Deep (>1 km depth) scientific boreholes are unique assets that can be used to address a variety of microbiological, hydrologic, and biogeochemical hypotheses. Few of these deep boreholes exist in oceanic crust. One of them, Deep Sea Drilling Project Hole 504B, reaches ∼190 ∘C at its base. We designed, fabricated, and laboratory-tested the Multi-Temperature Fluid Sampler (MTFS), a non-gas-tight, titanium syringe-style fluid sampler for borehole applications that is tolerant of such high temperatures. Each of the 12 MTFS units collects a single 1 L sample at a predetermined temperature, which is defined by the trigger design and a shape memory alloy (SMA). SMAs have the innate ability to be deformed and only return to their initial shapes when their activation temperatures are reached, thereby triggering a sampler at a predetermined temperature. Three SMA-based trigger mechanisms, which do not rely on electronics, were tested. Triggers were released at temperatures spanning from 80 to 181 ∘C. The MTFS was set for deployment on International Ocean Discovery Program Expedition 385T, but hole conditions precluded its use. The sampler is ready for use in deep oceanic or continental scientific boreholes with minimal training for operational success.
The remineralization of organic material via heterotrophy in the marine environment is performed by a diverse and varied group of microorganisms that can specialize in the type of organic material degraded and the niche they occupy. The marine Dadabacteria are cosmopolitan in the marine environment and belong to a candidate phylum for which there has not been a comprehensive assessment of the available genomic data to date. Here in, we assess the functional potential of the marine pelagic Dadabacteria in comparison to members of the phylum that originate from terrestrial, hydrothermal, and subsurface environments. Our analysis reveals that the marine pelagic Dadabacteria have streamlined genomes, corresponding to smaller genome sizes and lower nitrogen content of their DNA and predicted proteome, relative to their phylogenetic counterparts. Collectively, the Dadabacteria have the potential to degrade microbial dissolved organic matter, specifically peptidoglycan and phospholipids. The marine Dadabacteria belong to two clades with apparent distinct ecological niches in global metagenomic data: a clade with the potential for photoheterotrophy through the use of proteorhodopsin, present predominantly in surface waters up to 100 m depth; and a clade lacking the potential for photoheterotrophy that is more abundant in the deep photic zone.
Chloroflexi are widespread in subsurface environments, and recent studies indicate that they represent a major fraction of the communities in subseafloor sediment. Here, we compare the abundance, diversity, metabolic potential and gene expression of Chloroflexi from three abyssal sediment cores from the western North Atlantic Gyre (water depth >5400 m) covering up to 15 million years of sediment deposition, where Chloroflexi were found to represent major components of the community at all sites. Chloroflexi communities die off in oxic red clay over 10–15 million years, and gene expression was below detection. In contrast, Chloroflexi abundance and gene expression at the anoxic abyssal clay site increase below the seafloor and peak in 2–3 million-year-old sediment, indicating a comparably higher activity. Metatranscriptomes from the anoxic site reveal increased expression of Chloroflexi genes involved in cell wall biogenesis, protein turnover, inorganic ion transport, defense mechanisms and prophages. Phylogenetic analysis shows that these Chloroflexi are closely related to homoacetogenic subseafloor clades and actively transcribe genes involved in sugar fermentations, gluconeogenesis and Wood–Ljungdahl pathway in the subseafloor. Concomitant expression of cell division genes indicates that these putative homoacetogenic Chloroflexi are actively growing in these million-year-old anoxic abyssal sediments.
Shallow-water hydrothermal vents are ubiquitous ecosystems distributed worldwide between the inter-tidal zone and ~ 200 m depth. Important factors differentiating shallow-water vents from their deep-sea counterparts include the presence of sunlight, tidal/wave forcing, meteoric water sources, terrigenous inputs of organic matter and abundant free gas. The presence of sunlight and high organic carbon loads together with differences in availability of electron donors and acceptors defines unique habitats that are exploited by microbial communities. We summarize here the current knowledge of the microbiology of shallow-water vents worldwide, highlighting gaps in our understanding of these unique environments.
The availability of microbiological and geochemical data from island-based and high-arsenic hydrothermal systems is limited. Here, the microbial diversity in island-based hot springs on Ambitle Island (Papua New Guinea) was investigated using culture-dependent and -independent methods. Waramung and Kapkai are alkaline springs high in sulfide and arsenic, related hydrologically to previously described hydrothermal vents in nearby Tutum Bay. Enrichments were carried out at 24 conditions with varying temperature (45, 80 °C), pH (6.5, 8.5), terminal electron acceptors (O2, SO4 2−, S0, NO3 −), and electron donors (organic carbon, H2, AsIII). Growth was observed in 20 of 72 tubes, with media targeting heterotrophic metabolisms the most successful. 16S ribosomal RNA gene surveys of environmental samples revealed representatives in 15 bacterial phyla and 8 archaeal orders. While the Kapkai 4 bacterial clone library is primarily made up of Thermodesulfobacteria (74 %), no bacterial taxon represents a majority in the Kapkai 3 and Waramung samples (40 % Proteobacteria and 39 % Aquificae, respectively). Deinococcus/Thermus and Thermotogae are observed in all samples. The Thermococcales dominate the archaeal clone libraries (65–85 %). Thermoproteales, Desulfurococcales, and uncultured Eury- and Crenarchaeota make up the remaining archaeal taxonomic diversity. The culturing and phylogenetic results are consistent with the geochemistry of the alkaline, saline, and sulfide-rich fluids. When compared to other alkaline, island-based, high-arsenic, or shallow-sea hydrothermal communities, the Ambitle Island archaeal communities are unique in geochemical conditions, and in taxonomic diversity, richness, and evenness.
We studied microbially mediated diagenetic processes driven by carbon mineralization in subseafloor sediment of the northeastern Bering Sea Slope to a depth of 745 meters below seafloor (mbsf). Sites U1343, U1344 and U1345 were drilled during Integrated Ocean Drilling Program (IODP) Expedition 323 at water depths of 1008 to 3172 m. They are situated in the high productivity “Green Belt” region, with organic carbon burial rates typical of the high-productivity upwelling domains on western continental margins. The three sites show strong geochemical similarities. The downward sequence of microbially mediated processes in the sediment encompasses (1) organoclastic sulfate reduction, (2) anaerobic oxidation of methane (AOM) coupled to sulfate reduction, and (3) methanogenesis. The sediment contains two distinct zones of diagenetic carbonate formation, located at the sulfate–methane transition zone (SMTZ) and between 300 and 400 mbsf. The SMTZ at the three sites is located between 6 and 9 mbsf. The upward methane fluxes into the SMTZ are similar to fluxes in SMTZs underlying high-productivity surface waters off Chile and Namibia. Our Bering Sea results show that intense organic carbon mineralization drives high ammonium and dissolved inorganic carbon (DIC) production rates (> 4.2 mmol m− 3 y− 1) in the uppermost 10 mbsf and strongly imprints on the stable carbon isotope composition of DIC, driving it to a minimum value of − 27‰ (VPDB) at the SMTZ. Pore-water calcium and magnesium profiles demonstrate formation of diagenetic Mg-rich calcite in the SMTZ. Below the SMTZ, methanogenesis results in 13C-enrichment of pore-water DIC, with a maximum value of + 11.9‰. The imprint of methanogenesis on the DIC carbon isotope composition is evident down to a depth of 150 mbsf. Below this depth, slow or absent microbially mediated carbon mineralization leaves DIC isotope composition unaffected. Ongoing carbonate formation between 300 and 400 mbsf strongly influences pore-water DIC and magnesium concentration profiles. The linked succession of organic carbon mineralization and carbonate dissolution and precipitation patterns that we observe in the Bering Sea Slope sediment may be representative of passive continental margin settings in high-productivity areas of the world’s ocean.
Advances in sequencing, assembly, and assortment of contigs into species-specific bins has enabled the reconstruction of genomes from metagenomic data (MAGs). Though a powerful technique, it is difficult to determine whether assembly and binning techniques are accurate when applied to environmental metagenomes due to a lack of complete reference genome sequences against which to check the resulting MAGs. We compared MAGs derived from an enrichment culture containing ~20 organisms to complete genome sequences of 10 organisms isolated from the enrichment culture. Factors commonly considered in binning software—nucleotide composition and sequence repetitiveness—were calculated for both the correctly binned and not-binned regions. This direct comparison revealed biases in sequence characteristics and gene content in the not-binned regions. Additionally, the composition of three public data sets representing MAGs reconstructed from the Tara Oceans metagenomic data was compared to a set of representative genomes available through NCBI RefSeq to verify that the biases identified were observable in more complex data sets and using three contemporary binning software packages. Repeat sequences were frequently not binned in the genome reconstruction processes, as were sequence regions with variant nucleotide composition. Genes encoded on the not-binned regions were strongly biased towards ribosomal RNAs, transfer RNAs, mobile element functions and genes of unknown function. Our results support genome reconstruction as a robust process and suggest that reconstructions determined to be >90% complete are likely to effectively represent organismal function; however, population-level genotypic heterogeneity in natural populations, such as uneven distribution of plasmids, can lead to incorrect inferences.
