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Evidence of dark oxygen production at the abyssal seafloor
Sweetman, Andrew k.; Smith, Alycia J.; De Jonge, Danielle S. W.; Hahn, Tobias; Schroedl,
Peter; Silverstein, Michael; Andrade, Claire; Edwards, R. Lawrence; Lough, Alastair J. M.;
Woulds, Clare; Homoky, William B.; Koschinsky, Andrea; Fuchs, Sebastian; Kuhn, Thomas;
Geiger, Franz; Marlow, Jeffrey J.
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Nature Geoscience
Publication date:
2024
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Citation for published version (APA):
Sweetman, A. K., Smith, A. J., De Jonge, D. S. W., Hahn, T., Schroedl, P., Silverstein, M., Andrade, C.,
Edwards, R. L., Lough, A. J. M., Woulds, C., Homoky, W. B., Koschinsky, A., Fuchs, S., Kuhn, T., Geiger, F., &
Marlow, J. J. (2024). Evidence of dark oxygen production at the abyssal seafloor. Nature Geoscience.
https://doi.org/10.1038/s41561-024-01480-8




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nature geoscience

Brief Communication                                                                                    https://doi.org/10.1038/s41561-024-01480-8


Evidence of dark oxygen production at the
abyssal seafloor

Received: 24 January 2024                           Andrew K. Sweetman 1 , Alycia J. Smith2, Danielle S. W. de Jonge 1,
                                                    Tobias Hahn 3, Peter Schroedl 4, Michael Silverstein 5, Claire Andrade4,
Accepted: 6 June 2024
                                                    R. Lawrence Edwards6, Alastair J. M. Lough 7, Clare Woulds7,
Published online: xx xx xxxx                        William B. Homoky 7, Andrea Koschinsky 8, Sebastian Fuchs9, Thomas Kuhn9,
                                                    Franz Geiger10 & Jeffrey J. Marlow 4
   Check for updates


                                                    Deep-seafloor organisms consume oxygen, which can be measured by
                                                    in situ benthic chamber experiments. Here we report such experiments
                                                    at the polymetallic nodule-covered abyssal seafloor in the Pacific Ocean
                                                    in which oxygen increased over two days to more than three times the
                                                    background concentration, which from ex situ incubations we attribute to
                                                    the polymetallic nodules. Given high voltage potentials (up to 0.95 V) on
                                                    nodule surfaces, we hypothesize that seawater electrolysis may contribute
                                                    to this dark oxygen production.

Oxygen (O2) is prevalent in deep-sea surface sediments where its rate         rates of 1.7–18 mmol O2 m−2 d−1. Independent measurements of O2
of consumption reflects the sum of aerobic respiration and oxidation          concentration using the Winkler method also showed DOP (Extended
of reduced inorganic compounds produced by anaerobic decay. These             Data Fig. 2), providing evidence that the optodes were not malfunction-
processes define sediment community O2 consumption (SCOC), and                ing. No statistically significant difference in the total net O2 produced
quantifying SCOC is needed to estimate fluxes of major elemental cycles       (maximum [O2] – initial [O2]; Extended Data Table 2) was found between
through marine systems1–3. We undertook multiple in situ benthic cham-        chambers (ANOVA, F2,9 = 0.107, p = 0.900) or experimental treatments
ber lander experiments to measure abyssal SCOC in the Nauru Ocean             (ANOVA, F3,9 = 0.876, p = 0.489), ruling out any experimental bias. We
Resources Inc. (NORI)-D licence area of the Clarion–Clipperton Zone           found no difference in the total net O2 produced between cruises
(CCZ; Extended Data Fig. 1 and Extended Data Table 1) where polymetal-        (ANOVA, F2,12 = 0.391, p = 0.684), though DOP was correlated to the
lic nodules cover extensive areas of seafloor. Sediments and nodules          average surface area of the nodules (Spearman’s correlation, ⍴ = 0.664,
were exposed to different experimental treatments, which included the         p = 0.031). A re-evaluation of in situ O2 optode data collected from 36-h
addition of dead-algal biomass, dissolved inorganic carbon and ammo-          benthic chamber experiments in the abyssal eastern and western CCZ
nium (NH4+) or cold filtered surface seawater. No-injection controls were     (Extended Data Figs. 1 and 3) also showed DOP, indicating its occur-
also performed. In contrast to previous deep-sea O2 flux studies that only    rence in multiple locations across the CCZ. Our findings contrast with
showed SCOC, we consistently found that more O2 was accumulating in           all published deep-sea benthic O2 flux studies and suggest that DOP may
the chambers than was being consumed, resulting in net O2 production.         provide O2 for benthic respiration. Whereas the DOP measured was
     Constant linear decreases in O2 optode readings were observed in         greater than SCOC, we would urge caution when temporally upscaling
two experiments (Fig. 1), and SCOC determined by in situ O2 micropro-         our results, as the nonlinear production of O2 suggests that DOP may
filing was 0.7 mmol O2 m−2 d−1 indicating that SCOC occurs in NORI-D as       not be continuous in nature. Moreover, the variance in DOP activity
in many abyssal habitats2–4. However, O2 concentrations in 25 benthic         seen between experiments and its relationship to nodule surface area
chamber incubations started at 185.2 ± 2.9 µmol l−1 (1 standard error         suggests DOP activity may change with nodule spatial density and type
(SE)) and reached O2 maxima between 201 and 819 µmol l−1 over 47 h            (for example, diagenetic versus hydrogenetic), so upscaling our results
(Fig. 1), indicating net dark O2 production (DOP) corresponding to            by area is also imprudent without additional studies.


