Rachel L. Spietz

Seismic energy, like that released by earthquakes, can fracture rock and thereby alter subsurface fluid flow paths, release substrates from inclusions, and expose fresh mineral surfaces capable of reacting with water. However, it is unclear how such seismic-induced changes influence microbial communities. Volcanically active areas experience frequent seismic activity and thus represent ideal locations to examine the influence of seismic-induced geochemical change on subsurface microbial communities. Here, we demonstrate that energy released in an earthquake swarm in 2021 correlated with extensive temporal change in the geochemical and microbial composition of aquifer fluids sampled from ∼100 m depth in a borehole in Yellowstone National Park. Increased energy absorbed at the borehole over time was correlated with increased concentrations of hydrogen, dissolved organic carbon, and sulfide and was associated with depletion of δ13C in dissolved organic carbon, increased concentrations of cells, and increased abundances of chemolithotrophic, putative hydrogen-oxidizing Dethiobacteraceae and Desulfotomaculum bacteria. Dissipation of the earthquake swarm was associated with decreased concentrations of hydrogen, sulfide, and cells. These results suggest the subsurface biosphere dynamically responds to seismic-induced geochemical change at the level of activity and growth. Laboratory mechanical comminution of rhyolite, the primary bedrock in Yellowstone, released organic carbon and hydrogen and generated hydrogen when exposed to water. This indicates the presence of a large subsurface reservoir of organic carbon and hydrogen that can be released or generated by seismic induced bedrock fracturing. Taken together, these data indicate seismic-induced generation of chemical disequilibria can support the persistence of complex subsurface microbiomes.

Methanogens often inhabit sulfidic environments that favor the precipitation of transition metals such as iron (Fe) as metal sulfides, including mackinawite (FeS) and pyrite (FeS ₂ ). These metal sulfides have historically been considered biologically unavailable. Nonetheless, methanogens are commonly cultivated with sulfide (HS ^- ) as a sulfur source, a condition that would be expected to favor metal precipitation and thus limit metal availability. Recent studies have shown that methanogens can access Fe and sulfur (S) from FeS and FeS ₂ to sustain growth. As such, medium supplied with FeS ₂ should lead to higher availability of transition metals when compared to medium supplied with HS ^- . Here, we examined how transition metal availability under sulfidic (i.e., cells provided with HS ^- as sole S source) versus non-sulfidic (cells provided with FeS ₂ as sole S source) conditions impact the metalloproteome of Methanosarcina barkeri Fusaro. To achieve this, we employed size exclusion chromatography coupled with inductively coupled plasma mass spectrometry and shotgun proteomics. Significant changes were observed in the composition and abundance of iron, cobalt, nickel, zinc, and molybdenum proteins. Among the differences were alterations in the stoichiometry and abundance of multisubunit protein complexes involved in methanogenesis and electron transport chains. Our data suggest that M. barkeri utilizes the minimal iron-sulfur cluster complex and canonical cysteine biosynthesis proteins when grown on FeS ₂ but uses the canonical Suf pathway in conjunction with the tRNA-Sep cysteine pathway for iron-sulfur cluster and cysteine biosynthesis under sulfidic growth conditions. IMPORTANCE: Proteins that catalyze biochemical reactions often require transition metals that can have a high affinity for sulfur, another required element for life. Thus, the availability of metals and sulfur are intertwined and can have large impacts on an organismismal biochemistry. Methanogens often occupy anoxic, sulfide-rich (euxinic) environments that favor the precipitation of transition metals as metal sulfides, thereby creating presumed metal limitation. Recently, several methanogens have been shown to acquire iron and sulfur from pyrite, an abundant iron-sulfide mineral that was traditionally considered to be unavailable to biology. The work presented here provides new insights into the distribution of metalloproteins, and metal uptake of Methanosarcina barkeri Fusaro grown under euxinic or pyritic growth conditions. Thorough characterizations of this methanogen under different metal and sulfur conditions increase our understanding of the influence of metal availability on methanogens, and presumably other anaerobes, that inhabit euxinic environments.