The activity of individual microorganisms can be measured within environmental samples by detecting uptake of isotope‐labelled substrates using nano‐scale secondary ion mass spectrometry (nanoSIMS). Recent studies have demonstrated that sample preparation can decrease 13C and 15N enrichment in bacterial cells, resulting in underestimates of activity. Here, we explore this effect with a variety of preparation types, microbial lineages and isotope labels to determine its consistency and therefore potential for correction. Specifically, we investigated the impact of different protocols for fixation, nucleic acid staining and catalysed reporter deposition fluorescence in situ hybridization (CARD‐FISH) on >14 500 archaeal and bacterial cells (Methanosarcina acetivorans, Sulfolobus acidocaldarius and Pseudomonas putida) enriched in 13C, 15N, 18O, 2H and/or 34S. We found these methods decrease isotope enrichments by up to 80% – much more than previously reported – and that the effect varies by taxa, growth phase, isotope label and applied protocol. We make recommendations for how to account for this effect experimentally and analytically. We also re‐evaluate published nanoSIMS datasets and revise estimated microbial turnover times in the marine subsurface and nitrogen fixation rates in pelagic unicellular cyanobacteria. When sample preparation is accounted for, cell‐specific rates increase and are more consistent with modelled and bulk rates.
In 2016, temperature recorders were recovered, temperatures were measured, and fluid samples were collected from Vent 1, a high temperature (338°C) hydrothermal discharge site on the southern Cleft Segment of the Juan de Fuca Ridge. Coupled with previous sampling efforts, this collection represents a 32‐year record of discharge from a single chimney structure, the longest record to date. Remarkably, the fluid has remained brine‐dominated for more than three decades. This brine formed during phase separation and segregation prior to initial observations in 1984. Although the chloride concentration of the discharging fluid has decreased with time, the fluid temperature has remained nearly constant for at least 3.3 years and probably for 15 or even 22 years. Compositions of the discharging fluids are consistent with inputs from a deep‐sourced brine, which was last equilibrated at >400°C at a depth consistent with the base of the sheeted dikes and the brittle‐ductile transition. This brine mixed (diffusion or dispersion) with a likely non‐phase‐separated, hydrothermal fluid prior to discharge. A survey of hydrothermal endmember fluids with chlorinities in excess of 700 mmol/kg shows, with the exception of Fe, a single trend between major ion concentrations and chlorinity even though data are from a range of crustal compositions, spreading rates, and water and magma depths. Calculated deep‐sourced brines from hydrothermal fluid data are similar to data based on fluid inclusions and estimates of brine assimilation in magmas. A better understanding of brines is required given their potential duration of discharge and capacity for mobilizing metals.
How microbial metabolism is translated into cellular reproduction under energy-limited settings below the seafloor over long timescales is poorly understood. Here, we show that microbial abundance increases an order of magnitude over a 5 million-year-long sequence in anoxic subseafloor clay of the abyssal North Atlantic Ocean. This increase in biomass correlated with an increased number of transcribed protein-encoding genes that included those involved in cytokinesis, demonstrating that active microbial reproduction outpaces cell death in these ancient sediments. Metagenomes, metatranscriptomes, and 16S rRNA gene sequencing all show that the actively reproducing community was dominated by the candidate phylum “Candidatus Atribacteria,” which exhibited patterns of gene expression consistent with fermentative, and potentially acetogenic, metabolism. “Ca. Atribacteria” dominated throughout the 8 million-year-old cored sequence, despite the detection limit for gene expression being reached in 5 million-year-old sediments. The subseafloor reproducing “Ca. Atribacteria” also expressed genes encoding a bacterial microcompartment that has potential to assist in secondary fermentation by recycling aldehydes and, thereby, harness additional power to reduce ferredoxin and NAD+. Expression of genes encoding the Rnf complex for generation of chemiosmotic ATP synthesis were also detected from the subseafloor “Ca. Atribacteria,” as well as the Wood-Ljungdahl pathway that could potentially have an anabolic or catabolic function. The correlation of this metabolism with cytokinesis gene expression and a net increase in biomass over the million-year-old sampled interval indicates that the “Ca. Atribacteria” can perform the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in millions-of-years-old anoxic sediments.
Microbial life in marine sediment contributes substantially to global biomass and is a crucial component of the Earth system. Subseafloor sediment includes both aerobic and anaerobic microbial ecosystems, which persist on very low fluxes of bioavailable energy over geologic time. However, the taxonomic diversity of the marine sedimentary microbial biome and the spatial distribution of that diversity have been poorly constrained on a global scale. We investigated 299 globally distributed sediment core samples from 40 different sites at depths of 0.1 to 678 m below the seafloor. We obtained ∼47 million 16S ribosomal RNA (rRNA) gene sequences using consistent clean subsampling and experimental procedures, which enabled accurate and unbiased comparison of all samples. Statistical analysis reveals significant correlations between taxonomic composition, sedimentary organic carbon concentration, and presence or absence of dissolved oxygen. Extrapolation with two fitted species–area relationship models indicates taxonomic richness in marine sediment to be 7.85 × 103 to 6.10 × 105 and 3.28 × 104 to 2.46 × 106 amplicon sequence variants for Archaea and Bacteria, respectively. This richness is comparable to the richness in topsoil and the richness in seawater, indicating that Bacteria are more diverse than Archaea in Earth’s global biosphere.
The ribosome’s common core, comprised of ribosomal RNA (rRNA) and universal ribosomal proteins, connects all life back to a common ancestor and serves as a window to relationships among organisms. The rRNA of the common core is similar to rRNA of extant bacteria. In eukaryotes, the rRNA of the common core is decorated by expansion segments (ESs) that vastly increase its size. Supersized ESs have not been observed previously in Archaea, and the origin of eukaryotic ESs remains enigmatic. We discovered that the large ribosomal subunit (LSU) rRNA of two Asgard phyla, Lokiarchaeota and Heimdallarchaeota, considered to be the closest modern archaeal cell lineages to Eukarya, bridge the gap in size between prokaryotic and eukaryotic LSU rRNAs. The elongated LSU rRNAs in Lokiarchaeota and Heimdallarchaeota stem from two supersized ESs, called ES9 and ES39. We applied chemical footprinting experiments to study the structure of Lokiarchaeota ES39. Furthermore, we used covariation and sequence analysis to study the evolution of Asgard ES39s and ES9s. By defining the common eukaryotic ES39 signature fold, we found that Asgard ES39s have more and longer helices than eukaryotic ES39s. Although Asgard ES39s have sequences and structures distinct from eukaryotic ES39s, we found overall conservation of a three-way junction across the Asgard species that matches eukaryotic ES39 topology, a result consistent with the accretion model of ribosomal evolution.
Aquatic subglacial habitats occur throughout the cryosphere where basal melting is sufficient to produce aqueous environments (Priscu & Christner, 2004). Heat energy for melting of basal ice is produced by frictional heating due to glacier movement and geothermal heat flux (Fisher et al., 2015). These heat sources in concert with the lowering of the pressure melting point due to the weight and insulating properties of the overlying ice all contribute to basal ice melting.
Microbes need resources for energy and cellular building material. They also need access to clement conditions with liquid water and a cellular damage rate that is lower than repair. When deprived of resources and clement conditions, microbes often enter some form of dormancy (e.g., by ceasing cell division, slowing metabolic rate, or forming an endospore) until they can grow again (Lennon and Jones, 2011). For example, at night, phototrophs wait for the sun to return. In winter, soil microbes wait for warmer temperatures. Microbes that cause diseases like tuberculosis can stay dormant for years, waiting for the cessation of antibiotic or immune system bombardment (Alnimr, 2015). But what about longer timescales? Unlike multicellular life, microbes survive in an extremely broad range of conditions and can access an amazing variety of resources to maintain cellular functions in the absence of cell division (Finkel and Kolter, 1999). This means that they have the potential to be dormant for much longer than a few months or years. There is no theoretical reason microbes cannot survive on maintenance energy for hundreds or thousands of years, or longer, with little to no cell proliferation (Hoehler and Jørgensen, 2013; Lever et al., 2015). Given this lack of theoretical constraints on the length of microbial dormancy intervals, two questions arise, (i) is there evidence for the existence of organisms experiencing very long dormancies? And (ii) what could be the advantages of such long wait times?
The deep biosphere (subsurface life, including below the seafloor in rocks and sediments) makes up a substantial portion of the planet and harbors vast amounts of microbial life. The Center for Dark Energy Biosphere Investigations (C-DEBI) specializes in the exploration of microbial life, geochemistry, and hydrology in the subsurface (NSF-funded Science Technology Center). Since C-DEBI was established (2010), the number of scientists with a primary focus on deep biosphere research has increased within the last decade as a direct result of efforts from C-DEBI. The objective of this white paper is to present the broad ideas of what the future of deep biosphere research may look like, from the perspective of early career researchers (graduate students, postdoctoral scholars, pre-tenure faculty).
The advanced instrumented GeoMICROBE sleds (Cowen et al., 2012) facilitate the collection of hydrothermal fluids and suspended particles in the subseafloor (basaltic) basement through Circulation Obviation Retrofit Kits (CORKs) installed within boreholes of the Integrated Ocean Drilling Program. The main components of the GeoMICROBE can be converted into a mobile pumping system (MPS) that is installed on the front basket of a submersible or remotely-operated-vehicle (ROV). Here, we provide details of a hydrothermal fluid-trap used on the MPS, through which a gastight sampler can withdraw fluids. We also applied the MPS to demonstrate the value of fixing samples at the seafloor in order to determine redox-sensitive dissolved iron concentrations and speciation measurements. To make the best use of the GeoMICROBE sleds, we describe a miniature and mobile version of the GeoMICROBE sled, which permits rapid turn-over and is relatively easy for preparation and operation. Similar to GeoMICROBE sleds, the Mobile GeoMICROBE (MGM) is capable of collecting fluid samples, filtration of suspended particles, and extraction of organics. We validate this approach by demonstrating the seafloor extraction of hydrophobic organics from a large volume (247L) of hydrothermal fluids.
