The deep subseafloor sediment biosphere is one of Earth's largest microbial habitats, yet its global biomass estimate still rests on per-cell carbon conversions inherited from cultured or planktonic bacteria. Braun et al. (2016) provided the first quasi-direct constraint at one site (Landsort Deep, Baltic Sea): cells purified from the sediment by density centrifugation and fluorescence-activated cell sorting were measured for amino-acid carbon by HPLC and for cell volume by corrected fluorescence and electron microscopy, yielding 19–31 fg C per cell, an order-of-magnitude decrease of cell volume with depth, and a depth-increasing carbon density — but these numbers rest on assumptions that could not be tested directly on indigenous cells, chiefly that amino-acid carbon is 55% of total cell carbon (a ratio the authors defend from a ~61% amino-acid-carbon share measured in planktonic bacteria) and that volume correction factors derived from cultured reference strains apply to indigenous cells (supported by the agreement of the corrected fluorescence- and electron-microscopy volumes); these are residual systematic uncertainties rather than known errors. This problem asks for direct measurement of total cellular carbon, volume, and carbon density of indigenous deep-subseafloor cells, experimental testing of the conversion assumptions, and identification of the physiological adaptations responsible for the increasing carbon density. A subsequent direct-mass study of cultured prokaryotes (Khachikyan et al. 2019) found a nonlinear volume-to-carbon relation that conflicts with the low conversion values and would raise the estimated deep-biosphere biomass from 4.1 to about 13.3 Pg C, so the systematic uncertainty has grown rather than closed since 2016.

Background

Marine sediments buried below the seafloor host a vast population of mostly uncultured bacteria and archaea — the deep subseafloor biosphere — that persists under extreme energy limitation. Quantifying its biomass matters for Earth's carbon budget and for models of how slowly metabolizing life survives. The key conversion is the cell-specific carbon content (carbon mass per cell, in femtograms, fg = 10−1510^{-15} g): global inventories multiply counted cell abundances by an assumed fg C per cell. Historical assumptions came indirectly from culture or plankton studies: 65 fg C per cell (Parkes et al. 1994), 86 fg C per cell (Whitman et al. 1998, based partly on a single terrestrial-aquifer dry-weight measurement), lipid-biomarker-based estimates (Lipp et al. 2008), and 14 fg C per cell derived from cell volumes and a planktonic-bacteria protein-to-volume relation (Kallmeyer et al. 2012, who downscaled the global inventory to about 2.9×10292.9\times10^{29} cells, roughly 4.1 Pg C; Pg = 101510^{15} g). Braun et al. (2016) were the first to constrain the number quasi-directly for the deep biosphere: at Landsort Deep (Baltic Sea, IODP Expedition 347; IODP is the International Ocean Discovery Program, an ocean-drilling research collaboration), they separated cells from the sediment matrix by multi-layer density centrifugation, purified cell extracts by fluorescence-activated cell sorting (FACS, a method that physically sorts fluorescently labeled cells from detrital particles), and measured total hydrolyzable amino-acid carbon (THAA-C, the carbon contained in the amino acids released when cell protein is fully hydrolyzed) by high-performance liquid chromatography (HPLC). Total cell carbon was then obtained by assuming amino acids carry 55% of total cell carbon, a ratio taken from cultured Escherichia coli. Cell volumes were measured by epifluorescence microscopy (FM, light microscopy of DNA-stained cells) and scanning electron microscopy (SEM), then corrected for method artifacts: the fluorescence halo effect (a bright aureole around stained cells that inflates apparent size), shrinkage from fixation (chemical preservation of cells), and shrinkage from dehydration and critical point drying (CPD, replacing water with liquid CO2_2 evaporated at its critical point to avoid surface-tension collapse). All correction factors came from cultured E. coli and Micrococcus luteus reference cells or literature averages. Results: mean cell volumes decreased with depth (down to 60 meters below seafloor) by up to an order of magnitude, most deep cells being smaller than 0.05 μ\mum3^3; cell-specific carbon was 19–31 fg C per cell (mean 23); and carbon density (fg C per μ\mum3^3 of cell volume) rose from roughly 200 to 600, peaking near 1000 fg C μ\mum−3^{-3}, implying decreasing intracellular water content with depth. The authors themselves flagged the residual uncertainties this problem addresses: the 55% amino-acid-carbon share is untested for indigenous deep-biosphere cells (they discuss 40–75% scenarios shifting the deepest-sample estimate between about 13 and 23 fg C per cell); the culture-derived correction factors may misrepresent cells that probably hold less water than cultured cells; SEM verification succeeded only in high-abundance samples; and the physiological mechanism behind smaller, denser cells — reduced intracellular water, altered macromolecular composition, selection for minimal maintenance costs — remains uncharacterized. The measurements come from a single high-organic-carbon, high-sedimentation-rate site (Holocene clay younger than about 15,000 years), so whether the depth trends extend to contrasting regimes such as oligotrophic ocean gyres with million-year-old sediments has not been examined; the source itself, however, draws the opposite inference for the carbon content, arguing from a comparison with global estimates across very different sediment ages that cell-specific carbon content in marine sediments is a relatively constant parameter and "a general feature of subsurface microbial life". Super-resolution fluorescence microscopies (PALM, STORM, STED; techniques beating the diffraction limit of light) were noted as not yet used in deep biosphere research.