Iron (Fe) and sulfur (S) are required elements for life, and changes in their availability can limit the ecological distribution and function of microorganisms. In anoxic environments, soluble Fe typically exists as ferrous iron [Fe(II)] and S as sulfide (HS ⁻ ). These species exhibit a strong affinity that ultimately drives the formation of sedimentary pyrite (FeS ₂ ). Recently, paradigm-shifting studies indicate that Fe and S in FeS ₂ can be made bioavailable by methanogens through a reductive dissolution process. However, the impact of the utilization of FeS ₂ , as opposed to canonical Fe and S sources, on the phenotype of cells is not fully understood. Here, shotgun proteomics was utilized to measure changes in the phenotype of Methanosarcina barkeri MS grown with FeS ₂ , Fe(II)/HS ⁻ , or Fe(II)/cysteine. Shotgun proteomics tracked 1,019 proteins overall, with 307 observed to change between growth conditions. Functional characterization and pathway analyses revealed these changes to be systemic and largely tangential to Fe/S metabolism. As a final step, the proteomics data were viewed with respect to previously collected transcriptomics data to deepen the analysis. Presented here is evidence that M. barkeri adopts distinct phenotypes to exploit specific sources of Fe and S in its environment. This is supported by observed protein abundance changes across broad categories of cellular biology. DNA adjacent metabolism, central carbon metabolism methanogenesis, metal trafficking, quorum sensing, and porphyrin biosynthesis pathways are all features in the phenotypic differentiation. Differences in trace metal availability attributed to complexation with HS ⁻ , either as a component of the growth medium [Fe(II)/HS ⁻ ] or generated through reduction of FeS ₂ , were likely a major factor underpinning these phenotypic differences. IMPORTANCE: The methanogenic archaeon Methanosarcina barkeri holds great potential for industrial bio-mining and energy generation technologies. Much of the biochemistry of this microbe is poorly understood, and its characterization will provide a glimpse into biological processes that evolved close to life’s origin. The discovery of its ability to extract iron and sulfur from bulk, solid-phase minerals shifted a longstanding paradigm that these elements were inaccessible to biological systems. The full elucidation of this process has the potential to help scientists and engineers extract valuable metals from low-grade ore and mine waste generating energy in the form of methane while doing so.

Metallurgical coal mining generates significant amounts of waste rock, which can release nitrate and selenate upon atmospheric exposure, with resulting changes in surface water and ground water quality. Microorganisms residing on waste rock in the local subsurface have the metabolic potential to denitrifyand reduce selenium to treat mine-affected waters and stabilize waste rock. To support these processes, waste rock facilities have been designed to inject nutrients (e.g., methanol and phosphoric acid) in semi-passive water treatment of mine-impacted waters. Microbial community data regularly collected fromsubsurface semi-passive reactors throughout the startup and operations phases showed that native heterotrophic denitrifying bacteria and selenium reducing bacteria were enriched as a result of nutrient amendment. The microbial community was stable while the source water and geochemical parameters remained unchanged; however, a significant shift in the microbial community coincided with changes in the source of water treated. Metagenomic sequencing of microbial communities within the active treatment zone revealed multiple biochemical pathways of nitrate reduction. Geochemical and water quality data indicate near complete selenate reduction, yet a low abundance of in known selenate reduction genes were recovered. This may suggest that biologically mediated selenium reduction may be more widespread, both functionally and taxonomically. Further research into these pathways and mechanisms for nitrate and selenium reduction will help to strengthen our understanding of selenium reduction mechanisms and their application in mine water waste management.