We explore archaeal distributions in sedimentary subseafloor habitats of Guaymas Basin and the adjacent Sonora Margin, located in the Gulf of California, México. Sampling locations include (1) control sediments without hydrothermal or seep influence, (2) Sonora Margin sediments underlying oxygen minimum zone water, (3) compacted, highly reduced sediments from a pressure ridge with numerous seeps at the base of the Sonora Margin, and (4) sediments impacted by hydrothermal circulation at the off-axis Ringvent site. Generally, archaeal communities largely comprise Bathyarchaeal lineages, members of the Hadesarchaea, MBG-D, TMEG, and ANME-1 groups. Variations in archaeal community composition reflect locally specific environmental challenges. Background sediments are divided into surface and subsurface niches. Overall, the environmental setting and history of a particular site, not isolated biogeochemical properties out of context, control the subseafloor archaeal communities in Guaymas Basin and Sonora Margin sediments.
Distinct lineages of Gammaproteobacteria clade Woeseiales are globally distributed in marine sediments, based on metagenomic and 16S rRNA gene analysis. Yet little is known about why they are dominant or their ecological role in Arctic fjord sediments, where glacial retreat is rapidly imposing change. This study combined 16S rRNA gene analysis, metagenome-assembled genomes (MAGs), and genome-resolved metatranscriptomics uncovered the in situ abundance and transcriptional activity of Woeseiales with burial in four shallow sediment sites of Kongsfjorden and Van Keulenfjorden of Svalbard (79°N). We present five novel Woeseiales MAGs and show transcriptional evidence for metabolic plasticity during burial, including sulfur oxidation with reverse dissimilatory sulfite reductase (dsrAB) down to 4 cm depth and nitrite reduction down to 6 cm depth. A single stress protein, spore protein SP21 (hspA), had a tenfold higher mRNA abundance than any other transcript, and was a hundredfold higher on average than other transcripts. At three out of the four sites, SP21 transcript abundance increased with depth, while total mRNA abundance and richness decreased, indicating a shift in investment from metabolism and other cellular processes to build-up of spore protein SP21. The SP21 gene in MAGs was often flanked by genes involved in membrane-associated stress response. The ability of Woeseiales to shift from sulfur oxidation to nitrite reduction with burial into marine sediments with decreasing access to overlying oxic bottom waters, as well as enter into a dormant state dominated by SP21, may account for its ubiquity and high abundance in marine sediments worldwide, including those of the rapidly shifting Arctic.
Microbial cells buried in subseafloor sediments comprise a substantial portion of Earth’s biosphere and control global biogeochemical cycles; however, the rate at which they use energy (i.e., power) is virtually unknown. Here, we quantify organic matter degradation and calculate the power utilization of microbial cells throughout Earth’s Quaternary-age subseafloor sediments. Aerobic respiration, sulfate reduction, and methanogenesis mediate 6.9, 64.5, and 28.6% of global subseafloor organic matter degradation, respectively. The total power utilization of the subseafloor sediment biosphere is 37.3 gigawatts, less than 0.1% of the power produced in the marine photic zone. Aerobic heterotrophs use the largest share of global power (54.5%) with a median power utilization of 2.23 × 10−18 watts per cell, while sulfate reducers and methanogens use 1.08 × 10−19 and 1.50 × 10−20 watts per cell, respectively. Most subseafloor cells subsist at energy fluxes lower than have previously been shown to support life, calling into question the power limit to life.
Global marine sediments harbor a large and highly diverse microbial biosphere, but the mechanism by which this biosphere is established during sediment burial is largely unknown. During burial in marine sediments, concentrations of easily-metabolized organic compounds and total microbial cell abundance decrease. However, it is unknown whether some microbial clades increase with depth. We show total population increases in 38 microbial families over 3 cm of sediment depth in the upper 7.5 cm of White Oak River (WOR) estuary sediments. Clades that increased with depth were more often associated with one or more of the following: anaerobes, uncultured, or common in deep marine sediments relative to those that decreased. Maximum doubling times (in situ steady state growth rates could be faster to balance cell decay) were estimated as 2-25 years by combining sedimentation rate with either quantitative PCR (qPCR) or the product of the Fraction Read Abundance of 16S rRNA genes and total Cell counts (FRAxC). Doubling times were within an order of magnitude of each other in two adjacent cores, as well as in two laboratory enrichments of Cape Lookout Bight (CLB), NC, sediments (average difference of 28 ± 19%). qPCR and FRAxC in sediment cores and laboratory enrichments produced similar doubling times for key deep subsurface uncultured clades Bathyarchaeota (8.7 ± 1.9 years) and Thermoprofundales/MBG-D (4.1 ± 0.7 years). We conclude that common deep subsurface microbial clades experience a narrow zone of growth in shallow sediments, offering an opportunity for selection of long-term subsistence traits after resuspension events.
Microbial degradation of organic carbon in marine sediments is a key driver of global element cycles on multiple time scales. However, it is not known to what depth microorganisms alter organic carbon in marine sediments or how microbial rates of organic carbon processing change with depth, and thus time since burial, on a global scale. To better understand the connection between the dynamic carbon cycle and life’s limits in the deep subsurface, we have combined a number of global data sets with a reaction transport model (RTM) describing first, organic carbon degradation in marine sediments deposited throughout the Quaternary Period and second, a bioenergetic model for microbial activity. The RTM is applied globally, recognizing three distinct depositional environments – continental shelf, margin and abyssal zones. The results include the masses of particulate organic carbon, POC, stored in three sediment-depth layers: bioturbated Holocene (1.7 × 1017 g C), non-bioturbated Holocene (2.5 × 1018 g C) and Pleistocene (1.4 × 1020 g C) sediments. The global depth-integrated rates of POC degradation have been determined to be 1.3 × 1015, 1.3 × 1014 and 3.0 × 1014 g C yr-1 for the same three layers, respectively. A number of maps depicting the distribution of POC, as well as the fraction that has been degraded have also been generated. Using POC degradation as a proxy for microbial catabolic activity, total heterotrophic processing of POC throughout the Quaternary is estimated to be between 10-11 – 10-6 g C cm-3 yr-1, depending on the time since deposition and location. Bioenergetic modeling reveals that laboratory-determined microbial maintenance powers are poor predictors of sediment biomass concentration, but that cell concentrations in marine sediments can be accurately predicted by combining bioenergetic models with the rates of POC degradation determined in this study. Our model can be used to quantitatively describe both the carbon cycle and microbial activity on a global scale for marine sediments less than 2.59 million years old.
Sparse microbial populations persist from seafloor to basement in the slowly accumulating oxic sediment of the oligotrophic South Pacific Gyre (SPG). The physiological status of these communities, including their substrate metabolism, is previously unconstrained. Here we show that diverse aerobic members of communities in SPG sediments (4.3‒101.5 Ma) are capable of readily incorporating carbon and nitrogen substrates and dividing. Most of the 6986 individual cells analyzed with nanometer-scale secondary ion mass spectrometry (NanoSIMS) actively incorporated isotope-labeled substrates. Many cells responded rapidly to incubation conditions, increasing total numbers by 4 orders of magnitude and taking up labeled carbon and nitrogen within 68 days after incubation. The response was generally faster (on average, 3.09 times) for nitrogen incorporation than for carbon incorporation. In contrast, anaerobic microbes were only minimally revived from this oxic sediment. Our results suggest that microbial communities widely distributed in organic-poor abyssal sediment consist mainly of aerobes that retain their metabolic potential under extremely low-energy conditions for up to 101.5 Ma.
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Gas clathrates are both a resource and a hindrance. They store massive quantities of natural gas but also can clog natural gas pipelines, with disastrous consequences. Eco-friendly technologies for controlling and modulating gas clathrate growth are needed. Type I Antifreeze Proteins (AFPs) from cold-water fish have been shown to bind to gas clathrates via repeating motifs of threonine and alanine. We tested whether proteins encoded in the genomes of bacteria native to natural gas clathrates bind to and alter clathrate morphology. We identified putative clathrate-binding proteins (CBPs) with multiple threonine/alanine motifs in a putative operon (cbp) in metagenomes from natural clathrate deposits. We recombinantly expressed and purified five CbpA proteins, four of which were stable, and experimentally confirmed that CbpAs bound to tetrahydrofuran (THF) clathrate, a low-pressure analogue for structure II gas clathrate. When grown in the presence of CbpAs, the THF clathrate was polycrystalline and platelike instead of forming single, octahedral crystals. Two CbpAs yielded branching clathrate crystals, similar to the effect of Type I AFP, while the other two produced hexagonal crystals parallel to the [1 1 1] plane, suggesting two distinct binding modes. Bacterial CBPs may find future utility in industry, such as maintaining a platelike structure during gas clathrate transportation.
