Trophic interactions shape the spatial organization of medium-chain carboxylic acid producing granular biofilm communities.

Granular biofilms producing medium-chain carboxylic acids (MCCA) from carbohydrate-rich industrial feedstocks harbor highly streamlined communities converting sugars to MCCA either directly or via lactic acid as intermediate. We investigated the spatial organization and growth activity patterns of MCCA producing granular biofilms grown on an industrial side stream to test (i) whether key functional guilds (lactic acid producing Olsenella and MCCA producing Oscillospiraceae) stratified in the biofilm based on substrate usage, and (ii) whether spatial patterns of growth activity shaped the unique, lenticular morphology of these biofilms. First, three novel isolates (one Olsenella and two Oscillospiraceae species) representing over half of the granular biofilm community were obtained and used to develop FISH probes, revealing that key functional guilds were not stratified. Instead, the outer 150-500 µm of the granular biofilm consisted of a well-mixed community of Olsenella and Oscillospiraceae, while deeper layers were made up of other bacteria with lower activities. Second, nanoSIMS analysis of 15N incorporation in biofilms grown in normal and lactic acid amended conditions suggested Oscillospiraceae switched from sugars to lactic acid as substrate. This suggests competitive-cooperative interactions may govern the spatial organization of these biofilms, and suggests that optimizing biofilm size may be a suitable process engineering strategy. Third, growth activities were similar in the polar and equatorial biofilm peripheries, leaving the mechanism behind the lenticular biofilm morphology unexplained. Physical processes (e.g., shear hydrodynamics, biofilm life cycles) may have contributed to lenticular biofilm development. Together, this study develops an ecological framework of MCCA-producing granular biofilms that informs bioprocess development.

Physiological potential and evolutionary trajectories of syntrophic sulfate-reducing bacterial partners of anaerobic methanotrophic archaea.

Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we used comparative metagenomics and phylogenetics to investigate the metabolic adaptation among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distinguish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacterota. We show that the protein complexes involved in direct interspecies electron transfer (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic lineages. In contrast, the proteins involved in electron transfer within the SRB inner membrane differ between clades, indicative of convergent evolution in the adaptation to a syntrophic lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the acquisition of the DIET complexes in an ancestral clade and involve horizontal gene transfers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also provide evidence for unique adaptations within syntrophic SRB clades, which vary depending on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, subclades that specifically partner ANME-2a are missing the cobalamin synthesis pathway, suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a partners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features associated with DIET-based syntrophy and the adaptation of SRB towards it.

Determining resident microbial community members and their correlations with geochemistry in a serpentinizing spring.

Terrestrial serpentinizing systems allow us insight into the realm of alkaliphilic microbial communities driven by geology in a way that is frequently more accessible than their deep subsurface or marine counterparts. However, these systems are also marked by geochemical and microbial community variation due to the interactions of serpentinized fluids with host geology and the surface environment. To separate the transient from the endemic microbes in a hyperalkaline environment, we assessed the Ney Springs terrestrial serpentinizing system microbial community and geochemistry at six time points over the span of a year. Using 16S rRNA gene surveys we observed 93 amplicon sequence variants (ASVs) that were found at every sampling event. This is compared to ~17,000 transient ASVs that were detected only once across the six sampling events. Of the resident community members, 16 of these ASVs were regularly greater than 1% of the community during every sampling period. Additionally, many of these core taxa experienced statistically significant changes in relative abundance with time. Variation in the abundance of some core populations correlated with geochemical variation. For example, members of the Tindallia group, showed a positive correlation with variation in levels of ammonia at the spring. Investigating the metagenome assembled genomes of these microbes revealed evidence of the potential for ammonia generation via Stickland reactions within Tindallia. This observation offers new insight into the origin of high ammonia concentrations (>70 mg/L) seen at this site. Similarly, the abundance of putative sulfur-oxidizing microbes like Thiomicrospira, Halomonas, and a Rhodobacteraceae species could be linked to changes observed in sulfur-oxidation intermediates like tetrathionate and thiosulfate. While these data supports the influence of core microbial community members on a hyperalkaline spring's geochemistry, there is also evidence that subsurface processes affect geochemistry and may impact community dynamics as well. Though the physiology and ecology of these astrobiologically relevant ecosystems are still being uncovered, this work helps identify a stable microbial community that impacts spring geochemistry in ways not previously observed in serpentinizing ecosystems.