1
 The Scottish Association for Marine Science, (SAMS), Oban, UK. 2Heriot-Watt University, Edinburgh, UK. 3GEOMAR Helmholtz Centre for Ocean
Research Kiel, Kiel, Germany. 4Department of Biology, Boston University, Boston, MA, USA. 5Bioinformatics Program, Boston University, Boston, MA, USA.
6
  Department of Earth and Environmental Science, University of Minnesota, Minneapolis, MN, USA. 7Faculty of Environment, University of Leeds, Leeds, UK.
8
  School of Science, Physics and Earth Sciences, Constructor University Bremen, Bremen, Germany. 9Federal Institute for Geoscience and Natural
Resources (BGR), Hannover, Germany. 10Technological Institute, Northwestern University, Evanston, IL, USA.      e-mail: Andrew.Sweetman@sams.ac.uk


Nature Geoscience
Brief Communication                                                                                                https://doi.org/10.1038/s41561-024-01480-8

a               900                                                                       to purge air from the chambers as the lander sinks. Even if an air bubble
                800                                                                       could be trapped long enough to reach the seafloor, gaseous diffusion
                                                                        AKS268-Ch.2
                700                                                     AKS268-Ch.3
                                                                                          of O2 into the water phase would take < 1 s at 4,000 m depth (Extended
                600
                                                                        AKS271-Ch.3       Data Table 3), which is inconsistent with the steady increase in O2 over
                                                                        AKS273-Ch.3



O2 (µmol l–1)
                500
                                                                        AKS276-Ch.2       many hours seen in multiple experiments (Fig. 1). Third, intrusion of O2
                                                                        AKS276-Ch.3
                                                                        AKS279-Ch.3       from the plastic chambers into the water phase is unlikely (Methods) as
                400
                                                                        AKS282-Ch.3
                                                                        AKS286-Ch.2
                                                                                          they are built from polyoxymethylene, which is both highly inert and
                300
                                                                        AKS286-Ch.3       chemically stable in well-oxygenated settings and would not explain
                200                                                     AKS273-external
                                                                                          the variation in DOP because all experiments used identical materials.
                100
                                                                                          Last, DOP was also observed during 48-h ex situ sediment incubations
                 0                                                                        (Extended Data Fig. 4).
b                                                                                               Several lines of enquiry were pursued to explain the DOP. Subsur-
                900
                                                                                          face advection of oxic bottom water from seamount flanks into seafloor
                800
                                                                                          sediments5,6 and then into the chambers was discounted based on
                700                                                     AKS313-Ch.1
                                                                        AKS313-Ch.2
                                                                                          in situ O2 microprofiling that showed pore water was a net sink for O2
                600                                                     AKS316-Ch.1       and undersaturated compared with the O2 seen in the chambers. Fur-

O2 (µmol l–1)
                                                                        AKS316-Ch.3
                500                                                     AKS318-Ch.1       thermore, DOP was measured in sealed ex situ experiments (Extended
                                                                        AKS318-Ch.3
                400
                                                                        AKS321-Ch.1
                                                                                          Data Fig. 4) that prevented O2 intrusion from below. It is unlikely that
                300                                                     AKS321-Ch.3       biological mechanisms were responsible for the bulk of the DOP as
                                                                        AKS328-Ch.2
                200                                                     AKS328-Ch.3       ex situ core incubations revealed DOP in the presence of poison (HgCl2;
                                                                                          Extended Data Fig. 4). Whereas many microbes in the CCZ are able to
                100
                                                                                          detoxify Hg (II) to Hg (0)7, and some microhabitat pore spaces in the
                 0
                                                                                          core may have remained HgCl2 free, the taxa known to be capable of
c               900                                                                       DOP (for example, Nitrosopumilus maritimus) are killed by its addition8.
                800
                                                                                          We also observed weak statistical support between the relative abun-
                                                                                          dance of certain nitrogen-cycling microbial taxa and DOP (for example,
                700
                                                                                          Candidatus Nitrosopumilus ⍴ = 0.474, p = 0.420). The fact that DOP
                600