The degassing of primordial gases from Earth’s interior is evidenced by the high 3He/4He ratios in submarine hydrothermal plumes, vent fluids, and rock samples with mantle origin from active hydrothermal systems in mid-ocean ridges (MOR) and subduction zone volcanism. As the largest aquifer on Earth, the uppermost 40-500 m of permeable submarine ridge flank basement (1-65 million-years-old, Myr) holds ∼2% of the ocean volume and accounts for 70% of the seafloor hydrothermal heat flux. However, the degassing of primordial gases through the oceanic ridge flank crust has not yet been directly quantified. Here, we show that high integrity hydrothermal (65 °C) fluids from the sediment-buried 3.5 Myr basaltic crust from the eastern flank of the Juan de Fuca Ridge (JdFR) contain elevated 3He. The 3He/4He for the basaltic fluid is 4.5 ± 0.1 Ra (relative to the air ratio), which is greatly elevated when compared to deep seawater (1.05 Ra), but is half of that observed for high-temperature vent fluids (∼8 Ra) emitting from MOR. Only a small fraction of the 3He in ridge flank fluids is derived from the entrainment of high-temperature ridge-axis fluids and is better explained by degassing of the mantle through the mantle-crust boundary. The lower than MOR 3He/4He ratios indicate that radiogenic 4He originates from aged uranium and thorium decay within the mantle as well as from the ridge-flank basalts. The 3He outgassing through warm ridge flanks (4.9 to 36 mol/yr) accounts for 0.7-6% of the global 3He outgassing, exceeded only by degassing through mid-ocean ridges and subduction volcanism. The presence of mantle 3He suggests that the abiogenic methane present in the ridge flank fluids might be mantle-derived. Based on the 3He outgassing flux, a possibly mantle-derived abiotic methane production rate at the ridge flank is estimated to be 0.3-35 x 108 mol/yr.
Uncultured members of the Chloroflexi phylum are highly enriched in numerous subseafloor environments. Their metabolic potential was evaluated by reconstructing 31 Chloroflexi genomes from six different subseafloor habitats. The near ubiquitous presence of enzymes of the Wood–Ljungdahl pathway, electron bifurcation, and ferredoxin‐dependent transport‐coupled phosphorylation indicated anaerobic acetogenesis was central to their catabolism. Most of the genomes simultaneously contained multiple degradation pathways for complex carbohydrates, detrital protein, aromatic compounds, and hydrogen, indicating the coupling of oxidation of chemically diverse organic substrates to ubiquitous CO2 reduction. Such pathway combinations may confer a fitness advantage in subseafloor environments by enabling these Chloroflexi to act as primary fermenters and acetogens in one microorganism without the need for syntrophic H2 consumption. While evidence for catabolic oxygen respiration was limited to two phylogenetic clusters, the presence of genes encoding putative reductive dehalogenases throughout the phylum expanded the phylogenetic boundary for potential organohalide respiration past the Dehalococcoidia class.
Shallow-sea hydrothermal systems, like their deep-sea and terrestrial counterparts, can serve as relatively accessible portals into the microbial ecology of subsurface environments. In this study, we determined the chemical composition of 47 sediment porewater samples along a transect from a diffuse shallow-sea hydrothermal vent to a non-thermal background area in Paleochori Bay, Milos Island, Greece. These geochemical data were combined with thermodynamic calculations to quantify potential sources of energy that may support in situ chemolithotrophy. The Gibbs energies (ΔGr) of 730 redox reactions involving 23 inorganic H-, O-, C-, N-, S-, Fe-, Mn-, and As-bearing compounds were calculated. Of these reactions, 379 were exergonic at one or more sampling locations. The greatest energy yields were from anaerobic CO oxidation with NO2– (-136 to -162 kJ/mol e–), followed by reactions in which the electron acceptor/donor pairs were O2/CO, NO3–/CO, and NO2–/H2S. When expressed as energy densities (where the concentration of the limiting reactant is taken into account), a different set of redox reactions are the most exergonic: in sediments affected by hydrothermal input, sulfide oxidation with a range of electron acceptors or nitrite reduction with different electron donors provide 85~245 J per kg of sediment, whereas in sediments less affected or unaffected by hydrothermal input, various S0 oxidation reactions and aerobic respiration reactions with several different electron donors are most energy-yielding (80~95 J per kg of sediment). A model that considers seawater mixing with hydrothermal fluids revealed that there is up to ~50 times more energy available for microorganisms that can use S0 or H2S as electron donors and NO2– or O2 as electron acceptors compared to other reactions. In addition to revealing likely metabolic pathways in the near-surface and subsurface mixing zones, thermodynamic calculations like these can help guide novel microbial cultivation efforts to isolate new species.
As the importance of microbiome research continues to become more prevalent and essential to understanding a wide variety of ecosystems (e.g., marine, built, host-associated, etc.), there is a need for researchers to be able to perform highly reproducible and quality analysis of microbial genomes. MetaSanity incorporates analyses from eleven existing and widely used genome evaluation and annotation suites into a single, distributable workflow, thereby decreasing the workload of microbiologists by allowing for a flexible, expansive data analysis pipeline. MetaSanity has been designed to provide separate, reproducible workflows, that (1) can determine the overall quality of a microbial genome, while providing a putative phylogenetic assignment, and (2) can assign structural and functional gene annotations with varying degrees of specificity to suit the needs of the researcher. The software suite combines the results from several tools to provide broad insights into overall metabolic function. Importantly, this software provides built-in optimization for “big data” analysis by storing all relevant outputs in an SQL database, allowing users to query all the results for the elements that will most impact their research.
Subglacial Antarctic aquatic environments are important targets for scientific exploration due to the unique ecosystems they support and their sediments containing palaeoenvironmental records. Directly accessing these environments while preventing forward contamination and demonstrating that it has not been introduced is logistically challenging. The Whillans Ice Stream Subglacial Access Research Drilling (WISSARD) project designed, tested and implemented a microbiologically and chemically clean method of hot-water drilling that was subsequently used to access subglacial aquatic environments. We report microbiological and biogeochemical data collected from the drilling system and underlying water columns during sub-ice explorations beneath the McMurdo and Ross ice shelves and Whillans Ice Stream. Our method reduced microbial concentrations in the drill water to values three orders of magnitude lower than those observed in Whillans Subglacial Lake. Furthermore, the water chemistry and composition of microorganisms in the drill water were distinct from those in the subglacial water cavities. The submicron filtration and ultraviolet irradiation of the water provided drilling conditions that satisfied environmental recommendations made for such activities by national and international committees. Our approach to minimizing forward chemical and microbiological contamination serves as a prototype for future efforts to access subglacial aquatic environments beneath glaciers and ice sheets.
The upper oceanic crust is mainly composed of basaltic lava that constitutes one of the largest habitable zones on Earth. However, the nature of deep microbial life in oceanic crust remains poorly understood, especially where old cold basaltic rock interacts with seawater beneath sediment. Here we show that microbial cells are densely concentrated in Fe-rich smectite on fracture surfaces and veins in 33.5- and 104-million-year-old (Ma) subseafloor basaltic rock. The Fe-rich smectite is locally enriched in organic carbon. Nanoscale solid characterizations reveal the organic carbon to be microbial cells within the Fe-rich smectite, with cell densities locally exceeding 1010 cells/cm3. Dominance of heterotrophic bacteria indicated by analyses of DNA sequences and lipids supports the importance of organic matter as carbon and energy sources in subseafloor basalt. Given the prominence of basaltic lava on Earth and Mars, microbial life could be habitable where subsurface basaltic rocks interact with liquid water.
Microbial genomes have highly variable gene content, and the evolutionary history of microbial populations is shaped by gene gain and loss mediated by horizontal gene transfer and selection. To evaluate the influence of selection on gene content variation in hydrothermal vent microbial populations, we examined 22 metagenome-assembled genomes (MAGs) (70 to 97% complete) from the ubiquitous vent Epsilonbacteraeota genus Sulfurovum that were recovered from two deep-sea hydrothermal vent regions, Axial Seamount in the northeastern Pacific Ocean (13 MAGs) and the Mid-Cayman Rise in the Caribbean Sea (9 MAGs). Genes involved in housekeeping functions were highly conserved across Sulfurovum lineages. However, genes involved in environment-specific functions, and in particular phosphate regulation, were found mostly in Sulfurovum genomes from the Mid-Cayman Rise in the low-phosphate Atlantic Ocean environment, suggesting that nutrient limitation is an important selective pressure for these bacteria. Furthermore, genes that were rare within the pangenome were more likely to undergo positive selection than genes that were highly conserved in the pangenome, and they also appeared to have experienced gene-specific sweeps. Our results suggest that selection is a significant driver of gene gain and loss for dominant microbial lineages in hydrothermal vents and highlight the importance of factors like nutrient limitation in driving microbial adaptation and evolution.