Problem Statement

Establish, by direct measurement on indigenous deep-subseafloor microbial cells, their absolute cell volume, total cellular carbon content, and carbon density — replacing the assumption chain behind current global deep-biosphere biomass estimates — and determine how these quantities behave with sediment depth. The single scientific objective uniting these components is to convert the deep-subseafloor per-cell carbon conversion from an assumed constant into a directly measured, uncertainty-quantified quantity, and to establish the physiological basis of the Landsort Deep depth trends. Concretely, the open question comprises: (1) measuring total cell carbon of indigenous cells (and the ratio of amino-acid carbon to total cell carbon), directly testing the 55% conversion and the 19–31 fg C per cell range of Braun et al. (2016); (2) determining cell volumes and carbon densities with method artifacts (fixation, filtration, fluorescence halo, dehydration and critical-point-drying shrinkage) quantitatively controlled, with the adequacy of these controls for indigenous deep-biosphere cells directly assessed — by re-derivation on indigenous cells, cross-validation between independent methods, or equivalent means — rather than inferred from cultured reference strains alone; and (3) identifying which physiological adaptations (e.g. reduced intracellular water content, altered macromolecular composition, selection for small cells with low maintenance costs) cause the depth-increasing carbon density, via biochemical or structural characterization of indigenous deep-subseafloor taxa or long-term balanced-energy-limitation cultures, since acute stress responses of cultured cells are misleading analogues for a biosphere living under balanced conditions at extremely low metabolic rates. These three components are jointly required for a resolved answer, and none of them is sufficient alone: the measurement components must together establish the depth behaviour of cell volume, total cell carbon, and carbon density from directly measured, uncertainty-quantified values whose conversion and artifact-correction assumptions have been tested on indigenous cells, and the mechanism component must identify the physiological cause of the depth-increasing carbon density. Whether the Landsort Deep depth trends — cell volume decreasing by up to an order of magnitude, carbon density rising from roughly 200 to 600–1000 fg C μ\mum−3^{-3}, and cell-specific carbon staying comparatively low and constant — extend to contrasting sedimentary regimes such as oligotrophic ocean-gyre sediments and ancient, slowly accumulating deposits remains untested at the single source site and is a natural extension of, rather than a component of, this question. Consistency of the resulting per-cell carbon with the direct-mass relationship of Khachikyan et al. (2019), and the implied revision of the global subseafloor biomass inventory (currently spanning roughly 4–13 Pg C depending on the conversion adopted), are corollaries that a resolved measurement would inform.