                                                                         AKS329-Ch.1      was detected in ex situ controls containing only polymetallic nodules

O2 (µmol l–1)
                500                                                      AKS329-Ch.2
                                                                         AKS331-Ch.2      (Extended Data Fig. 4) suggested that the DOP was linked to their pres-
                400                                                      AKS331-Ch.3
                                                                         AKS334-Ch.2      ence. Hence, we estimated the potential contribution of radiolytic O2
                300                                                      AKS334-Ch.3      production using a kinetic model9 and found 0.18 μmol l−1 O2 would be
                                                                         AKS336-Ch.3
                200                                                                       generated by this process within 48 h. We also modelled the chemical
                100                                                                       reduction of manganese (IV) oxide at in situ temperature (1.6 °C) across
                  0                                                                       a range of pH and O2 conditions encountered at the seafloor to assess
                      0   10     20         30        40         50                       if this reaction (2MnO2 → 2MnO + O2;Extended Data Fig. 5) could liber-
                               Incubation time (h)                                        ate the O2 but found that <0.1 nmol of manganese (IV) oxide would be
Fig. 1 | Oxygen concentrations in μmol l−1 measured by calibrated O2 optodes              chemically reduced to manganese (II) at seafloor conditions. As such,
through time in h in the different benthic chamber incubations. a–c, The in situ          localized radiolytic O2 production from the sediments and nodules and
benthic chamber lander deployments were made during the 5D (a), 5E (b) and 7A             chemical dissolution explain only a negligible proportion (< 0.5%) of
(c) cruises to the NORI-D license area (Extended Data Fig. 1). Nodules were present       the DOP observed.
in all incubation experiments. The green hue, blue hue and red lines in the 5D figure           The oxygen evolution reaction requires an input voltage of 1.23 V
(a) denote dead-algal biomass, dissolved inorganic carbon + NH4+ and filtered             plus an overpotential of approximately 0.37 V to split seawater into
seawater treatments, respectively. The gap in the optode data in AKS279-Ch.3              H2 and O2 (ref. 10) at NORI-D’s seafloor mean pH (7.41). This value can
was caused by the optode periodically not logging data. The black line indicates
                                                                                          be lowered by several hundred millivolts if the reaction proceeds via
ambient O2 concentration measured on the outside of the benthic chambers
                                                                                          the lattice-oxygen-mediated mechanism11. Use of metal catalysts such
during AKS273 on the 5D cruise. The green and yellow hue lines in the 5E (b) and 7A
                                                                                          as Mn oxides enriched with transition metals (for example, Ni) found
(c) figures denote the dead-algal biomass and control (no injection) treatments,
respectively. The minor drops seen in some of the O2 concentration profiles at 28,
                                                                                          in nodules12 and characterized by large tunnel areas and abundant
38 and 47 h are caused by the dilution of the chamber water with 50 ml of seawater        defect sites can optimize the adsorption of reactants and enhance
that was entrained from the outside into the chamber through a 1.5 m (0.25 cm             conductivity and catalytic performance11,13,14. We tested the electrical
diameter) open tube when the syringe sampler collected seawater samples from              potential between two platinum electrodes at 153 sites on the sur-
within the chamber. The constant O2 concentration measured during the first 2 h           faces of 12 nodules (Fig. 2) from the UK1, NORI-D and Bundesanstalt
of the 5D and 7A experiments was due to the stirrers being turned off for 1 h to allow    für Geowissenschaften und Rohstoffe (BGR) license areas. Although
the substrates (for example, dead-algal biomass) to sink to the sediment surface.         the potentials between different positions on the nodules were
Stirrers were turned on during the 5E expedition from the moment the lander was           highly variable, - potentials up to 0.95 V were found and high mean
deployed until the lander returned and power to the stirrers was disconnected.            background-corrected potentials were detected under cold-water
                                                                                          conditions (Fig. 2 and Extended Data Table 4). On the basis of these
                                                                                          studies and DOP being observed in nodule-only ex situ incubations
     Several lines of evidence indicate that the DOP was not caused by                    (Extended Data Fig. 4), we hypothesize that the DOP may have partly
experimental artefacts. First, the total O2 change between the experi-                    resulted from seawater electrolysis, with the necessary energy coming
mental and control (non-injection) treatments was statistically indistin-                 from the potential difference between metal ions within the nodule
guishable, and a steady increase in O2 concentration was recorded over                    layers, leading to an internal redistribution of electrons. Whereas
many hours in multiple experiments; these observations demonstrate                        questions remain concerning this potential mechanism (such as the
that DOP was not attributable to the injection of exogenous fluids.                       identity of the energy source(s), longevity of DOP, catalytic stabilities,
Second, diffusion of O2 from trapped air bubbles within the chamber                       electrochemical conditions on exposed versus buried nodules surfaces
was unlikely because each chamber uses two one-way valves in the lid                      and the influence of different chemistries within the nodule layers),