Organic carbon in marine sediments is a critical component of the global carbon cycle, and its degradation influences a wide range of phenomena, including the magnitude of carbon sequestration over geologic timescales, the recycling of inorganic carbon and nutrients, the dissolution and precipitation of carbonates, the production of methane and the nature of the seafloor biosphere. Although much has been learned about the factors that promote and hinder rates of organic carbon degradation in natural systems, the controls on the distribution of organic carbon in modern and ancient sediments are still not fully understood. In this review, we summarize how recent findings are changing entrenched perspectives on organic matter degradation in marine sediments: a shift from a structurally-based chemical reactivity viewpoint towards an emerging acceptance of the role of the ecosystem in organic matter degradation rates. That is, organic carbon has a range of reactivities determined by not only the nature of the organic compounds, but by the biological, geochemical, and physical attributes of its environment. This shift in mindset has gradually come about due to a greater diversity of sample sites, the molecular revolution in biology, discoveries concerning the extent and limits of life, advances in quantitative modeling, investigations of ocean carbon cycling under a variety of extreme paleo-conditions (e.g. greenhouse environments, euxinic/anoxic oceans), the application of novel analytical techniques and interdisciplinary efforts. Adopting this view across scientific disciplines will enable additional progress in understanding how marine sediments influence the global carbon cycle.
Chemotrophic microorganisms gain energy for cellular functions by catalyzing oxidation‐reduction (redox) reactions that are out of equilibrium. Calculations of the Gibbs energy (∆Gr) can identify whether a reaction is thermodynamically favorable and quantify the accompanying energy yield at the temperature, pressure, and chemical composition in the system of interest. Based on carefully calculated values of ∆Gr, we predict a novel microbial metabolism—sulfur comproportionation (3H2S + SO42‐ + 2H+ ⇌ 4S0 + 4H2O). We show that at elevated concentrations of sulfide and sulfate in acidic environments over a broad temperature range, this putative metabolism can be exergonic (∆Gr<0), yielding ~30‐50 kJ/mol. We suggest that this may be sufficient energy to support a chemolithotrophic metabolism currently missing from the literature. Other versions of this metabolism, comproportionation to thiosulfate (H2S + SO42‐ ⇌ S2O32‐ + H2O) and to sulfite (H2S + 3SO42‐ ⇌ 4SO32‐ + 2H+), are only moderately exergonic or endergonic even at ideal geochemical conditions. Natural and impacted environments, including sulfidic karst systems, shallow‐sea hydrothermal vents, sites of acid mine drainage, and acid‐sulfate crater lakes, may be ideal hunting grounds for finding microbial sulfur comproportionators.
Phage–host interactions likely play a major role in the composition and functioning of many microbiomes, yet remain poorly understood. Here, we employed single cell genomics to investigate phage–host interactions in a diffuse-flow, low-temperature hydrothermal vent that may be reflective of a broadly distributed biosphere in the subseafloor. We identified putative prophages in 13 of 126 sequenced single amplified genomes (SAGs), with no evidence for lytic infections, which is in stark contrast to findings in the surface ocean. Most were distantly related to known prophages, while their hosts included bacterial phyla Campylobacterota, Bacteroidetes, Chlorobi, Proteobacteria, Lentisphaerae, Spirochaetes, and Thermotogae. Our results suggest the predominance of lysogeny over lytic interaction in diffuse-flow, deep-sea hydrothermal vents, despite the high activity of the dominant Campylobacteria that would favor lytic infections. We show that some of the identified lysogens have co-evolved with their host over geological time scales and that their genes are transcribed in the environment. Functional annotations of lysogeny-related genes suggest involvement in horizontal gene transfer enabling host’s protection against toxic metals and antibacterial compounds.
Numerous archaeal lineages are known to inhabit marine subsurface sediments, although their distributions, metabolic capacities, and interspecies interactions are still not well understood. Abundant and diverse archaea were recently reported in Costa Rica (CR) margin subseafloor sediments recovered during IODP Expedition 334. Here, we recover metagenome-assembled genomes (MAGs) of archaea from the CR margin and compare them to their relatives from shallower settings. We describe 31 MAGs of six different archaeal lineages (Lokiarchaeota, Thorarchaeota, Heimdallarchaeota, Bathyarcheota, Thermoplasmatales, and Hadesarchaea) and thoroughly analyze representative MAGs from the phyla Lokiarchaeota and Bathyarchaeota. Our analysis suggests the potential capability of Lokiarchaeota members to anaerobically degrade aliphatic and aromatic hydrocarbons. We show it is genetically possible and energetically feasible for Lokiarchaeota to degrade benzoate if they associate with organisms using nitrate, nitrite, and sulfite as electron acceptors, which suggests a possibility of syntrophic relationships between Lokiarchaeota and nitrite and sulfite reducing bacteria. The novel Bathyarchaeota lineage possesses an incomplete methanogenesis pathway lacking the methyl coenzyme M reductase complex and encodes a noncanonical acetogenic pathway potentially coupling methylotrophy to acetogenesis via the methyl branch of Wood–Ljungdahl pathway. These metabolic characteristics suggest the potential of this Bathyarchaeota lineage to be a transition between methanogenic and acetogenic Bathyarchaeota lineages. This work expands our knowledge about the metabolic functional repertoire of marine benthic archaea.
Zetaproteobacteria create extensive iron (Fe) oxide mats at marine hydrothermal vents, making them an ideal model for microbial Fe oxidation at circumneutral pH. Comparison of neutrophilic Fe oxidizer isolate genomes has revealed a hypothetical Fe oxidation pathway, featuring a homolog of the Fe oxidase Cyc2 from Acidithiobacillus ferrooxidans. However, Cyc2 function is not well verified in neutrophilic Fe oxidizers, particularly in Fe-oxidizing environments. Toward this, we analyzed genomes and metatranscriptomes of Zetaproteobacteria, using 53 new high-quality metagenome-assembled genomes reconstructed from Fe mats at Mid-Atlantic Ridge, Mariana Backarc, and Loihi Seamount (Hawaii) hydrothermal vents. Phylogenetic analysis demonstrated conservation of Cyc2 sequences among most neutrophilic Fe oxidizers, suggesting a common function. We confirmed the widespread distribution of cyc2 and other model Fe oxidation pathway genes across all represented Zetaproteobacteria lineages. High expression of these genes was observed in diverse Zetaproteobacteria under multiple environmental conditions and in incubations. The putative Fe oxidase gene cyc2 was highly expressed in situ, often as the top expressed gene. The cyc2 gene showed increased expression in Fe(II)-amended incubations, with corresponding increases in carbon fixation and central metabolism gene expression. These results substantiate the Cyc2-based Fe oxidation pathway in neutrophiles and demonstrate its significance in marine Fe-mineralizing environments.
The crustal sub-seafloor covers a large portion of the Earth’s surface but is poorly understood as a habitat for life. It is unclear what metabolisms support the microscopic cells that have been observed, and how they survive under resource limitation. As the deep crustal subsurface represents a significant portion of the Earth’s surface, microbially mediated reactions may therefore be significant contributors to biogeochemical cycling. In the present study, we used electrochemical techniques to investigate the possibility that crustal subsurface microbial groups can use the solid rock matrix (basalts, etc.) as a source of electrons for redox reactions via extracellular electron transfer (EET). Subsurface crustal fluids and mineral colonization experiments from the cool and oxic basaltic crustal subsurface at the North Pond site on the western flank of the Mid-Atlantic Ridge were used as inocula in cathodic poised potential experiments. Electrodes in oxic microbial fuel cells (MFCs) were poised at −200 mV versus a standard hydrogen electrode to mimic the delivery of electrons in an energy range equivalent to iron oxidation. In this way, microbes that use reduced iron in solid minerals for energy were selected for from the general community onto the electrode surface for interrogation of EET activity, and potential identification by scanning electron microscopy (SEM) and DNA sequencing. The results document that there are cathodic EET-capable microbial groups in the low biomass crustal subsurface at this site. The patterns of current generation in the experiments indicate that these microbial groups are active but likely not growing under the low-resource condition of the experiments, consistent with other studies of activity versus growth in the deep biosphere. Lack of growth stymied attempts to determine the phylogeny of EET-capable microbial groups from this habitat, but the results indicate that these microbial groups are a small part of the overall crustal deep biosphere community. This first demonstration of using electromicrobiology techniques to investigate microbial metabolic potential in the crustal deep biosphere reveals the challenges and opportunities for studying EET in the crustal deep biosphere.
Understanding the emergence of metabolic pathways is key to unraveling the factors that promoted the origin of life. One popular view is that protein cofactors acted as catalysts prior to the evolution of the protein enzymes with which they are now associated. We investigated the stability of acetyl coenzyme A (Acetyl Co-A, the group transfer cofactor in citric acid synthesis in the TCA cycle) under early Earth conditions, as well as whether Acetyl Co-A or its small molecule analogs thioacetate or acetate can catalyze the transfer of an acetyl group onto oxaloacetate in the absence of the citrate synthase enzyme. Several different temperatures, pH ranges, and compositions of aqueous environments were tested to simulate the Earth’s early ocean and its possible components; the effect of these variables on oxaloacetate and cofactor chemistry were assessed under ambient and anoxic conditions. The cofactors tested are chemically stable under early Earth conditions, but none of the three compounds (Acetyl Co-A, thioacetate, or acetate) promoted synthesis of citric acid from oxaloacetate under the conditions tested. Oxaloacetate reacted with itself and/or decomposed to form a sequence of other products under ambient conditions, and under anoxic conditions was more stable; under ambient conditions the specific chemical pathways observed depended on the environmental conditions such as pH and presence/absence of bicarbonate or salt ions in early Earth ocean simulants. This work demonstrates the stability of these metabolic intermediates under anoxic conditions. However, even though free cofactors may be stable in a geological environmental setting, an enzyme or other mechanism to promote reaction specificity would likely be necessary for at least this particular reaction to proceed.