Mcr-dependent methanogenesis in Archaeoglobaceae enriched from a terrestrial hot spring.

The preeminent source of biological methane on Earth is methyl coenzyme M reductase (Mcr)-dependent archaeal methanogenesis. A growing body of evidence suggests a diversity of archaea possess Mcr, although experimental validation of hypothesized methane metabolisms has been missing. Here, we provide evidence of a functional Mcr-based methanogenesis pathway in a novel member of the family Archaeoglobaceae, designated Methanoglobus nevadensis, which we enriched from a terrestrial hot spring on the polysaccharide xyloglucan. Our incubation assays demonstrate methane production that is highly sensitive to the Mcr inhibitor bromoethanesulfonate, stimulated by xyloglucan and xyloglucan-derived sugars, concomitant with the consumption of molecular hydrogen, and causing a deuterium fractionation in methane characteristic of hydrogenotrophic and methylotrophic methanogens. Combined with the recovery and analysis of a high-quality M. nevadensis metagenome-assembled genome encoding a divergent Mcr and diverse potential electron and carbon transfer pathways, our observations suggest methanogenesis in M. nevadensis occurs via Mcr and is fueled by the consumption of cross-fed byproducts of xyloglucan fermentation mediated by other community members. Phylogenetic analysis shows close affiliation of the M. nevadensis Mcr with those from Korarchaeota, Nezhaarchaeota, Verstraetearchaeota, and other Archaeoglobales that are divergent from well-characterized Mcr. We propose these archaea likely also use functional Mcr complexes to generate methane on the basis of our experimental validation in M. nevadensis. Thus, divergent Mcr-encoding archaea may be underestimated sources of biological methane in terrestrial and marine hydrothermal environments.

McrD binds asymmetrically to methyl-coenzyme M reductase improving active-site accessibility during assembly.

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane, and its activity accounts for nearly all biologically produced methane released into the atmosphere. The assembly of MCR is an intricate process involving the installation of a complex set of posttranslational modifications and the unique Ni-containing tetrapyrrole called coenzyme F430. Despite decades of research, details of MCR assembly remain largely unresolved. Here, we report the structural characterization of MCR in two intermediate states of assembly. These intermediate states lack one or both F430 cofactors and form complexes with the previously uncharacterized McrD protein. McrD is found to bind asymmetrically to MCR, displacing large regions of the alpha subunit and increasing active-site accessibility for the installation of F430-shedding light on the assembly of MCR and the role of McrD therein. This work offers crucial information for the expression of MCR in a heterologous host and provides targets for the design of MCR inhibitors.

Metabolic Strategies Shared by Basement Residents of the Lost City Hydrothermal Field.

Alkaline fluids venting from chimneys of the Lost City hydrothermal field flow from a potentially vast microbial habitat within the seafloor where energy and organic molecules are released by chemical reactions within rocks uplifted from Earth's mantle. In this study, we investigated hydrothermal fluids venting from Lost City chimneys as windows into subseafloor environments where the products of geochemical reactions, such as molecular hydrogen (H2), formate, and methane, may be the only available sources of energy for biological activity. Our deep sequencing of metagenomes and metatranscriptomes from these hydrothermal fluids revealed a few key species of archaea and bacteria that are likely to play critical roles in the subseafloor microbial ecosystem. We identified a population of Thermodesulfovibrionales (belonging to phylum Nitrospirota) as a prevalent sulfate-reducing bacterium that may be responsible for much of the consumption of H2 and sulfate in Lost City fluids. Metagenome-assembled genomes (MAGs) classified as Methanosarcinaceae and Candidatus Bipolaricaulota were also recovered from venting fluids and represent potential methanogenic and acetogenic members of the subseafloor ecosystem. These genomes share novel hydrogenases and formate dehydrogenase-like sequences that may be unique to hydrothermal environments where H2 and formate are much more abundant than carbon dioxide. The results of this study include multiple examples of metabolic strategies that appear to be advantageous in hydrothermal and subsurface alkaline environments where energy and carbon are provided by geochemical reactions. IMPORTANCE The Lost City hydrothermal field is an iconic example of a microbial ecosystem fueled by energy and carbon from Earth's mantle. Uplift of mantle rocks into the seafloor can trigger a process known as serpentinization that releases molecular hydrogen (H2) and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon. This study provides an initial glimpse into the kinds of microbes that live deep within the seafloor where serpentinization takes place, by sampling hydrothermal fluids exiting from the Lost City chimneys. The metabolic strategies that these microbes appear to be using are also shared by microbes that inhabit other sites of serpentinization, including continental subsurface environments and natural springs. Therefore, the results of this study contribute to a broader, interdisciplinary effort to understand the general principles and mechanisms by which serpentinization-associated processes can support life on Earth and perhaps other worlds.