Nature Geoscience
 Brief Communication                                                                                                  https://doi.org/10.1038/s41561-024-01480-8

              0.30                                                                          References
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                                                                                                  opportunities and challenges. ACS Energy Lett. 4, 933–942 (2019).
 nodule surfaces. The nodules were collected from the NORI-D (1-5), UK1 (6-8)
 and the BGR (9-12) license areas. Potentials were measured at 21 °C (nodules 1–12)
                                                                                            11.   He, Y. et al. Recent progress of manganese dioxide based
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                                                                                                  nanosheets as highly efficient electrocatalysts for water
                                                                                                  oxidation. Adv. Funct. Mater. 28, 1802463 (2018).
 the ‘geo-battery’ hypothesis was supported by the link between DOP                         15.   Wegorzewski, A. V. & Kuhn, T. The influence of suboxic diagenesis
 and nodule average surface area. This connection could be due to an                              on the formation of manganese nodules in the Clarion Clipperton
 increased abundance of anode and cathode sites or a greater abun-                                nodule belt of the Pacific Ocean. Mar. Geol. 357, 123–138 (2014).
 dance of high Ni and Cu dendritic porous layers in larger nodules15.                       16.   Robins, L. J. et al. Manganese oxides, Earth surface oxygenation,
 Assuming the ‘geo-battery’ is partly responsible for the DOP observed,                           and the rise of oxygenic photosynthesis. Earth Sci. Rev. 239,
 the initial high DOP rate may have been related to the ‘bow-wave’ of the                         104368 (2023).
 lander removing sediments from the surface of the nodules and expos-                       17.   Chyba, C. F. & Had, K. P. Life without photosynthesis. Science 292,
 ing electrochemically active sites on the nodules. The slowdown in DOP                           2026–2027 (2001).
 seen later in the incubations could have then been caused by a reduc-
 tion in voltage potential and/or degradation of metal-oxide catalysts                      Publisher’s note Springer Nature remains neutral with regard to
 that has been observed in Mn oxide catalysts previously10. Whereas                         jurisdictional claims in published maps and institutional affiliations.
 this process requires further investigation, if true, DOP activity may
 fluctuate with sediment coverage on the nodules inviting the urgent                        Open Access This article is licensed under a Creative Commons
 question of how sediment remobilization and distribution over large                        Attribution 4.0 International License, which permits use, sharing,
 areas during deep-sea mining may influence DOP.                                            adaptation, distribution and reproduction in any medium or format,
      Understanding the mechanism(s) behind DOP, its temporal nature                        as long as you give appropriate credit to the original author(s) and the
 and its spatial distribution will allow its role in abyssal ocean ecosys-                  source, provide a link to the Creative Commons licence, and indicate
 tems to be better understood. Future studies of DOP in the deep sea                        if changes were made. The images or other third party material in this
 may also shed light on broader relationships between metal-oxide                           article are included in the article’s Creative Commons licence, unless
 deposition, biological evolution and the oxygenation of Earth16,17.                        indicated otherwise in a credit line to the material. If material is not
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Brief Communication                                                                                       https://doi.org/10.1038/s41561-024-01480-8