Methanogens inhabit euxinic (sulfide-rich) or ferruginous (iron-rich) environments that promote the precipitation of transition metals as metal sulfides, such as pyrite, reducing metal or sulfur availability. Such environments have been common throughout Earth’s history raising the question as to how anaerobes obtain(ed) these elements for the synthesis of enzyme cofactors. Here, we show a methanogen can synthesize molybdenum nitrogenase metallocofactors from pyrite as the source of iron and sulfur, enabling nitrogen fixation. Pyrite-grown, nitrogen-fixing cells grow faster and require 25-fold less molybdenum than cells grown under euxinic conditions. Growth yields are 3 to 8 times higher in cultures grown under ferruginous relative to euxinic conditions. Physiological, transcriptomic, and geochemical data indicate these observations are due to sulfide-promoted metal limitation, in particular molybdenum. These findings suggest that molybdenum nitrogenase may have originated in a ferruginous environment that titrated sulfide to form pyrite, facilitating the availability of sufficient iron, sulfur, and molybdenum for cofactor biosynthesis.

Metagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of methyl-coenzyme M reductase subunit A (mcrA) gene amplicons demonstrated a wide diversity of Mcr-encoding archaea inhabit geothermal features with differing physicochemical regimes across YNP. From three selected hot springs we recovered twelve Mcr-encoding metagenome assembled genomes (MAGs) affiliated with lineages of cultured methanogens as well as Candidatus (Ca.) Methanomethylicia, Ca. Hadesarchaeia, and Archaeoglobi. These MAGs encoded the potential for hydrogenotrophic, aceticlastic, hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represent minor fractions of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in the stimulation of methanogenic activity and the enrichment of lineages affiliated with Methanosaeta and Methanothermobacter as well as with uncultured Mcr-encoding archaea including Ca. Korarchaeia, Ca. Nezhaarchaeia, and Archaeoglobi. We revealed that diverse Mcr-encoding archaea with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP and can be enriched under methanogenic conditions. This study highlights the importance of combining environmental metagenomics with laboratory-based experiments to expand our understanding of uncultured Mcr-encoding archaea and their potential impact on microbial carbon transformations in geothermal environments and beyond.

Metagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated the methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of gene amplicons of methyl-coenzyme M reductase subunit A ( mcrA ) indicated a wide diversity of Mcr-encoding archaea across geothermal features with differing physicochemical regimes. From three selected hot springs we recovered twelve Mcr - encoding metagenome assembled genomes (MAGs) affiliated with lineages of cultured methanogens as well as Candidatus ( Ca .) Methanomethylicia, Ca . Hadesarchaeia, and Archaeoglobi. These MAGs encoded the potential for hydrogenotrophic, aceticlastic, or hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represented a minor fraction of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in stimulation of methanogenic activity and the enrichment of lineages affiliated with Methanosaeta and Methanothermobacter as well as with uncultured Mcr-encoding archaea including Ca . Korarchaeia, Ca . Nezhaarchaeia, and Archaeoglobi. Altogether, we revealed that diverse Mcr-encoding populations with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP. This study highlights the importance of combining environmental metagenomics with laboratory-based experiments to expand our understanding of uncultured Mcr-encoding archaea and their potential impact on microbial carbon transformations in geothermal environments and beyond.

The SUP05 clade of gammaproteobacteria (Thioglobaceae) comprises both primary producers and primary consumers of organic carbon in the oceans. Host-associated autotrophs are a principal source of carbon and other nutrients for deep-sea eukaryotes at hydrothermal vents, and their free-living relatives are a primary source of organic matter in seawater at vents and in marine oxygen minimum zones. Similar to other abundant marine heterotrophs, such as SAR11 and Roseobacter, heterotrophic Thioglobaceae use the dilute pool of osmolytes produced by phytoplankton for growth, including methylated amines and sulfonates. Heterotrophic members are common throughout the ocean, and autotrophic members are abundant at hydrothermal vents and in anoxic waters; combined, they can account for more than 50% of the total bacterial community. Studies of both cultured and uncultured representatives from this diverse family are providing novel insights into the shifting biogeochemical roles of autotrophic and heterotrophic bacteria that cross oxic–anoxic boundary layers in the ocean.

FeS ₂ is the most abundant sulfide mineral in the Earth’s crust and is common in environments inhabited by methanogenic archaea. FeS ₂ can be reduced by methanogens, yielding aqueous FeS _aq clusters that are thought to be a source of Fe and S.

Summary: 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.