Investigation of microbial metabolisms in an extremely high pH marine-like terrestrial serpentinizing system: Ney Springs.

Ney Springs, a continental serpentinizing spring in northern California, has an exceptionally high reported pH (12.4) for a naturally occurring water source. With high conductivity fluids, it is geochemically more akin to marine serpentinizing systems than other terrestrial locations. Our geochemical analyses also revealed high sulfide concentrations (544 mg/L) and methane emissions (83% volume gas content) relative to other serpentinizing systems. Thermodynamic calculations were used to investigate the potential for substrates resulting from serpentinization to fuel microbial life, and were found to support the energetic feasibility of sulfate reduction, anaerobic methane oxidation, denitrification, and anaerobic sulfide oxidation within this system. Assessment of the microbial community via 16S rRNA taxonomic gene surveys and metagenome sequencing revealed a community composition dominated by poorly characterized members of the Izemoplasmatales and Clostridiales. The genomes of these dominant taxa point to a fermentative lifestyle, though other highly complete (>90%) metagenome assembled genomes support the potential for organisms to perform sulfate reduction, sulfur disproportionation and/or sulfur oxidation (aerobic and anaerobic). Two chemolithoheterotrophs identified in the metagenome, a Halomonas sp. and a Rhodobacteraceae sp., were isolated and shown to oxidize thiosulfate and were capable of growth in conditions up to pH 12.4. Despite being characteristic products of serpentinization reactions, little evidence was seen for hydrogen and methane utilization in the Ney Springs microbial community. Hydrogen is not highly abundant and could be consumed prior to reaching the spring community. Other metabolic strategies may be outcompeted by more energetically favorable heterotrophic or fermentation reactions, or even inhibited by other compounds in the spring such as ammonia. The unique geochemistry of Ney Springs provides an opportunity to study how local geology interacts with serpentinized fluids, while its microbial community can better inform us of the metabolic strategies employed in hyperalkaline environments.

Comparative Genomics on Cultivated and Uncultivated Freshwater and Marine "Candidatus Manganitrophaceae" Species Implies Their Worldwide Reach in Manganese Chemolithoautotrophy.

Chemolithoautotrophic manganese oxidation has long been theorized but only recently demonstrated in a bacterial coculture. The majority member of the coculture, "Candidatus Manganitrophus noduliformans," is a distinct but not yet isolated lineage in the phylum Nitrospirota (Nitrospirae). Here, we established two additional MnCO3-oxidizing cultures using inocula from Santa Barbara (California) and Boetsap (South Africa). Both cultures were dominated by strains of a new species, designated "Candidatus Manganitrophus morganii." The next most abundant members differed in the available cultures, suggesting that while "Ca. Manganitrophus" species have not been isolated in pure culture, they may not require a specific syntrophic relationship with another species. Phylogeny of cultivated "Ca. Manganitrophus" and related metagenome-assembled genomes revealed a coherent taxonomic family, "Candidatus Manganitrophaceae," from both freshwater and marine environments and distributed globally. Comparative genomic analyses support this family being Mn(II)-oxidizing chemolithoautotrophs. Among the 895 shared genes were a subset of those hypothesized for Mn(II) oxidation (Cyc2 and PCC_1) and oxygen reduction (TO_1 and TO_2) that could facilitate Mn(II) lithotrophy. An unusual, plausibly reverse complex 1 containing 2 additional pumping subunits was also shared by the family, as were genes for the reverse tricarboxylic acid carbon fixation cycle, which could enable Mn(II) autotrophy. All members of the family lacked genes for nitrification found in Nitrospira species. The results suggest that "Ca. Manganitrophaceae" share a core set of candidate genes for the newly discovered manganese-dependent chemolithoautotrophic lifestyle and likely have a broad, global distribution. IMPORTANCE Manganese (Mn) is an abundant redox-active metal that cycles in many of Earth's biomes. While diverse bacteria and archaea have been demonstrated to respire Mn(III/IV), only recently have bacteria been implicated in Mn(II) oxidation-dependent growth. Here, two new Mn(II)-oxidizing enrichment cultures originating from two continents and hemispheres were examined. By comparing the community composition of the enrichments and performing phylogenomic analysis on the abundant Nitrospirota therein, new insights are gleaned on cell interactions, taxonomy, and machineries that may underlie Mn(II)-based lithotrophy and autotrophy.