Methods                                                                         immediately fixed for microWinkler titration. The sample was then
A benthic chamber lander was deployed in the NORI-D license area six            mixed thoroughly using a glass bead placed in the exetainer and placed
times in May–June 2021 (5D cruise), five times in November–December             in the dark in a 4 °C refrigerator for 30–45 min to allow the precipitate to
2021 (5E cruise) and five times in August–September 2022 (7A cruise)            settle. Once the precipitate had sedimented, the exetainers were shaken
(Extended Data Fig. 1 and Extended Data Table 1). The lander comprised          again and left for 2–3 h before Winkler titrations were performed. All
three independent, autonomous, square benthic chambers (484 cm2)                titrations were completed within 12 h after sampling to determine dis-
separated by approximately <0.5 m. After arriving at the seafloor,              solved O2 concentrations. Each Winkler sample (approximately 5 ml)
the lander waited for 0.07–1.34 d before the chambers were pushed               was titrated twice, and duplicate measurements showed minor differ-
into the sediment to create an enclosed microcosm of the seafloor.              ences in O2 concentration (5D cruise error: 3.5 ± 0.3 μmol l−1, n = 71; 5E
Ten minutes into the incubation period, the enclosed chambers were              cruise error: 1.3 ± 0.2 μmol l−1, n = 69; 7A cruise error: 2.8 ± 0.4 μmol l−1,
injected with 50 ml of one of three solutions: (1) 0.45-µm-filtered, cold       n = 84). Winkler O2 concentration data were averaged for each syringe
surface seawater containing 79.2 mg of freeze-dried Phaeodactylum tri-          sample. The O2 concentrations estimated by Winkler analysis were
cornutum algae, (2) 32 µM Na2HCO3 and 40 µM NH4Cl dissolved in cold             22 ± 1% (n = 42, SE, 5D cruise), 8 ± 4% (n = 39, SE, 5E cruise) and 24 ± 2%
artificial seawater (salinity 35) and (3) 0.45-µm-filtered, cold surface        (n = 40, SE, 7A cruise) lower than the concentrations measured by the
seawater. On some occasions, the injection mechanism failed allowing            optodes at the same time point in the same incubations most likely
the response to control (no injection) conditions to be measured. The           due to out gassing of supersaturated O2 caused by depressurization
seafloor in the study area had a temperature of 1.6 °C ± 0.006 °C (SE,          and warming of the externally mounted syringes (whose samples were
n = 28) and a pH of 7.41 ± 0.05 (SE, n = 17). Immediately after the injec-      used for Winkler analyses) during the lander recovery to the surface.
tion, the overlying water was mixed with a submersible stirrer at 60 rpm             Back on shore, the final O2 concentration values were calculated
for 1 min before the stirrer was turned off that allowed any particulate        following Bittig et al. (ref. 18) from the optode, calibration and in situ
substrates to settle for 1 h. After 1 h, the stirrer was then turned on again   pressure data that was derived from the depth where each lander
for the remainder of the experiments. During the 5E expedition, the             deployment was made. Time stamps in the optode data were compared
stirrers were programmed to continually stir the overlying water even           to the lander computer programme times so the optode readings
immediately after injection.                                                    could be aligned to the schedule of the chamber experiment. The total
      The syringe samplers removed approximately 50 ml of seawater              change in O2 concentration in each chamber was then calculated from
from the water phase of each chamber at 0.1 or 0.03, 1, 3, 9, 28, 38 and        the volume of the water phase above the sediment and the difference in
47 h into the incubation experiment. Oxygen optodes (CONTROS                    O2 concentration from when the chambers started to seal off the sedi-
HydroFlash O2 manufactured by Kongsberg Maritime Contros GmbH)                  ment to the point when the maximum O2 concentration was reached.
mounted in the lid of each chamber logged O2 concentrations in the
chamber every 10 seconds throughout each experiment. Two days                   Benthic O2 microprofiling
before the first lander deployment of each cruise, the optodes under-           Benthic O2 microprofiles were made during lander deployments
went a two-point, multi-temperature calibration using 0 and 100% O2             AKS313, AKS316, AKS318 and AKS321 during the 5E cruise using a
calibration solutions at 1.2, 7, 18 and 30 °C following the recommen-           UNISENSE deep-sea microprofiling unit mounted <0.5 m from the
dations of Bittig et al. (ref. 18). On the 5D cruise, we also calibrated        benthic chambers. The microprofiles were made using 20 cm O2
the sensors 2 d after the last lander experiment so we could estimate           microsensors that penetrated the sediment in 0.05 mm steps. The
optode drift, which was negligible (0.27 µmol l−1 d−1) over the course          microsensors were calibrated 2 h before the lander deployments at
of the six-week cruise. The 0% and 100% O2 saturation solutions were            in situ temperature (1.6 °C) at 0% and 100% O2 saturation (above). At
created by bubbling 0.45-µm-filtered surface seawater in a bottle sitting       each sampling depth, the microsensor stopped for 5 s before each
in a water-chilling/heating unit with N2 gas (0%) or an aquarium air bub-       measurement was made. The sensor then recorded five individual O2
bling unit (100%) for 30 min. The O2 concentration of the calibration           concentration measurements. The average of these five measurements
solutions was confirmed in triplicate by Winkler titration. After incubat-      was taken for each depth point. The sediment surface was determined
ing seafloor sediments for 47 h, the lander chambers were closed by a           manually based on the turning point in the slope of O2 concentration
shutter door at the base of the chambers, and the chambers were then            with depth where O2 started to become depleted. SCOC was determined
pulled slowly out of the sediment, which took 1 h. The lander was then          from Fick’s first law of diffusion.
recalled from the seafloor. In eight instances, the lander programme
did not finish and the doors did not shut, preventing the sampling              Microbiology sampling
of sediment and determination of the volume of the water phase in               Nodule and sediment samples for microbial community analyses were
the chambers (Extended Data Table 2). Once the lander was back and              collected from the 5D experimental chambers. Approximately 30 g
secured on deck, the chambers were opened and the water above the               of sediment from each of the 0–2 cm and 2–5 cm horizons and 50 g
sediment removed via syphoning into a bucket. The distance from the             of intact nodules were placed in separate sterile Whirlpak bags with
top of the sediment to the base of the chamber lid was then measured in         a pre-sterilized spatula and then transferred to a −80 °C freezer. DNA
four places to get an accurate water depth for water volume estimates.          from approximately 10 g of nodules and 250 mg of sediment were
Whenever possible, a photograph was then taken of the chamber sedi-             extracted using the Qiagen PowerMax soil and PowerSoil extraction
ment and nodules from directly above the opening of the chamber. All            kits, respectively. Extracted DNA was then shipped on dry ice to Lara-
syringes containing water samples were removed and taken to the                 gen Inc. and sequenced using a proprietary in-house method. The V4
shipboard lab for immediate processing or stored in a cold lab (4 °C)           region of 16 S rRNA genes were amplified using the Earth Microbiome
before processing. The optodes were removed and their onboard data              Project protocol19 with the 515 F (5′‐GTGYCAGCMGCCGCGGTAA20)
downloaded to a computer. Finally, the nodules were removed from                and 806 R (5′-GGACTACNVGGGTWTCTAAT21) primers. Raw fastq
the chambers and washed of attached organic debris with cold (4 °C),            files were processed using a custom pipeline (https://github.com/
0.45-µm-filtered surface seawater and placed in sterile Whirlpak bags to        Boston-University-Microbiome-Initiative/BU16s) built with QIIME
be weighed in the laboratory later. The number of polymetallic nodules          2020.2 (https://www.nature.com/articles/s41587-019-0209-9). Adap-
at the seafloor determined from chamber counts was 1170 ± 97 m−2.               tor sequences were removed using cutadapt (https://doi.org/10.14806/
      Unfiltered syringe sample seawater was carefully transferred              ej.17.1.200), read truncation positions were determined by mineer
from each 50 ml syringe to a 12 ml exetainer via a 10 cm tube attached          (more below), amplicon sequence variants (ASVs) were generated
to the syringe nozzle, ensuring no air bubbles were introduced and              using dada2 (trunc-len-r20) (https://doi.org/10.1038/nmeth.3869)


