Anoxic subsurface sediments contain communities of heterotrophic microorganisms that metabolize organic carbon at extraordinarily slow rates. In order to assess the mechanisms by which subsurface microorganisms access detrital sedimentary organic matter, we measured kinetics of a range of extracellular peptidases in anoxic sediments of the White Oak River estuary, NC. Nine distinct peptidase substrates were enzymatically hydrolyzed at all depths. Potential peptidase activities (Vmax) decreased with increasing sediment depth, although Vmaxexpressed on a per cell basis was approximately the same at all depths. Half-saturation constants (Km) decreased with depth, indicating peptidases that functioned more efficiently at low substrate concentrations. Potential activities of extracellular peptidases acting on molecules that are enriched in degraded organic matter (D-phenylalanine and L-ornithine) increased relative to enzymes that act on L-phenylalanine, further suggesting microbial community adaptation to access degraded organic matter. Nineteen classes of predicted, exported peptidases were identified in genomic data from the same site, of which genes for class C25 (gingipain-like) peptidases represented more than 40% at each depth. Methionine aminopeptidases, zinc carboxypeptidases, and class S24-like peptidases, which are involved in single-stranded DNA repair, were also abundant. These results suggest a subsurface heterotrophic microbial community that primarily accesses low-quality detrital organic matter via a diverse suite of well-adapted extracellular enzymes.
Energy-starved microbes in deep marine sediments subsist at near-zero growth for thousands of years, yet the mechanisms for their subsistence are unknown because no model strains have been cultivated from most of these groups. We investigated Baltic Sea sediments with single-cell genomics, metabolomics, metatranscriptomics, and enzyme assays to identify possible subsistence mechanisms employed by uncultured Atribacteria, Aminicenantes, Actinobacteria group OPB41, Aerophobetes, Chloroflexi, Deltaproteobacteria, Desulfatiglans, Bathyarchaeota, and Euryarchaeota marine group II lineages. Some functions appeared to be shared by multiple lineages, such as trehalose production and NAD+-consuming deacetylation, both of which have been shown to increase cellular life spans in other organisms by stabilizing proteins and nucleic acids, respectively. Other possible subsistence mechanisms differed between lineages, possibly providing them different physiological niches. Enzyme assays and transcripts suggested that Atribacteria and Actinobacteria group OPB41 catabolized sugars, whereas Aminicenantes and Atribacteria catabolized peptides. Metabolite and transcript data suggested that Atribacteria utilized allantoin, possibly as an energetic substrate or chemical protectant, and also possessed energy-efficient sodium pumps. Atribacteria single-cell amplified genomes (SAGs) recruited transcripts for full pathways for the production of all 20 canonical amino acids, and the gene for amino acid exporter YddG was one of their most highly transcribed genes, suggesting that they may benefit from metabolic interdependence with other cells. Subsistence of uncultured phyla in deep subsurface sediments may occur through shared strategies of using chemical protectants for biomolecular stabilization, but also by differentiating into physiological niches and metabolic interdependencies.
Globally, marine sediments are a vast repository of organic matter which is degraded through various microbial pathways, including polymer hydrolysis and monomer fermentation. The sources, abundances, and quality (i.e. labile or recalcitrant) of the organic matter and the composition of the microbial assemblages vary between sediments. Here, we examine new and previously published sediment metagenomes from the Baltic Sea and the nearby Kattegat to determine connections between geochemistry and the community potential to degrade organic carbon. Diverse organic matter hydrolysis encoding genes were present in sediments between 0.25 to 67 meters below seafloor, and were in higher relative abundances in those sediments that contained more organic matter. New analysis of previously published metatranscriptomes demonstrated that many of these genes were transcribed in two organic-rich Holocene sediments. Some of the variation in deduced pathways in the metagenomes correlated to carbon content and depositional conditions. Fermentation-related genes were found in all samples, and encoded for multiple fermentation strategies. Notably, genes conferring alcohol metabolism were amongst the most abundant of these genes, indicating this is an important but underappreciated aspect of sediment carbon cycling. This study is a step towards a more complete understanding of microbial food webs and the impacts of depositional facies on present sedimentary microbial communities.