Zetaproteobacteria are obligate chemolithoautotrophs that oxidize Fe(II) as an electron and energy source, and play significant roles in nutrient cycling and primary production in the marine biosphere. Zetaproteobacteria thrive under microoxic conditions near oxic–anoxic interfaces, where they catalyze Fe(II) oxidation faster than the abiotic reaction with oxygen. Neutrophilic Fe(II) oxidizing bacteria produce copious amounts of insoluble iron oxyhydroxides as a by-product of their metabolism. Oxygen consumption by aerobic respiration and formation of iron oxyhydroxides at oxic–anoxic interfaces can result in periods of oxygen limitation for bacterial cells. Under laboratory conditions, all Zetaproteobacteria isolates have been shown to strictly require oxygen as an electron acceptor for growth, and anaerobic metabolism has not been observed. However, genomic analyses indicate a range of potential anaerobic pathways present in Zetaproteobacteria. Heterologous expression of proteins from Mariprofundus ferrooxydans PV-1, including pyruvate formate lyase and acetate kinase, further support a capacity for anaerobic metabolism. Here we define auxiliary anaerobic metabolism as a mechanism to provide maintenance energy to cells and suggest that it provides a survival advantage to Zetaproteobacteria in environments with fluctuating oxygen availability.
The isotopic composition of lipid biomarkers and biomass in sedimentary environments are widely used to infer microbial metabolisms and constrain carbon cycling processes. It has been observed that metabolic energy availability in the form of H2 impacts the stable carbon isotopes of CH4 during hydrogenotrophic methanogenesis, but it is unknown whether this relationship extends to lipids and amino acids. Since lipids and amino acids are long-lived, they can be used to reconstruct past conditions over geologic timescales. Therefore, the controls on their isotopic signatures are important to constrain to better interpret past environments.
In this study, the isotopic distributions of carbon metabolized and reduced by the hyperthermophile Methanocaldococcus jannaschii were quantified following growth at 82 °C in a chemostat with high (80–83 µM) and low (15–27 µM) H2 concentrations. As has been shown previously, the stable carbon isotope fractionation factors for CH4 were >15‰ larger in low H2 experiments than in high H2 experiments. Lipid biomarkers and amino acids were similarly impacted with approximately 10‰ larger fractionation factors under low H2 conditions. The increase in fractionation factors can be related to the lower availability of thermodynamic energy, suggesting that even larger fractionation factors would be observed in methanogens living close to their threshold energy needs, as they do in most environments. The resulting isotopic signatures of long-lived lipid biomarkers synthesized by hydrogenotrophic methanogens may become as ‘superlight’ as those synthesized by archaea carrying out the anaerobic oxidation of methane. These results help to describe the underlying mechanisms that determine the isotopic composition of long-lived biomarkers and provide constraints for interpreting these signatures in the environment.
Diazotrophic microorganisms regulate marine productivity by alleviating nitrogen limitation. However, we know little about the identity and activity of diazotrophs in deep-sea sediments, a habitat covering nearly two-thirds of the planet. Here, we identify candidate diazotrophs from Pacific Ocean sediments collected at 2893 m water depth using 15N-DNA stable isotope probing and a novel pipeline for nifH sequence analysis. Together, these approaches detect an unexpectedly diverse assemblage of active diazotrophs, including members of the Acidobacteria, Firmicutes, Nitrospirae, Gammaproteobacteria, and Deltaproteobacteria. Deltaproteobacteria, predominately members of the Desulfobacterales and Desulfuromonadales, are the most abundant diazotrophs detected, and display the most microdiversity of associated nifH sequences. Some of the detected lineages, including those within the Acidobacteria, have not previously been shown to fix nitrogen. The diazotrophs appear catabolically diverse, with the potential for using oxygen, nitrogen, iron, sulfur, and carbon as terminal electron acceptors. Therefore, benthic diazotrophy may persist throughout a range of geochemical conditions and provide a stable source of fixed nitrogen over geologic timescales. Our results suggest that nitrogen-fixing communities in deep-sea sediments are phylogenetically and catabolically diverse, and open a new line of inquiry into the ecology and biogeochemical impacts of deep-sea microorganisms.
The permeability, connectivity, and reactivity of fluid reservoirs in oceanic crust are poorly constrained, yet these reservoirs are pathways for about a quarter of the Earth’s heat loss and seawater‐rock exchange within them impact ocean chemical cycles. We present results from the second‐ever cross‐hole tracer experiment within oceanic crust and the first conducted during a single expedition and in slow‐spreading crust west of the Mid‐Atlantic Ridge at North Pond. Here we employed boreholes that were drilled by the Integrated Ocean Drilling Program (IODP Sites U1382 and U1383) that were instrumented and sealed. A cesium‐salt solution and bottom seawater tracer experiment provided a measure of the minimum Darcy fluid velocity (2 to 41 m d‐1) within the upper volcanic crust, constraining the minimum permeability of 10‐11 to 10‐9 m2. We also document chemical heterogeneities in crustal fluid compositions, rebound from drilling disturbances, and nitrification within the basaltic crust, based on systematic differences in borehole fluid compositions over a 5‐year period. These results also show heterogeneous fluid compositions with depth in the borehole, indicating that hydrothermal circulation is not vigorous enough to homogenize the fluid composition in the upper permeable basaltic basement, at least not on the time scale of 5 years. Our work verifies the potential for future manipulative experiments to characterize hydrologic, biogeochemical, and microbial process within the upper basaltic crust.
Genus assignment is fundamental in the characterization of microbes, yet there is currently no unambiguous way to demarcate genera solely using standard genomic relatedness indices. Here, we propose an approach to demarcate genera that relies on the combined use of the average nucleotide identity, genome alignment fraction, and the distinction between type- and non-type species. More than 3,500 genomes representing type strains of species from >850 genera of either bacterial or archaeal lineages were tested. Over 140 genera were analyzed in detail within the taxonomic context of order/family. Significant genomic differences between members of a genus and type species of other genera in the same order/family were conserved in 94% of the cases. Nearly 90% (92% if polyphyletic genera are excluded) of the type strains were classified in agreement with current taxonomy. The 448 type strains that need reclassification directly impact 33% of the genera analyzed in detail. The results provide a first line of evidence that the combination of genomic indices provides added resolution to effectively demarcate genera within the taxonomic framework that is currently based on the 16S rRNA gene. We also identify the emergence of natural breakpoints at the genome level that can further help in the circumscription of taxa, increasing the proportion of directly impacted genera to at least 43% and pointing at inaccuracies on the use of the 16S rRNA gene as a taxonomic marker, despite its precision. Altogether, these results suggest that genomic coherence is an emergent property of genera in Bacteria and Archaea.
Interest in extracting mineral resources from the seafloor through deep‐sea mining has accelerated in the past decade, driven by consumer demand for various metals like zinc, cobalt, and rare earth elements. While there are ongoing studies evaluating potential environmental impacts of deep‐sea mining activities, these focus primarily on impacts to animal biodiversity. The microscopic spectrum of seafloor life and the services that this life provides in the deep sea are rarely considered explicitly. In April 2018, scientists met to define the microbial ecosystem services that should be considered when assessing potential impacts of deep‐sea mining, and to provide recommendations for how to evaluate and safeguard these services. Here, we indicate that the potential impacts of mining on microbial ecosystem services in the deep sea vary substantially, from minimal expected impact to loss of services that cannot be remedied by protected area offsets. For example, we (1) describe potential major losses of microbial ecosystem services at active hydrothermal vent habitats impacted by mining, (2) speculate that there could be major ecosystem service degradation at inactive massive sulfide deposits without extensive mitigation efforts, (3) suggest minor impacts to carbon sequestration within manganese nodule fields coupled with potentially important impacts to primary production capacity, and (4) surmise that assessment of impacts to microbial ecosystem services at seamounts with ferromanganese crusts is too poorly understood to be definitive. We conclude by recommending that baseline assessments of microbial diversity, biomass, and, importantly, biogeochemical function need to be considered in environmental impact assessments of deep‐sea mining.
International Ocean Discovery Program (IODP) Expedition 385T aimed to take advantage of a transit of the R/V JOIDES Resolution from Antofagasta, Chile, to San Diego, California (USA), to accomplish new sampling and data collection from legacy borehole observatories in Deep Sea Drilling Project (DSDP) and Ocean Drilling Program (ODP) Holes 504B and 896A on the southern flank of the Costa Rica Rift. In addition, the US Science Support Program organized the participation of 3 Outreach Officers to evaluate the performance of the JOIDES Resolution Outreach Officer program as well as 2 educators and 12 undergraduate students for a shipboard “JR Academy.” Our scientific objectives were to collect (1) new Formation MicroScanner logs from Hole 504B for improving lithologic interpretations of crustal architecture at this archetype deep oceanic crust hole and (2) fluid samples from both holes for evaluating the crustal deep biosphere in deep and warm oceanic crust. These operations in Holes 504B and 896A have direct relevance to Challenges 5, 6, 9, 10, 13, and 14 of the IODP 2013–2023 Science Plan. Accomplishing both of these scientific objectives required the removal of old wireline CORK observatories, including associated inflatable packers that were installed in the cased boreholes in 2001. The fluid sampling plan also included testing a new Multi-Temperature Fluid Sampler. Despite successfully removing the CORK wellhead platforms from both holes, we were unable to remove the packers stuck in casing at both locations after 48 h of milling operations in Hole 504B and 2 h of milling operations in Hole 896A, thus precluding accomplishing any of the scientific objectives of the expedition. We provide an assessment of the final state of the holes and recommendations for possible future operations.