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Brief Communication                                                                                    https://doi.org/10.1038/s41561-024-01480-8

and ASVs were clustered to 99% identity with the SILVA 132 database            dish and the platinum probes placed on the nodule at random loca-
(https://academic.oup.com/nar/article/42/D1/D643/1061236) using                tions, ensuring contact in one of two ways. We either carefully drilled
the vsearch cluster-features-closed-reference (https://doi.org/10.7717/        a hole into some nodules so one platinum wire could be fixed inside it
peerj.2584). Due to drops in sequencing quality, all reverse reads were        while the second platinum wire was firmly pressed against the nodule
truncated by 49 bases (from a length of 301 to 252) as determined by           surface using a clamp. Alternatively, the platinum wires were pressed
minERR, an algorithm for determining optimal sequence length based             firmly against two different spots on the nodule surface and held in
on sequence quality scores (https://github.com/michaelsilverstein/             place using a clamp. Voltages were then recorded for 1–2 min until the
mineer). Family- and genus-level abundance was computed by sum-                signal was stable. This procedure was repeated up to 20 times in differ-
ming the relative abundance of all ASVs with the same family/genus             ent randomly selected regions of the nodules depending on their size.
classification within each sample. Spearman correlations were then             Measurements were undertaken on 12 nodules at 21 °C (n = 153) and a
computed between family- and genus-level abundance and observed                single control rock composed of metamorphosed carbonate (n = 10).
optode-derived total O2 changes. Sequences have been archived at               Two nodules from UK1 were also retested after being cooled to 5 °C
National Centre for Biotechnology Information GenBank under the                (n = 18) by placing them in Instant Ocean water in a refrigerator over-
Bioproject ID PRJNA1117483.                                                    night. Voltage potentials (n = 20) between two nodules were measured
                                                                               using four nodules collected from UK1. Potentials measured during
Polymetallic nodule surface area measurements                                  each measurement were averaged and corrected for the background
Photographs of the surface sediment and nodules in the chambers                seawater voltage measured using only Instant Ocean seawater in the
were imported into Image J. The outline of each nodule in each cham-           absence of a nodule. Measured resistances inside some of the nodules
ber photograph was then traced and the surface area of the nodule              that were broken up were in the kΩ to 100s of kΩ range, though it is
automatically calculated in Image J (assuming each surface nodule              unclear if these resistivities change at the nano- or microscale requir-
was flat in shape) and logged as an Image J file before being exported         ing further investigation.
and saved as an Excel file.
                                                                               Geochemistry modelling
Radiolysis O2 production estimates                                             The chemical stability and solubility of manganese (IV) oxide (birnes-
To estimate the potential radiolytic O2 production, published concen-          site) to dissolved Mn2+ as a function of pH and O2 activity was modelled
trations of 238U, 235U, 232Th, 40K (refs. 22–26) in seawater were used (Sup-   using the Geochemist Workbench Professional (version 12) software,
plementary Table 1). For nodules, 238U, 235U and 232Th isotopes of three       with the in-built and internally consistent THERMO database. The
nodules from chamber experiments from the 5D cruise were measured              conditions used for generating the phase diagram (Extended Data
by Multicollector-Inductively Coupled Plasma Mass Spectrometer                 Fig. 5) represent bottom seawater as measured in the eastern CCZ with
using previously described methods27–29 and averaged; 40K values were          a temperature of 1.6 °C and chlorine and manganese concentrations
derived from the literature12. Nodule and seawater contributions were          of 0.55 M Cl and 2e−10 M Mn, respectively.
calculated using a kinetic model developed by ref. 9 that incorpo-
rates 32 reactions (equation (1) in ref. 30). The nodule boundary layer        Ex situ core incubations
was assumed to be fully integrated with the seawater, surpassing the           Opportunistic ex situ experiments were undertaken during the 5D
respective ~23 to ~452 μm stopping power distance of alpha and beta            cruise using sediment cores retrieved by a multi-corer from the CTA
particles used to model geologic materials31. Sediment radiolytic O2           area (Extended Data Fig. 1). Immediately after the multi-corer arrived
was calculated as half of the previously quantified H2 production rates        back at the surface, cores were removed and transferred to a cold lab
in equatorial Pacific subsurface sediment32, given the stoichiometry of        held at in situ temperature. The cores were then exposed to the fol-
water’s radiolytic decomposition (an equivalency that probably offers          lowing five treatments (administered using a 60 ml syringe), which
an overestimate of derived O2). Contributions from these three com-            included (1) Na2HCO3 (0.3 μM final concentration, n = 3), (2) NH4Cl
ponents (nodules, sediment and seawater) were scaled by the benthic            (10 μM final concentration, n = 3) and (3) NH4Cl (50 μM final concen-
chamber’s size and contents to produce an estimate of 0.18 μmol l−1 of         tration, n = 3), (4) 0.3 μM Na2HCO3 + 10 μM NH4Cl (final concentration,
O2 generated over 48 h according to the following expression.                  n = 3) and (5) HgCl2 (1.1 μM final concentration, n = 3). No-injection
                                                                               controls (n = 3) were also performed and separate core experiments
                                                   −1       −2
         (O2 )t = (Qiz × Ea × G(O2 ) × MO2 × Aiz        × 10 ) × (1 − e−λt )   in which four nodules were incubated for 48 h by themselves with no
                                                                               additions. After addition, the water phase of each core was stirred and
Here (O2)t is the mass (kg) of O2 produced over a given time t (yr), Qiz       a 50-ml sample of top water was taken for microWinkler analysis (as
is the mass (g) of the isotope, Ea is the average energy (eV) released         above). Stoppers were then placed on the top of the cores, ensuring
from the decay of one atom; G(O2) is the radiation chemical yield of           no air bubbles were present. The stoppers were secured tightly and the
molecules per 100 eV of the radiation energy; MO2 is the O2 molecular          cores fully submerged in a large bucket containing 0.45-µm-filtered,
mass (g), Aiz is the isotope atomic mass (g) and λ is the isotope-specific     cold, surface seawater (salinity 35). The bucket was covered with five
decay constant (y−1). The overall (O2)t value summed the contributions         black plastic bags and secured in the cold room with the lights turned
from 238U, 235U, 232Th and 40K across water, nodule and sediment sources.      off. After 48 h, the cores were removed from the bucket, and the cores
                                                                               were inspected for the presence of air bubbles. Only one core, a HgCl2
Electrochemistry measurements                                                  treatment, had a gas bubble beneath the bung, which was rejected from
Voltage potentials were measured using a Keithley DMM6500 digital              further analysis, leaving n = 2 for this treatment. The other cores were
multimeter on nodules previously collected by coring in the UK1,               then re-sampled for dissolved O2 and analysed as before. Core-specific
NORI-D and BGR license areas. Nodules were initially immersed for              water volume measurements were used together with the change in O2
seven days in Instant Ocean artificial seawater (salinity 35). To measure      concentration to calculate the total net O2 change per core.
the potentials, two electrodes (platinum wire, 99.9% purity) were first              To determine if our ex situ DOP detection was affected by intrusion
washed in perchloric acid, rinsed in Milli-Q water and dried before            of O2 from the atmosphere into the core tube, two controls were per-
being attached to alligator clamps attached to the multimeter. The             formed: a shipboard test with an O2 microprofiler and a lab-based test
platinum wires were then immersed in Instant Ocean artificial seawater         using the Winkler method. Shipboard, a clean core tube was filled with
in a glass petri dish to measure background voltages (0.003 ± 0.001 V,         Milli-Q water and sparged with N2 for 10 min before beginning the test. A
SE, n = 17) until stable. Once stable, a nodule was placed in the petri        Metrohm 8663 Multimeter was inserted through a predrilled hole in the