Difficulty quantifying biogeochemically significant microbes in marine sediments limits our ability to assess interspecific interactions, population turnover times, and niches of uncultured taxa. We incubated surface sediments from Cape Lookout Bight, North Carolina USA, anoxically at 21°C for 122 days. Sulfate decreased until day 68, after which methane increased, with hydrogen concentration consistent with predicted values of an electron donor exerting thermodynamic control. We measured turnover times using two relative quantification methods, quantitative PCR (qPCR) and the product of 16S gene read abundance and total cell abundance (FRAxC, for fraction of read abundance times cells), to estimate population turnover rates of uncultured clades. Most 16S rRNA reads were from deeply-branching uncultured groups and ∼ 98% of 16S rRNA genes did not abruptly shift in relative abundance when sulfate reduction gave way to methanogenesis. Uncultured Methanomicrobiales and Methanosarcinales increased at the onset of methanogenesis with population turnover times estimated from quantitative PCR (qPCR) at 9.7 ± 3.9 and 12.6 ± 4.1 days, respectively. These were consistent with FRAxC turnover times of 9.4 ± 5.8 and 9.2 ± 3.5 days, respectively. Uncultured Syntrophaceae, which are possibly fermentative syntrophs of methanogens, and uncultured Kazan-3A-21 archaea also increased at the onset of methanogenesis with FRAxC turnover times of 14.7 ± 6.9 and 10.6 ± 3.6 days. Kazan-3A-21 may therefore either perform methanogenesis or form a fermentative syntrophy with methanogens. Three genera of sulfate reducing bacteria, Desulfovibrio sp., Desulfobacter sp., and Desulfobacterium sp. increased in the first 19 days before declining rapidly during sulfate reduction. We conclude that population turnover times on the order of days can be measured robustly in organic-rich marine sediment, and the transition from sulfate-reducing to methanogenic conditions only stimulates growth in a few clades directly involved in methanogenesis, rather than the whole microbial community.
Microbial life in the deep subsurface biosphere is taxonomically and metabolically diverse, but it is vigorously debated whether the resident organisms are thriving (metabolizing, maintaining cellular integrity, and expressing division genes) or just surviving. As part of Integrated Ocean Drilling Program (IODP) Expedition 347: Baltic Sea Paleoenvironment, we extracted and sequenced RNA from organic carbon-rich, nutrient-replete, and permanently anoxic sediment. In stark contrast to the oligotrophic subsurface biosphere, Baltic Sea Basin samples provided a unique opportunity to understand the balance between metabolism and other cellular processes. Targeted sequencing of 16S rRNA transcripts showed Atribacteria (an uncultured phylum) and Chloroflexi to be among the dominant and the active members of the community. Metatranscriptomic analysis identified methane cycling, sulfur cycling, and halogenated compound utilization as active in situ respiratory metabolisms. Genes for cellular maintenance, cellular division, motility, and antimicrobial production were also transcribed. This indicates that microbial life in deep subsurface Baltic Sea Basin sediments was not only alive, but thriving.
| URL | https://www.bco-dmo.org/dataset/649751 |
|---|---|
| Download URL | https://www.bco-dmo.org/dataset/649751/data/download |
| Media Type | text/tab-separated-values |
| Created | June 20, 2016 |
| Modified | August 19, 2016 |
| State | Final no updates expected |
| Brief Description | Methane and sulfate concentration profiles - sediment cores from White Oak River estuary Station H, October 2012 |
The cores were sequentially cut into 3 cm section from the topmost to bottommost depth. For methane measurements, 3 ml of sediments were taken via cut-off syringe immediately after each section was sliced and quickly added to 60 ml serum vials containing 1 ml of 0.1 M KOH, which were stoppered and crimp-sealed with butyl rubber stoppers to minimize gas loss. After being shaken for 1 min to release methane from sediments (> 99.5% of the methane equilibrated in the headspace), a 5 ml headspace aliquot was displaced with an equal volume of anaerobic distilled water, injected into a 1 ml sample loop, and then analyzed on an Agilent 7890a gas chromatograph equipped with flame ionization detector. For sulfate measurements, plastic 15 ml tubes filled completely with sediment were centrifuged and the resulting porewater was filtered at 0.2 µm, acidified with 10% HCl and measured using a 2010i Dionex ion chromatograph.