International Ocean Discovery Program (IODP) Expedition 385T will revisit two Deep Sea Drilling Project (DSDP) and Ocean Drilling Program (ODP) legacy sites—Holes 504B and 896A on the Costa Rica Rift flank—to advance lithostratigraphic, hydrogeological, and deep biosphere studies of upper oceanic crust. Hole 504B has served as a standard reference site for upper oceanic crust for decades despite low core recovery during drilling operations. Hole 896A serves as an analog site of crustal alteration for examining biogeography in the crustal deep biosphere. During Expedition 385T, we will advance lithostratigraphic records of in situ crustal architecture through Formation MicroScanner (FMS) logging, with priority for these operations in Hole 504B. The new logs from Hole 504B will reveal whether unrecovered intervals are highly fractured and/or brecciated and whether alteration style and intensity are correlated to volcanic architecture, which will allow for assessment of the hypothesis that hydrothermal alteration and mineralization style are spreading-rate dependent. We will also advance crustal hydrogeological and deep biosphere research through temperature logging and water sampling in both holes, with priority for these operations in Hole 896A. The new FMS-based lithostratigraphy coupled with new fluid assessment will also allow for improvements on the thermal limits of microbial life and seawater-basalt reactions. These operations in Holes 504B and 896A have direct relevance to Challenges 5, 6, 9, 10, 13, and 14 of the IODP 2013–2023 Science Plan. To achieve these data and sample recoveries from these legacy sites, existing wireline observatories installed in both holes will be removed and the remaining cased holes will be left open for possible future installation of next-generation observatories. The expedition will be implemented as an abbreviated (10 operational days) expedition with no new coring.
The Methanosarcinales, a lineage of cytochrome-containing methanogens, have recently been proposed to participate in direct extracellular electron transfer interactions within syntrophic communities. To shed light on this phenomenon, we applied electrochemical techniques to measure electron uptake from cathodes by Methanosarcina barkeri, which is an important model organism that is genetically tractable and utilizes a wide range of substrates for methanogenesis. Here, we confirm the ability of M. barkeri to perform electron uptake from cathodes and show that this cathodic current is linked to quantitative increases in methane production. The underlying mechanisms we identified include, but are not limited to, a recently proposed association between cathodes and methanogen-derived extracellular enzymes (e.g., hydrogenases) that can facilitate current generation through the formation of reduced and diffusible methanogenic substrates (e.g., hydrogen). However, after minimizing the contributions of such extracellular enzymes and using a mutant lacking hydrogenases, we observe a lower-potential hydrogen-independent pathway that facilitates cathodic activity coupled to methane production in M. barkeri. Our electrochemical measurements of wild-type and mutant strains point to a novel and hydrogenase-free mode of electron uptake with a potential near −484 mV versus standard hydrogen electrode (SHE) (over 100 mV more reduced than the observed hydrogenase midpoint potential under these conditions). These results suggest that M. barkeri can perform multiple modes (hydrogenase-mediated and free extracellular enzyme-independent modes) of electrode interactions on cathodes, including a mechanism pointing to a direct interaction, which has significant applied and ecological implications.
This chapter presents an overview of the motivation for the volume, emphasizing why studying deep carbon is relevant to the goal of understanding carbon quantities, movements, forms, and origins. It contains brief highlights of the new instruments, cross-cutting research initiatives, and deep carbon science over the past decade (spurred in large part by the international Deep Carbon Observatory) that have expanded our understanding of carbon on Earth.
Work aboard E/V Nautilus and at the Inner Space Center in 2018 may assist in the search for extraterrestrial life, as exploration of iron-rich hydrothermal vent systems on Lō‘ihi Seamount (Figure 1) informs the design of future science-focused missions across our solar system. From August 21 to September 12, 2018, this research program was conducted by the SUBSEA (Systematic Underwater Biogeochemical Science and Exploration Analog) team, which is supported by NASA’s Science Mission Directorate and NOAA’s Office of Ocean Exploration and Research. Our mission comprises three research elements: science, operations, and technology. Both natural and social sciences anchor the SUBSEA program, providing the basis for the operations and technology domains to design and implement their studies and supporting capabilities.
Methanogenic archaea can be integrated into a sustainable, carbon-neutral cycle for producing organic chemicals from C1 compounds if the rate, yield, and titer of product synthesis can be improved using metabolic engineering. However, metabolic engineering techniques are limited in methanogens by insufficient methods for controlling cellular protein levels. We conducted a systematic approach to tune protein levels in Methanosarcina acetivorans C2A, a model methanogen, by regulating transcription and translation initiation. Rationally designed core promoter and ribosome binding site mutations in M. acetivorans C2A resulted in a predicable change in protein levels over a 60 fold range. The overall range of protein levels was increased an additional 3 fold by introducing the 5′ untranslated region of the mcrB transcript. This work demonstrates a wide range of precisely controlled protein levels in M. acetivorans C2A, which will help facilitate systematic metabolic engineering efforts in methanogens.
The oceanic magnesium budget is important to our understanding of Earth’s carbon cycle, because similar processes control both (e.g., weathering, volcanism, and carbonate precipitation). However, dolomite sedimentation and low-temperature hydrothermal circulation remain enigmatic oceanic Mg sinks. In recent years, magnesium isotopes (δ26Mg) have provided new constraints on the Mg cycle, but the lack of data for the low-temperature hydrothermal isotope fractionation has hindered this approach. Here we present new δ26Mg data for low-temperature hydrothermal fluids, demonstrating preferential 26Mg incorporation into the oceanic crust, on average by εsolid-fluid ≈ 1.6‰. These new data, along with the constant seawater δ26Mg over the past ~20 Myr, require a significant dolomitic sink (estimated to be 1.5–2.9 Tmol yr−1; 40–60% of the oceanic Mg outputs). This estimate argues strongly against the conventional view that dolomite formation has been negligible in the Neogene and points to the existence of significant hidden dolomite formation.
Stable isotope compositions of methane (δ13C and δD) and of short-chain alkanes are commonly used to trace the origin and fate of carbon in the continental crust. In continental sedimentary systems, methane is typically produced through thermogenic cracking of organic matter and/or through microbial methanogenesis. However, secondary processes such as mixing, migration or biodegradation can alter the original isotopic and composition of the gas, making the identification and the quantification of primary sources challenging. The recently resolved methane ‘clumped’ isotopologues Δ13CH3D and Δ12CH2D2 are unique indicators of whether methane is at thermodynamic isotopic equilibrium or not, thereby providing insights into formation temperatures and/or into kinetic processes controlling methane generation processes, including microbial methanogenesis.
In this study, we report the first systematic use of methane Δ13CH3D and Δ12CH2D2 in the context of continental sedimentary basins. We investigated sedimentary formations from the Southwest Ontario and Michigan Basins, where the presence of both microbial and thermogenic methane was previously proposed. Methane from the Silurian strata coexist with highly saline brines, and clumped isotopologues exhibit large offsets from thermodynamic equilibrium, with Δ12CH2D2 values as low as −23‰. Together with conventional δ13C and δD values, the variability in Δ13CH3D and Δ12CH2D2 to first order reflects a mixing relationship between near-equilibrated thermogenic methane similar to gases from deeper Cambrian and Middle Ordovician units, and a source characterized by a substantial departure from equilibrium that could be associated with microbial methanogenesis. In contrast, methane from the Devonian-age Antrim Shale, associated with less saline porewaters, reveals Δ13CH3D and Δ12CH2D2 values that are approaching low temperature thermodynamic equilibrium. While microbial methanogenesis remains an important contributor to the methane budget in the Antrim Shale, it is suggested that Anaerobic Oxidation of Methane (AOM) could contribute to reprocessing methane isotopologues, yielding Δ13CH3D and Δ12CH2D2 signatures approaching thermodynamic equilibrium.
A multinational research team drilled into the seafloor to see whether chemical processes in exposed shallow mantle rocks could generate nutrients to support life in the subsurface.
Earth’s oceans are immense, covering much of our planet’s surface and filled with microbial communities that make up ∼70% of all marine biomass. These microbes thrive from the sunlit coral reefs to the frigid Arctic ice to hot hydrothermal vents, yet sparingly few are growing in our laboratories. Extreme gradients in temperature, pH, nutrient availability, and pressure present innumerable challenges to growing and studying these microbes on land. However, innovations in creative culturing are opening up the high seas to microbiologists, revealing new metabolisms, novel branches on the tree of life, and clever strategies for surviving at the extremes. Consider SAR11, the most abundant bacteria in the plankton, finally coaxed into lab growth using specific methods developed for slow-growing, oligotrophs with unusual nutrient requirements or the anaerobic methane oxidizing archaea, maintained and studied in the lab via mixed enrichments with syntrophic partners. Still others may never grow on terra firma, including the deep sediment microbe with estimates for cell division over millennia or the tubeworm endosymbiont that operates as efficiently as a laboratory chemostat, but only with its host. For these especially wild types, microbiologists will continue to study them in their salty homes, using new instruments and analytical tools to measure their activity and decipher their genomes. Whether we study them growing solo in our labs or in their watery milieu, the coming years are certain to expand our understanding of how these amazing organisms thrive in our vast ocean world.