Comparative genomics reveals electron transfer and syntrophic mechanisms differentiating methanotrophic and methanogenic archaea.

The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota, none of which have been isolated in pure culture. Here, we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features that separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor.

Sulfate differentially stimulates but is not respired by diverse anaerobic methanotrophic archaea.

Sulfate-coupled anaerobic oxidation of methane (AOM) is a major methane sink in marine sediments. Multiple lineages of anaerobic methanotrophic archaea (ANME) often coexist in sediments and catalyze this process syntrophically with sulfate-reducing bacteria (SRB), but the potential differences in ANME ecophysiology and mechanisms of syntrophy remain unresolved. A humic acid analog, anthraquinone 2,6-disulfonate (AQDS), could decouple archaeal methanotrophy from bacterial sulfate reduction and serve as the terminal electron acceptor for AOM (AQDS-coupled AOM). Here in sediment microcosm experiments, we examined variations in physiological response between two co-occurring ANME-2 families (ANME-2a and ANME-2c) and tested the hypothesis of sulfate respiration by ANME-2. Sulfate concentrations as low as 100 µM increased AQDS-coupled AOM nearly 2-fold matching the rates of sulfate-coupled AOM. However, the SRB partners remained inactive in microcosms with sulfate and AQDS and neither ANME-2 families respired sulfate, as shown by their cellular sulfur contents and anabolic activities measured using nanoscale secondary ion mass spectrometry. ANME-2a anabolic activity was significantly higher than ANME-2c, suggesting that ANME-2a was primarily responsible for the observed sulfate stimulation of AQDS-coupled AOM. Comparative transcriptomics showed significant upregulation of ANME-2a transcripts linked to multiple ABC transporters and downregulation of central carbon metabolism during AQDS-coupled AOM compared to sulfate-coupled AOM. Surprisingly, genes involved in sulfur anabolism were not differentially expressed during AQDS-coupled AOM with and without sulfate amendment. Collectively, this data indicates that ANME-2 archaea are incapable of respiring sulfate, but sulfate availability differentially stimulates the growth and AOM activity of different ANME lineages.

Evidence of a Streamlined Extracellular Electron Transfer Pathway from Biofilm Structure, Metabolic Stratification, and Long-Range Electron Transfer Parameters.

A strain of Geobacter sulfurreducens, an organism capable of respiring solid extracellular substrates, lacking four of five outer membrane cytochrome complexes (extABCD+ strain) grows faster and produces greater current density than the wild type grown under identical conditions. To understand cellular and biofilm modifications in the extABCD+ strain responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS (nanoscale secondary ion mass spectrometry) to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild-type and extABCD+ biofilms spanning 5-µm gaps. Our results reveal that extABCD+ biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to the wild type (based on increased rates of 15N incorporation). We also observed an increased rate of electron transfer through extABCD+ versus wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multiheme cytochromes streamline electron transfer to electrodes. The combination of imaging, physiological, and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than the wild type. IMPORTANCE Current-producing biofilms in microbial electrochemical systems could potentially sustain technologies ranging from wastewater treatment to bioproduction of electricity if the maximum current produced could be increased and current production start-up times after inoculation could be reduced. Enhancing the current output of microbial electrochemical systems has been mostly approached by engineering physical components of reactors and electrodes. Here, we show that biofilms formed by a Geobacter sulfurreducens strain producing ∼1.4× higher current than the wild type results from a combination of denser cell packing and higher anabolic activity, enabled by an increased rate of electron diffusion through the biofilms. Our results confirm that it is possible to engineer electrode-specific G. sulfurreducens strains with both faster growth on electrodes and streamlined electron transfer pathways for enhanced current production.

An ecophysiological explanation for manganese enrichment in rock varnish.