Methane concentrations (mmol per litre of porewater) were calculated using the following equation:
[CH4] = (ρ(CH4)Vheadspace)/(RTφVsed1000)
where p(CH4) is the partial pressure of methane (in ppmv), Vheadspace is the volume of the serum vial headspace (ml) after the sediment and KOH are added, R is the universal gas constant, T is the temperature at time of measurement in Kelvin and Vsed is the volume (ml) of whole sediment added to the serum vial.
Porosity, φ, was calculated using the formula:
φ = (mw/ ρw)/(mw/ρw+((md-S*mw/1000)/ρds))
where mw is the mass of the water lost on drying, md is the mass of the dried sediment, ρw is the density of pure water, ρds is the density of dry sediment (assumed to be 2.5 g cm−3), and S is salinity in grams per kilogram (assumed to be 19 grams per kilogram for all samples).
Standards at sulfate concentrations 0, 0.1, 0.5, 1, 5, 10 mM measure prior to samples from each core and sample peak areas were converted to sulfate concentrations using the standard curves after accounting for the dilution ((peak area * slope + intercept) * 0.7 / 0.6) by the 10% HCl.
No samples have been flagged as below the detection limit.
BCO-DMO Processing:
- added conventional header with dataset name, PI name, version date
- renamed parameters to BCO-DMO and BODC standards
- replaced NaN with nd
- removed CH4_mod data
Agilent 7890a gas chromatograph equipped with flame ionization detector
2010i Dionex ion chromatograph
Ion chromatography is a form of liquid chromatography that measures concentrations of ionic species by separating them based on their interaction with a resin. Ionic species separate differently depending on species type and size. Ion chromatographs are able to measure concentrations of major anions, such as fluoride, chloride, nitrate, nitrite, and sulfate, as well as major cations such as lithium, sodium, ammonium, potassium, calcium, and magnesium in the parts-per-billion (ppb) range. (from http://serc.carleton.edu/microbelife/research_methods/biogeochemical/ic.html)
latitude, in decimal degrees, North is positive, negative denotes South; Reported in some datasets as degrees, minutes
longitude, in decimal degrees, East is positive, negative denotes West; Reported in some datsets as degrees, minutes
depth of core sample
Observation/sample depth below the sea surface. Units often reported as: meters, feet.
When used in a JGOFS/GLOBEC dataset the depth is a best estimate; usually but not always calculated from pressure; calculated either from CTD pressure using Fofonoff and Millard (1982; UNESCO Tech Paper #44) algorithm adjusted for 1980 equation of state for seawater (EOS80) or simply equivalent to nominal depth as recorded during sampling if CTD pressure was unavailable.
methane concentration in porewater
Concentration of sulfate (SO4) per unit volume
unique sample identification or number; any combination of alpha numeric characters; precise definition is file dependent
???methane concentration in porewater - modified in some way???
| Name | Affiliation | Contact |
|---|---|---|
| Karen G. Lloyd | University of Tennessee | ✓ |
| Jordan T. Bird | University of Tennessee Knoxville (UTK) | |
| Nancy Copley | Woods Hole Oceanographic Institution (WHOI BCO-DMO) |
| Project Title | Quantifying the contribution of the deep biosphere in the marine sediment carbon cycle using deep-sea sediment cores from the Baltic Sea |
|---|---|
| Acronym | IODP-347 Microbial Quantification |
| URL | https://www.bco-dmo.org/project/639417 |
| Created | February 29, 2016 |
| Modified | December 6, 2017 |
Marine sediments contain a microbial population large enough to rival that of Earth's oceans, but much about this vast community is unknown. Innovations in total cell counting methods have refined estimates of cell concentrations, but tell us nothing about specific taxa. Isotopic data provides evidence that a majority of subsurface microorganisms survive by breaking down organic matter, yet measurable links between specific microbial taxa and their organic matter substrates are untested. The proposed work overcomes these limitations, with a particular focus on the degradation of proteins and carbohydrates, which comprise the bulk of classifiable sedimentary organic matter. The project will link specific taxa to potential extracellular enzyme activity in the genomes of single microbial cells, apply newly-identified, optimal methods for counting viable cells belonging to specific taxa using catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH), and measure the potential activity of their enzymes in situ. The resulting data will provide key evidence about the strategies subsurface life uses to overcome extreme energy limitation and contribute to the long-term carbon cycle.