Last year we launched our first “Early-Career Special Issue” to highlight and share the work of some of the pioneers in systems microbiology science who are just stepping into their first independent research positions. The special issue—published in March/April 2018 (https://msystems.asm.org/content/3/2)—was, we believe, a great success. First, it provided a platform for these early-career researchers to highlight their work and their vision, in a format frequently reserved for people further along in their careers. Having such a large collection all in a single place allows one to get an excellent perspective on the future of microbial systems biology. Second, we believe the collection also serves as an important networking and communication resource. For example, conference organizers, companies, department chairs, and others looking for early-career scholars working across the breadth of microbial systems biology can use these articles. In addition, the collection provides an opportunity to see the bigger picture of the vision driving the work of key people in the field, which in turn could be a key resource for those looking for new collaborators, or possible mentors for graduate school or postdoctoral training.
Energy is continuously transformed in the environment through the metabolic activities of organisms. Catabolic reactions generate energy (energy-yielding) which are used to fuel anabolic reactions for maintenance and growth (energy-requiring). These transformations of energy (i.e., bioenergetics) underpin most biogeochemical cycles on Earth and allow the delivery of a wide range of life-supporting ecosystem services. It has long been understood that the amount and types of energy available in an environment influence the rates of biological activity and the complexity of interactions in that system. Traditionally, energy fluxes and stocks have not been described in a quantitative manner, and it is not well-understood how physicochemical theorems such as thermodynamic principles are manifested in environmental systems. Theoretical ecological frameworks (Odum, 1969; Addiscott, 1995) have suggested that the more complex ecosystems become in terms of their food webs, the more efficient they are, i.e., relatively less energy is wasted when utilizing resources. However, this has not been rigorously tested experimentally, but in recent years, scientists in a number of fields have increasingly shown interest in quantifying how bioenergetics constrain and define ecosystem functioning. For example, organic matter in soils has distinct energetic signatures, e.g., energy densities and activation energies (Barré et al., 2016; Williams et al., 2018), and microbial bioenergetics provides empirical data for mechanistic models of carbon turnover in soils, work that is relevant to climate change (Sparling, 1983; Herrmann et al., 2014; Barros et al., 2016; Bölscher et al., 2017). Furthermore, geochemists have quantified the amount of chemolithotrophic energy available for microorganisms in a number of extreme environments to infer the dominant metabolic activities (e.g., McCollom and Shock, 1997; Shock et al., 2010; Osburn et al., 2014). These activities are challenging to monitor due to their inaccessibility and incredibly slow rates of energy processing. Although all of these efforts represent significant progress in the field of biogeochemistry, bioenergetics analysis of natural systems is still in its infancy. Nonetheless, there is increasing interest in using bioenergetics tools to better characterize biogeochemical cycling in water, soils, and sediments in terrestrial, freshwater, and marine ecosystems.
Marine microorganisms play a fundamental role in the global carbon cycle by mediating the sequestration of organic matter in ocean waters and sediments. A better understanding of how biological factors, such as microbial community composition, influence the lability and fate of organic matter is needed. Here, we explored the extent to which organic matter remineralization is influenced by species‐specific metabolic capabilities. We carried out aerobic time‐series incubations of Guaymas Basin sediments to quantify the dynamics of carbon utilization by two different heterotrophic marine isolates (Vibrio splendidus 1A01; Pseudoalteromonas sp. 3D05). Continuous measurement of respiratory CO2 production and its carbon isotopic compositions (13C and 14C) shows species‐specific differences in the rate, quantity, and type of organic matter remineralized. Each species was incubated with hydrothermally‐influenced vs. unimpacted sediments, resulting in a ~2‐fold difference in respiratory CO2 yield across the experiments. Genomic analysis indicated that the observed carbon utilization patterns may be attributed in part to the number of gene copies encoding for extracellular hydrolytic enzymes. Our results demonstrate that the lability and remineralization of organic matter in marine environments is not only a function of chemical composition and/or environmental conditions, but also a function of the microorganisms that are present and active.
Natural fluids with a pH (25°C) up to 12.3 were collected from a sub-seafloor borehole observatory (Ocean Drilling Program (ODP) Hole 1200C) on South Chamorro Seamount, a serpentinite mud volcano in the Mariana forearc. We used systematic differences in the chemical compositions of pore waters from drilling operations during ODP Leg 195 and borehole fluids collected subsequently from Hole 1200C to define two endmember solutions, one of which was a sulfate-rich fluid with a methane concentration of 50 mM that ascends from the subduction channel and the other was a low-sulfate fluid. The sequence of sample collection and fluid compositions constrain subsurface hydrologic conditions. Deep-sourced, sulfate- and methane-rich, sterile fluids from the subduction channel can reach the seafloor unchanged within the central conduit, whereas other fluid pathways likely intersect the pelagic sediment that underlies the serpentinite mud volcano, providing potentially suitable conditions and inoculum for microbial anaerobic oxidation of methane (AOM). These AOM-affected, low-sulfate fluids also make it to the seafloor where they discharge. The source of the sulfate- and methane-rich fluid in the subduction channel is attributed to abiotic methane production fueled by hydrogen production from serpentinization and carbonate dissolution. This methane production includes a mechanism to raise the pH above values from serpentinization alone. Results from South Chamorro Seamount represent an end member along a transect defined by the distance from the trench. Results from this site are applied to other serpentinite mud volcanoes along this transect to speculate on likely chemical conditions within shallower and cooler portions of the subduction channel.
Microorganisms possess a variety of survival mechanisms, including the production of antimicrobials that function to kill and/or inhibit the growth of competing microorganisms. Studies of antimicrobial production have largely been driven by the medical community in response to the rise in antibiotic-resistant microorganisms and have involved isolated pure cultures under artificial laboratory conditions neglecting the important ecological roles of these compounds. The search for new natural products has extended to biofilms, soil, oceans, coral reefs, and shallow coastal sediments; however, the marine deep subsurface biosphere may be an untapped repository for novel antimicrobial discovery. Uniquely, prokaryotic survival in energy-limited extreme environments force microbial populations to either adapt their metabolism to outcompete or produce novel antimicrobials that inhibit competition. For example, subsurface sediments could yield novel antimicrobial genes, while at the same time answering important ecological questions about the microbial community.
The structure and function of microbial communities inhabiting the subseafloor near hydrothermal systems are influenced by fluid geochemistry, geologic setting, and fluid flux between vent sites, as well as biological interactions. Here we used genome‐resolved metagenomics and metatranscriptomics to examine patterns of gene abundance and expression and assess potential niche differentiation in microbial communities in venting fluids from hydrothermal vent sites at the Mid‐Cayman Rise. We observed similar patterns in gene and transcript abundance between two geochemically distinct vent fields at the community level, but found that each vent site harbors a distinct microbial community with differing transcript abundances for individual microbial populations. Through an analysis of metabolic pathways in 64 metagenome‐assembled genomes (MAGs), we show that MAG transcript abundance can be tied to differences in metabolic pathways and to potential metabolic interactions between microbial populations, allowing for niche‐partitioning and divergence in both population distribution and activity. Our results illustrate that most microbial populations have a restricted distribution within the seafloor, and that the activity of those microbial populations is tied to both genome content and abiotic factors.
Hydrothermal circulation of low temperature fluids within oceanic crust affects global biogeochemical cycles. We present data from a warm temperature (64°C) hydrothermal system on the eastern flank of the Juan de Fuca Ridge to assess whether chemical fluxes to the ocean from these systems arise from the basaltic crust or the overlying sediment pore waters. Extensive sampling of this system included fluid chemical data from deep sea drilling, gravity coring, and submersible operations from five sites on a buried ridge that is parallel to the spreading center to the west. These data were subjected to a transport (advection‐diffusion) model to constrain chemical and fluid fluxes along this five‐site transect. Solutes (K, Cl, sulfate, Ba, Sr, Cs, Mo, and Y) that are non reactive within the basaltic crust constrain the volumetric fluid flux per unit width within the basaltic crust from 0.05 to 0.2 m3 y‐1 cm‐1, consistent with a recent tracer study. Using this average fluid flux, reactive fluxes were determined for twenty‐four solutes and partitioned among seawater, sediment and basaltic sources and sinks. Only Ca, Ce, and Gd were released from basaltic basement to the ocean, whereas other solutes (sulfate, Mg, K, Li, Rb, Cd, U, Y, Yb, Gd, and La) were consumed in the basaltic crust, and still others (Cl, Ba, Sr, Cs, Mo, Mn, Fe, Co, NH3, and Zn) had a sediment origin with a net flux to the ocean. Diffusive exchange with the overlying sediment had a greater impact than seawater‐basalt reactions for some solutes.
Building on the synthesis of carbon reservoirs in Earth’s subsurface, this chapter focuses on the forms, cycling, and fate of the carbon supporting microbial life in the terrestrial and marine subsurface. As the subsurface is estimated to host a vast reservoir of life on Earth, identifying the carbon compounds that life uses for energy and growth is key to understanding ecosystem functioning in the past and at present, and also for extrapolating these findings to the search for life in the universe. This chapter highlights advances in quantifying small carbon compounds, measuring rates of carbon turnover, and the fate of carbon in the deep biosphere.