Desert varnish is a dark rock coating that forms in arid environments worldwide. It is highly and selectively enriched in manganese, the mechanism for which has been a long-standing geological mystery. We collected varnish samples from diverse sites across the western United States, examined them in petrographic thin section using microscale chemical imaging techniques, and investigated the associated microbial communities using 16S amplicon and shotgun metagenomic DNA sequencing. Our analyses described a material governed by sunlight, water, and manganese redox cycling that hosts an unusually aerobic microbial ecosystem characterized by a remarkable abundance of photosynthetic Cyanobacteria in the genus Chroococcidiopsis as the major autotrophic constituent. We then showed that diverse Cyanobacteria, including the relevant Chroococcidiopsis taxon, accumulate extraordinary amounts of intracellular manganese-over two orders of magnitude higher manganese content than other cells. The speciation of this manganese determined by advanced paramagnetic resonance techniques suggested that the Cyanobacteria use it as a catalytic antioxidant-a valuable adaptation for coping with the substantial oxidative stress present in this environment. Taken together, these results indicated that the manganese enrichment in varnish is related to its specific uptake and use by likely founding members of varnish microbial communities.

Controls on Interspecies Electron Transport and Size Limitation of Anaerobically Methane-Oxidizing Microbial Consortia.

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.

Draft Genome Sequence of the Free-Living, Iridescent Bacterium Tenacibaculum mesophilum Strain ECR.

Here, we report the genome sequence of Tenacibaculum mesophilum strain ECR, which was isolated from the river/ocean interface at Trunk River in Falmouth, Massachusetts. The isolation and sequencing were performed as part of the 2016 and 2018 Microbial Diversity courses at the Marine Biological Laboratory in Woods Hole, Massachusetts.

Peptidoglycan Production by an Insect-Bacterial Mosaic.

Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.

NanoSIMS imaging reveals metabolic stratification within current-producing biofilms.

Metal-reducing bacteria direct electrons to their outer surfaces, where insoluble metal oxides or electrodes act as terminal electron acceptors, generating electrical current from anaerobic respiration. Geobacter sulfurreducens is a commonly enriched electricity-producing organism, forming thick conductive biofilms that magnify total activity by supporting respiration of cells not in direct contact with electrodes. Hypotheses explaining why these biofilms fail to produce higher current densities suggest inhibition by formation of pH, nutrient, or redox potential gradients; but these explanations are often contradictory, and a lack of direct measurements of cellular growth within biofilms prevents discrimination between these models. To address this fundamental question, we measured the anabolic activity of G. sulfurreducens biofilms using stable isotope probing coupled to nanoscale secondary ion mass spectrometry (nanoSIMS). Our results demonstrate that the most active cells are at the anode surface, and that this activity decreases with distance, reaching a minimum 10 µm from the electrode. Cells nearest the electrode continue to grow at their maximum rate in weeks-old biofilms 80-µm-thick, indicating nutrient or buffer diffusion into the biofilm is not rate-limiting. This pattern, where highest activity occurs at the electrode and declines with each cell layer, is present in thin biofilms (<5 µm) and fully grown biofilms (>20 µm), and at different anode redox potentials. These results suggest a growth penalty is associated with respiring insoluble electron acceptors at micron distances, which has important implications for improving microbial electrochemical devices as well as our understanding of syntrophic associations harnessing the phenomenon of microbial conductivity.

Carbon isotopic heterogeneity of coenzyme F430 and membrane lipids in methane-oxidizing archaea.

Archaeal ANaerobic MEthanotrophs (ANME) facilitate the anaerobic oxidation of methane (AOM), a process that is believed to proceed via the reversal of the methanogenesis pathway. Carbon isotopic composition studies indicate that ANME are metabolically diverse and able to assimilate metabolites including methane, methanol, acetate, and dissolved inorganic carbon (DIC). Our data support the interpretation that ANME in marine sediments at methane seeps assimilate both methane and DIC, and the carbon isotopic compositions of the tetrapyrrole coenzyme F430 and the membrane lipids archaeol and hydroxy-archaeol reflect their relative proportions of carbon from these substrates. Methane is assimilated via the methyl group of CH3 -tetrahydromethanopterin (H4 MPT) and DIC from carboxylation reactions that incorporate free intracellular DIC. F430 was enriched in 13 C (mean δ13 C = -27‰ for Hydrate Ridge and -80‰ for the Santa Monica Basin) compared to the archaeal lipids (mean δ13 C = -97‰ for Hydrate Ridge and -122‰ for the Santa Monica Basin). We propose that depending on the side of the tricarboxylic acid (TCA) cycle used to synthesize F430, its carbon was derived from 76% DIC and 24% methane via the reductive side or 57% DIC and 43% methane via the oxidative side. ANME lipids are predicted to contain 42% DIC and 58% methane, reflecting the amount of each assimilated into acetyl-CoA. With isotope models that include variable fractionation during biosynthesis for different carbon substrates, we show the estimated amounts of DIC and methane can result in carbon isotopic compositions of - 73‰ to - 77‰ for F430 and - 105‰ for archaeal lipids, values close to those for Santa Monica Basin. The F430 δ13 C value for Hydrate Ridge was 13 C-enriched compared with the modeled value, suggesting there is divergence from the predicted two carbon source models.