The Principal Investigators are employing novel,improved methods to quantify cells of specific taxa in the marine subsurface and to determine the biogeochemical functions of those uncultured taxa, including:
1) Determine the pathway of organic carbon degradation in single cell genomes of uncultured, numerically dominant subsurface microorganisms.
2) Quantify viable bacteria and archaea in the deep subsurface using an improvement on the existing technology of CARD-FISH.
3 )Measure the potential activities (Vmax values) of enzymes in deep Baltic Sea sediments, and use the abundances of enzyme-producing microorganisms to calculate depth profiles of cell-specific Vmax values.
The project combines these methods in order to identify and quantify the cells capable of degrading organic matter in deep sediments of the Baltic Sea, obtained from Integrated Ocean Drilling Program (IODP) expedition 347. These results will greatly expand our knowledge of the function and activity of uncultured microorganisms in the deep subsurface.
This project is associated with C-DEBI account number 157595.
| Name | Affiliation | Role | Contact |
|---|---|---|---|
| Karen G. Lloyd | University of Tennessee Knoxville (UTK) | Principal Investigator | ✓ |
| Andrew D. Steen | University of Tennessee Knoxville (UTK) | Co-Principal Investigator |
A genomic reconstruction belonging to the genus Methanosarcina was assembled from metagenomic data from a methane-producing enrichment of Antarctic permafrost. This is the first methanogen genome reported from permafrost of the Dry Valleys and can help shed light on future climate-affected methane dynamics.
The recently proposed candidatus order Altiarchaeales remains an uncultured archaeal lineage composed of genetically diverse, globally widespread organisms frequently observed in anoxic subsurface environments. In spite of 15 years of studies on the psychrophilic biofilm-producingCandidatus Altiarchaeum hamiconexum and its close relatives, very little is known about the phylogenetic and functional diversity of the widespread free-living marine members of this taxon. From methanogenic sediments in the White Oak River Estuary, NC, USA, we sequenced a single cell amplified genome (SAG), WOR_SM1_SCG, and used it to identify and refine two high-quality genomes from metagenomes, WOR_SM1_79 and WOR_SM1_86-2, from the same site. These three genomic reconstructions form a monophyletic group, which also includes three previously published genomes from metagenomes from terrestrial springs and a SAG from Sakinaw Lake in a group previously designated as pMC2A384. A synapomorphic mutation in the Altiarchaeales tRNA synthetase β subunit, pheT, caused the protein to be encoded as two subunits at non-adjacent loci. Consistent with the terrestrial spring clades, our estuarine genomes contained a near-complete autotrophic metabolism, H2 or CO as potential electron donors, a reductive acetyl-CoA pathway for carbon fixation, and methylotroph-like NADP(H)-dependent dehydrogenase. Phylogenies based on 16S rRNA genes and concatenated conserved proteins identified two distinct sub-clades of Altiarchaeales, Alti-1 populated by organisms from actively flowing springs, and Alti-2 which was more widespread, diverse, and not associated with visible mats. The core Alti-1 genome suggested Alti-1 is adapted for the stream environment with lipopolysaccharide production capacity and extracellular hami structures. The core Alti-2 genome suggested members of this clade are free-living with distinct mechanisms for energy maintenance, motility, osmoregulation, and sulfur redox reactions. These data suggested that the hamus structures found in Candidatus Altiarchaeum hamiconexum are not present outside of stream-adapted Altiarchaeales. Homologs to a Na+transporter and membrane bound coenzyme A disulfide reductase that were unique to the brackish sediment Alti-2 genomes, could indicate adaptations to the estuarine, sulfur-rich environment.
Marine sediments host a large population of diverse, heterotrophic, uncultured microorganisms with unknown physiologies that control carbon flow through organic matter decomposition. Recently, single-cell genomics uncovered new key players in these processes, such as the miscellaneous crenarchaeotal group. These widespread archaea encode putative intra- and extracellular proteases for the degradation of detrital proteins present in sediments. Here, we show that one of these enzymes is a self-compartmentalizing tetrameric aminopeptidase with a preference for cysteine and hydrophobic residues at the N terminus of the hydrolyzed peptide. The ability to perform detailed characterizations of enzymes from native subsurface microorganisms, without requiring that those organisms first be grown in pure culture, holds great promise for understanding key carbon transformations in the environment as well as identifying new enzymes for biomedical and biotechnological applications.