References

  1. Braun S, Morono Y, Littmann S, Kuypers M, Aslan H, Dong M, Jørgensen BB, Lomstein BA (2016) Size and Carbon Content of Sub-seafloor Microbial Cells at Landsort Deep, Baltic Sea. Frontiers in Microbiology 7:1375. DOI: 10.3389/fmicb.2016.01375. https://doi.org/10.3389/fmicb.2016.01375
  2. Khachikyan A, Milucka J, Littmann S, Ahmerkamp S, Meador T, Könneke M, Burg T, Kuypers MMM (2019) Direct Cell Mass Measurements Expand the Role of Small Microorganisms in Nature. Applied and Environmental Microbiology 85:e00493-19. DOI: 10.1128/AEM.00493-19. https://doi.org/10.1128/AEM.00493-19
  3. Kallmeyer J, Pockalny R, Adhikari RR, Smith DC, D'Hondt S (2012) Global distribution of microbial abundance and biomass in subseafloor sediment. Proceedings of the National Academy of Sciences USA 109:16213–16216. DOI: 10.1073/pnas.1203849109. https://doi.org/10.1073/pnas.1203849109
  4. Whitman WB, Coleman DC, Wiebe WJ (1998) Prokaryotes: The unseen majority. Proceedings of the National Academy of Sciences USA 95:6578–6583. DOI: 10.1073/pnas.95.12.6578. https://doi.org/10.1073/pnas.95.12.6578
  5. Parkes RJ, Cragg BA, Bale SJ, Getlifff JM, Goodman K, Rochelle PA, Fry JC, Weightman AJ, Harvey SM (1994) Deep bacterial biosphere in Pacific Ocean sediments. Nature 371:410–413. DOI: 10.1038/371410a0. https://doi.org/10.1038/371410a0
  6. Lipp JS, Morono Y, Inagaki F, Hinrichs K-U (2008) Significant contribution of Archaea to extant biomass in marine subsurface sediments. Nature 454:991–994. DOI: 10.1038/nature07174. https://doi.org/10.1038/nature07174
  7. Bar-On YM, Phillips R, Milo R (2018) The biomass distribution on Earth. Proceedings of the National Academy of Sciences USA 115:6506–6511. DOI: 10.1073/pnas.1711842115. https://doi.org/10.1073/pnas.1711842115
  8. Braun S, Mhatre SS, Jaussi M, Røy H, Kjeldsen KU, Pearce C, Seidenkrantz M-S, Jørgensen BB, Lomstein BA (2017) Microbial turnover times in the deep seabed studied by amino acid racemization modelling. Scientific Reports 7:5680. DOI: 10.1038/s41598-017-05972-z. https://doi.org/10.1038/s41598-017-05972-z
  9. Mhatre SS, Kaufmann S, Marshall IPG, Obrochta S, Andrèn T, Jørgensen BB, Lomstein BA (2019) Microbial biomass turnover times and clues to cellular protein repair in energy-limited deep Baltic Sea sediments. FEMS Microbiology Ecology 95:fiz068. DOI: 10.1093/femsec/fiz068. https://doi.org/10.1093/femsec/fiz068
  10. Hoehler TM, Mankel DJ, Girguis PR, McCollom TM, Kiang NY, Jørgensen BB (2023) The metabolic rate of the biosphere and its components. Proceedings of the National Academy of Sciences USA 120:e2303764120. DOI: 10.1073/pnas.2303764120. https://doi.org/10.1073/pnas.2303764120
  11. Beulig F, Schubert F, Adhikari RR, Glombitza C, Heuer VB, Hinrichs K-U, Homola KL, Inagaki F, Jørgensen BB, Kallmeyer J, Krause SJE, Morono Y, Sauvage J, Spivack AJ, Treude T (2022) Rapid metabolism fosters microbial survival in the deep, hot subseafloor biosphere. Nature Communications 13:312. DOI: 10.1038/s41467-021-27802-7. https://doi.org/10.1038/s41467-021-27802-7
  12. Avcı B, Panagiotou K, Albertsen M, Ettema TJG, Schramm A, Kjeldsen KU (2025) Peculiar morphology of Asgard archaeal cells close to the prokaryote-eukaryote boundary. mBio 16:e00327-25. DOI: 10.1128/mbio.00327-25. https://doi.org/10.1128/mbio.00327-25