Divergent methyl-coenzyme M reductase genes in a deep-subseafloor Archaeoglobi.

The methyl-coenzyme M reductase (MCR) complex is a key enzyme in archaeal methane generation and has recently been proposed to also be involved in the oxidation of short-chain hydrocarbons including methane, butane, and potentially propane. The number of archaeal clades encoding the MCR continues to grow, suggesting that this complex was inherited from an ancient ancestor, or has undergone extensive horizontal gene transfer. Expanding the representation of MCR-encoding lineages through metagenomic approaches will help resolve the evolutionary history of this complex. Here, a near-complete Archaeoglobi metagenome-assembled genome (MAG; Ca. Polytropus marinifundus gen. nov. sp. nov.) was recovered from the deep subseafloor along the Juan de Fuca Ridge flank that encodes two divergent McrABG operons similar to those found in Ca. Bathyarchaeota and Ca. Syntrophoarchaeum MAGs. Ca. P. marinifundus is basal to members of the class Archaeoglobi, and encodes the genes for ß-oxidation, potentially allowing an alkanotrophic metabolism similar to that proposed for Ca. Syntrophoarchaeum. Ca. P. marinifundus also encodes a respiratory electron transport chain that can potentially utilize nitrate, iron, and sulfur compounds as electron acceptors. Phylogenetic analysis suggests that the Ca. P. marinifundus MCR operons were horizontally transferred, changing our understanding of the evolution and distribution of this complex in the Archaea.

Widespread nitrogen fixation in sediments from diverse deep-sea sites of elevated carbon loading.

Nitrogen fixation, the biological conversion of N2 to NH3 , is critical to alleviating nitrogen limitation in many marine ecosystems. To date, few measurements exist of N2 fixation in deep-sea sediments. Here, we conducted > 400 bottle incubations with sediments from methane seeps, whale falls and background sites off the western coast of the United States from 600 to 2893 m water depth to investigate the potential rates, spatial distribution and biological mediators of benthic N2 fixation. We found that N2 fixation was widespread, yet heterogeneously distributed with sediment depth at all sites. In some locations, rates exceeded previous measurements by > 10×, and provided up to 30% of the community anabolic growth requirement for nitrogen. Diazotrophic activity appeared to be inhibited by pore water ammonium: N2 fixation was only observed if incubation ammonium concentrations were ≤ 25 µM, and experimental additions of ammonium reduced diazotrophy. In seep sediments, N2 fixation was dependent on CH4 and coincident with sulphate reduction, consistent with previous work showing diazotrophy by microorganisms mediating sulphate-coupled methane oxidation. However, the pattern of diazotrophy was different in whale-fall and associated reference sediments, where it was largely unaffected by CH4 , suggesting catabolically different diazotrophs at these sites.

Convergent evolution of unusual complex I homologs with increased proton pumping capacity: energetic and ecological implications.

Respiratory complex I is part of a large family of homologous enzymes that carry out the transfer of electrons between soluble cytoplasmic electron carriers and membrane-bound electron carriers. These complexes are vital bioenergetic enzymes that serve as the entry points into electron transport chains for a wide variety of microbial metabolisms, and electron transfer is coupled to proton translocation. The core complex of this enzyme is made up of 11 protein subunits, with three major proton pumping subunits. Here, we document a large number of modified complex I gene cassettes found in genome sequences from diverse cultured bacteria, shotgun metagenomics, and environmentally derived archaeal fosmids all of which encode a fourth proton pumping subunit. The incorporation of this extra subunit into a functional protein complex is supported by large amino acid insertions in the amphipathic helix that runs the length of the protein complex. Phylogenetic analyses reveal that these modified complexes appear to have arisen independently multiple times in a remarkable case of convergent molecular evolution. From an energetic perspective, we hypothesize that this modification on the canonical complex I architecture allows for the translocation of a fifth proton per reaction cycle-the physiological utility of this modified complex is discussed.