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Brief Communication                                                                                  https://doi.org/10.1038/s41561-024-01480-8

rubber stopper, allowing for O2 concentration to be recorded every 5 s.      22. Choppin, G. et al. Radiochemistry and Nuclear Chemistry (Elsevier,
An increase from 39 to 69 µmol l−1 was observed over ~5 h, correspond-           2002).
ing to a rate of 0.14 mmol m−2 d−1 or 4% of the 3.5 mmol m−2 d−1 mean net    23. Katz, J. J. et al. The Chemistry of the Actinide Elements 2nd edn
DOP measured in the ex situ experiments. Back in the home laboratory,            (Springer, 1986).
three of the original core tubes were filled with 4 °C, 0.2-µm-filtered      24. Lide, D. R. CRC Handbook of Chemistry and Physics Vol. 85 (CRC
artificial seawater (salinity 35) and sparged with N2 for 8 min through          Press, 2004).
a filtered pipette tip to achieve an initial dissolved O2 concentration of   25. Nier, A. O. A redetermination of the relative abundances of the
~100 µmol l−1 (for example, the approximate starting O2 concentrations           isotopes of carbon, nitrogen, oxygen, argon, and potassium. Phys.
for the shipboard experiments). The tubes were sealed with rubber                Rev. 77, 789 (1950).
stoppers and electrical tape, being careful to avoid bubble forma-           26. Stumm, W. and Morgan, J. J. Aquatic Chemistry: An Introduction
tion. They were then submerged in a 32-gallon plastic garbage can of             Emphasizing Chemical Equilibria in Natural Waters 2nd edn (John
unfiltered seawater (O2 concentration: 228.12 µmol l−1) in a dark cold           Wiley & Sons, 1981).
room (8 °C) for 48 h. After 48 h, the tubes were quickly unsealed and        27. Cheng, H. et al. Improvements in 230Th dating, 230Th and
analysed one at a time to prevent additional O2 dissolution from the             234U half-life values, and U–Th isotopic measurements by
air. A 50-ml sterile syringe was used to slowly collect 10 ml of seawater        multi-collector inductively coupled plasma mass spectrometry.
from the centre of the core tube, being sure to avoid bubble entrain-            Earth Planet. Sci. Lett. 371, 82–91 (2013).
ment into the syringe. The sample was carefully expelled into a 10-ml        28. Edwards, R. L. et al. 238U, 234U, 230Th, 232Th systematics and the
reaction vial and fixed using the adjusted values for a 10-ml sample             precise measurement of time over the past 500,000 years. Earth
according to a volume-scaled Winkler titration protocol33 and the                Planet. Sci. Lett. 81, 175–192 (1987).
reagents from the LaMotte Dissolved Oxygen Test Kit. The fixation            29. Shen, C. C. et al. Uranium and thorium isotopic and concentration
of each collected sample was done in less than 2 min in a fume hood.             measurements by magnetic sector inductively coupled plasma
Dissolved O2 increased by 0.11 mmol m−2 d−1 during the 48 h, which               mass spectrometry. Chem. Geol. 185, 165–178 (2002).
corresponds to between 3.2% of the mean net DOP rate observed in             30. Ershov, B. G. & Gordeev, A. V. A model for radiolysis of water and
the ex situ experiments (3.5 mmol m−2 d−1). Both of our control experi-          aqueous solutions of H2, H2O2 and O2. Radiat. Phys. Chem. 77,
ments provide high confidence that the diffusion of external O2 into             928–935 (2008).
the core tubes did not cause the O2 production measured in the ex situ       31. DeWitt, J. et al. The effect of grain size on porewater
core incubations.                                                                radiolysis.Earth Space Sci. 9, e2021EA002024 (2021).
                                                                             32. Blair, C. C. et al. Radiolytic hydrogen and microbial respiration in
Calculations to quantify intrusion of O2 from the                                subsurface sediments. Astrobiology 7, 951–970 (2007).
polyoxymethylene chambers and lids                                           33. Shriwastav, A. et al. A modified Winkler’s method for
Oxygen intrusion was estimated from Stephens34 who calculated that               determination of dissolved oxygen concentration in water:
20.66 µmol l−1 of O2 could diffuse out of 428 cm2 of polyoxymethylene            dependence of method accuracy on sample volume.
plastic when immersed for 48 h in hypoxic water (O2 diffusion rate:              Measurement 106, 190–195 (2017).
0.02 µmol O2 cm−2 d−1). To determine the total area of plastic that would    34. Stevens, E. D. Use of plastic materials in oxygen-measuring
be available for diffusion (869–1,584 cm2), we added the surface area            systems. J. Appl. Physiol. 72, 801–804 (1992).
of the lid to the surface area of the four walls that would be exposed       35. Sweetman, A. K. Data collected from replicate benthic chamber
at the seafloor (based on the depth of the water phase—above). The               experiments conducted at abyssal depths across the Clarion
minimum and maximum areas available for diffusion were multiplied                Clipperton Zone (CCZ), Pacific Ocean. Dryad https://doi.org/
by 0.02 µmol O2 cm−2 d−1 to estimate that 41.9–76.5 µmol O2 l−1 would            10.5061/dryad.tdz08kq6w (2024).
diffuse out of the polyoxymethylene chamber walls and lid in 48 h under
hypoxic conditions. Thus, we are highly confident that O2 leakage from       Acknowledgements
the plastic chambers could not replicate the high O2 concentration seen      We would like to thank S. Wilson, E. Holsting, F. Mann and L. Carrera
in some of our oxygenated experiments (Fig. 1).                              at Maersk Supply Service, the captain and crew of the research
                                                                             vessels ‘Maersk Launcher’ and ‘Island Pride’ for all their help preparing
Data availability                                                            for the research expeditions and their excellent assistance at sea.
Source data are provided with this paper. These data are also available      We are grateful to R. Davis for help with the lander deployments and
via Dryad at https://doi.org/10.5061/dryad.tdz08kq6w (ref. 35), and          D. Anderson, M. Delgado and M. Cecchetto for help at sea. We thank
geological samples were exported in accordance with relevant permits.        Y. Maierhaba, C. Momjian and A. Shukla for their assistance with
The nucleotide sequences generated by metagenome sequencing have             lab-based molecular analyses and R. Merrifield for his help with the
been deposited in the National Centre for Biotechnology Information          electrochemistry analysis. We would like to acknowledge and give our
database under BioProject ID PRJNA1117483.                                   thanks to K. M. Allen, M. Clarke, A. O’Sullivan, P. Clarke, L. Marsh and
                                                                             J. Smith for helping to initiate the research. The work was funded by The
References                                                                   Metals Company Inc. through its subsidiary Nauru Ocean Resources
18. Bittig, H. C. et al. Oxygen optode sensors: principle,                   Inc. (NORI). NORI holds exploration rights to the NORI-D contract
    characterization, calibration, and application in the ocean. Front.      area in the CCZ and is regulated by the International Seabed Authority
    Mar. Sci. 4, 429 (2018).                                                 and sponsored by the government of Nauru (A.K.S., C.W., W.B.H.).
19. Caporaso J. G. et al. EMP 16 S Illumina amplicon protocol V.1.           UK Seabed Resources funded the research expedition to the UK1 and
    protocols.io. https://doi.org/10.17504/protocols.io.nuudeww (2018).      OMS license areas in 2015 (A.K.S.), and the Gordon and Betty Moore
20. Parada, A. E. et al. Every base matters. Assessing small subunit         Foundation provided funding for the research cruise to APEIs 1, 4 and
    rRNA primers for marine microbiomes with mock communities,               7 in 2018 (A.K.S.). Research support from the Natural Environment
    time series and global field samples. Environ. Microbiol. 18,            Research Council SMARTEX (Seabed Mining And Resilience To
    1403–1414 (2016).                                                        Experimental impact) project (grant number NE/T003537/1) and the
21. Apprill, A. et al. Minor revision to V4 region SSU rRNA 806 R gene       European Commission project iAtlantic (grant number 818123) to
    primer greatly increases detection of SAR11 bacterioplankton.            A.K.S. is also acknowledged. We thank K. Mizell at the US Geological
    Aquat. Microb. Ecol. 75, 129–137 (2015).                                 Survey for comments on our manuscript.


