Pescadero Basin within the Gulf of California. During the cruise expedition FK181031 in 2018, 33 sediment cores were collected from various sites within the Pescadero vent fields and processed for 16S rRNA amplicon sequence variants (ASVs) and geochemical analysis. Correlative analysis of the chemical composition of hydrothermal pore fluids and microbial abundances identified several sediment-associated phyla, including Thermotogota, that appear to be enriched in sediment horizons impacted by hydrothermal fluid flow. Comparative analysis of Thermotogota with the previously exploredAukahydrothermal vent field situated 2 km away displayed broad similarity between the two locations, although at finer scales (e.g., ASV level), there were notable differences that point to core-to-core and site-level factors revealing distinct patterns of distribution and abundance within these two sediment-hosted hydrothermal vent fields. These patterns are intricately linked to the specific physical and geochemical conditions defining each vent, illuminating the complexity of this unique deep ocean chemosynthetic ecosystem. 

ABSTRACT About 382 Tg yr −1 of methane rising through the seafloor is oxidized anaerobically (W. S. Reeburgh, Chem Rev 107:486–513, 2007, https://doi.org/10.1021/cr050362v ), preventing it from reaching the atmosphere, where it acts as a strong greenhouse gas. Microbial consortia composed of anaerobic methanotrophic archaea and sulfate-reducing bacteria couple the oxidation of methane to the reduction of sulfate under anaerobic conditions via a syntrophic process. Recent experimental studies and modeling efforts indicate that direct interspecies electron transfer (DIET) is involved in this syntrophy. Here, we explore a fluorescent in situ hybridization-nanoscale secondary ion mass spectrometry data set of large, segregated anaerobic oxidation of methane (AOM) consortia that reveal a decline in metabolic activity away from the archaeal-bacterial interface and use a process-based model to identify the physiological controls on rates of AOM. Simulations reproducing the observational data reveal that ohmic resistance and activation loss are the two main factors causing the declining metabolic activity, where activation loss dominated at a distance of <8 μm. These voltage losses limit the maximum spatial distance between syntrophic partners with model simulations, indicating that sulfate-reducing bacterial cells can remain metabolically active up to ∼30 μm away from the archaeal-bacterial interface. Model simulations further predict that a hybrid metabolism that combines DIET with a small contribution of diffusive exchange of electron donors can offer energetic advantages for syntrophic consortia. IMPORTANCE Anaerobic oxidation of methane is a globally important, microbially mediated process reducing the emission of methane, a potent greenhouse gas. In this study, we investigate the mechanism of how a microbial consortium consisting of archaea and bacteria carries out this process and how these organisms interact with each other through the sharing of electrons. We present a process-based model validated by novel experimental measurements of the metabolic activity of individual, phylogenetically identified cells in very large (>20-μm-diameter) microbial aggregates. Model simulations indicate that extracellular electron transfer between archaeal and bacterial cells within a consortium is limited by potential losses and suggest that a flexible use of electron donors can provide energetic advantages for syntrophic consortia. 

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. 

Abstract Archaeal anaerobic methanotrophs (“ANME”) and sulfate-reducing Deltaproteobacteria (“SRB”) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH4 and 15N2 revealed differences in cellular 15N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in 15N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph. 

The microbial ecology of the deep biosphere is difficult to characterize, owing in part to sampling challenges and poorly understood response mechanisms to environmental change. Pre-drilled wells, including oil wells or boreholes, offer convenient access, but sampling is frequently limited to the water alone, which may provide only a partial view of the native diversity. Mineral heterogeneity demonstrably affects colonization by deep biosphere microorganisms, but the connections between the mineral-associated and planktonic communities remain unclear. To understand the substrate effects on microbial colonization and the community response to changes in organic carbon, we conducted an 18-month series of in situ experiments in a warm (57°C), anoxic, fractured carbonate aquifer at 752 m depth using replicate open, screened cartridges containing different solid substrates, with a proteinaceous organic matter perturbation halfway through this series. Samples from these cartridges were analyzed microscopically and by Illumina (iTag) 16S rRNA gene libraries to characterize changes in mineralogy and the diversity of the colonizing microbial community. The substrate-attached and planktonic communities were significantly different in our data, with some taxa (e.g., Candidate Division KB-1) rare or undetectable in the first fraction and abundant in the other. The substrate-attached community composition also varied significantly with mineralogy, such as with two Rhodocyclaceae OTUs, one of which was abundant on carbonate minerals and the other on silicic substrates. Secondary sulfide mineral formation, including iron sulfide framboids, was observed on two sets of incubated carbonates. Notably, microorganisms were attached to the framboids, which were correlated with abundant Sulfurovum and Desulfotomaculum sp. sequences in our analysis. Upon organic matter perturbation, mineral-associated microbial diversity differences were temporarily masked by the dominance of putative heterotrophic taxa in all samples, including OTUs identified as Caulobacter , Methyloversatilis , and Pseudomonas . Subsequent experimental deployments included a methanogen-dominated stage ( Methanobacteriales and Methanomicrobiales ) 6 months after the perturbation and a return to an assemblage similar to the pre-perturbation community after 9 months. Substrate-associated community differences were again significant within these subsequent phases, however, demonstrating the value of in situ time course experiments to capture a fraction of the microbial assemblage that is frequently difficult to observe in pre-drilled wells.