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Brief Communication                                                                                 https://doi.org/10.1038/s41561-024-01480-8

Author contributions                                                        S.F. and T.K. also work for the Federal Institute for Geoscience and
A.K.S., C.W., W.B.H. and J.J.M. generated the funding. A.K.S.               Natural Resources, which holds exploration rights in the CCZ.
conceived the study and led the benthic chamber lander
investigations with A.J.S. A.K.S., A.J.S., D.S.W.d.J., C.A., P.S. and       Additional information
J.J.M. conducted the Winkler analysis and ex situ core incubations.         Extended data is available for this paper at
A.K.S., A.J.S., D.S.W.d.J. and T.H. carried out the in situ oxygen optode   https://doi.org/10.1038/s41561-024-01480-8.
calibrations and analysis. M.S., P.S. and J.J.M. led the microbiology
analysis, whereas P.S. and R.L.E. undertook the radio­activity              Supplementary information The online version contains supplementary
measurements and radiolysis calculations. A.K., S.F., T.K. and A.K.S.       material available at https://doi.org/10.1038/s41561-024-01480-8.
did the solubility assessments, and F.G. and A.K.S. undertook the
electro­chemistry measurements. A.K.S., J.J.M. and W.B.H. drafted the       Correspondence and requests for materials should be addressed to
paper, and all authors contributed further ideas and approved the           Andrew K. Sweetman.
final version.
                                                                            Peer review information Nature Geoscience thanks Bo Barker
Competing interests                                                         Jørgensen and Kira Mizell for their contribution to the peer
A.K.S., C.W. and W.B.H. received research support (funding) from            review of this work. Primary Handling Editor: Stefan Lachowycz,
The Metals Company, and A.K.S. also received research support               in collaboration with the Nature Geoscience team.
from UK Seabed Resources to carry out part of the work. The Metals
Company and UK Seabed Resources aided in the selection of study             Reprints and permissions information is available at
sites and operational scheduling at sea in a collaborative effort.          www.nature.com/reprints.




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Extended Data Fig. 1 | Benthic chamber lander and multi-corer deployment    (b–d) of NORI-D in the central abyssal Pacific. The deployment location for
locations across the CCZ. Benthic chamber lander (BCL) locations in APEIs   the multi-corer (MUC) that sampled sediments for the ex situ experiments
1, 4, and 7 (western CCZ), UK1 and OMS and NORI-D (stars) (a) and both      conducted during the 5D cruise is also shown (c).
areas (Collector Test Area or CTA and Preservation Reference Zone or PRZ)




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Extended Data Fig. 2 | Oxygen concentrations measured from water                dead-algal biomass during expeditions 5D (A), 5E (E), and 7A (G), DIC + NH4+
samples by Winkler titration during the NORI-D benthic chamber lander           during expedition 5D (B), 0.45-μm filtered seawater during expedition 5D (C),
experiments. Mean O2 concentration (μmol L−1) measured by micro-Winkler         and control (no injection) during expeditions 5D (D), 5E (F), and 7A (H).
analysis conducted on water samples that were collected periodically from the   Each datapoint is the mean of two Winkler measurements.
chambers through time (hr) under different treatments. The treatments were




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Brief Communication                                                                                        https://doi.org/10.1038/s41561-024-01480-8




Extended Data Fig. 3 | Oxygen optode concentrations measured during             carried out at NORI-D. The O2 concentrations recorded by the optodes in the 2015
benthic chamber lander experiments in the UK1 and OMS license areas             and 2018 experiments were derived from factory calibrations undertaken 4–6
and APEIs 1, 4, and 7. Oxygen optode readings through time (hr) from 36-hour    months prior to the expeditions as in-situ temperature could not be replicated
abyssal (4037-5216m) in-situ benthic chamber lander experiments conducted       onboard during the optode calibration process. As such, only relative changes in
in the UK1 and OMS license areas in 2015 and APEIs 1, 4, and 7 in the western   O2 concentrations can be interpreted.
CCZ in June 2018. The experiments that were conducted were identical to those




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Brief Communication                                                                                         https://doi.org/10.1038/s41561-024-01480-8




Extended Data Fig. 4 | Bar chart showing total net O2 production in ex situ      difference in O2 concentration of the water phase overlying the sediment
sediment cores. Mean total net O2 production (μmol O2 core−1) measured on        between t = 0 hours and 48 hours accounting for the core volume. Error bars refer
sediment cores (n=1-3) exposed to a variety of treatments during 48-hr ex situ   to ± 1 standard deviation. Individual fluxes from the ex-situ incubations are also
incubations that were carried out on the ship at in-situ temperature and in      shown as data points overlying the bars.
the dark during the 5D cruise. Oxygen production was determined from the




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Brief Communication                                                                                               https://doi.org/10.1038/s41561-024-01480-8




Extended Data Fig. 5 | Phase stability and solubility of birnessite in seawater        in MUC cores, and the lowest O2 condition (average bottom seawater); the red
as a function of O2 activity and pH. The phase stability and solubility of             point indicates the predominant manganese form at the lowest pH (measured
birnessite (manganese [IV] oxide) in seawater as a function of O2 activity and pH      in MUC cores) and highest O2 concentration measured in the in-situ benthic
at a temperature of 1.6 °C, 0.55M Cl, and 2e−10M Mn. The bold black line illustrates   chamber experiments at NORI-D with the arrows showing their range. Under
the phase boundary between birnessite and dissolved Mn2+; the dashed lines the         the latter conditions, a vanishing small amount of birnessite would dissolve into
solubility of birnessite into seawater. The green point indicates the predominant      seawater to form Mn2+.
manganese form that would be experienced at the highest pH that was measured




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Extended Data Table 1 | In-situ benthic chamber lander deployment locations and depths in NORI-D




In-situ benthic chamber lander deployment locations and depths from the 3 cruises to the NORI-D license area. Deployment areas are shown in Extended Data Fig. 1 and are defined as the
Collector Test Area (CTA) and Preservation Reference Zone (PRZ).




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Brief Communication                                                                                                            https://doi.org/10.1038/s41561-024-01480-8


Extended Data Table 2 | Total net oxygen change measured by O2 optodes during the in-situ benthic chamber lander
experiments at NORI-D




Total net oxygen change measured by O2 optodes during the in-situ benthic chamber lander experiments at NORI-D. Stars (*) denote the incubations where the optodes did not start to log
O2 concentrations immediately or stopped logging before 47 hr meaning the total O2 change could not be determined (designated by a dash [-]). The total net O2 change could also not be
determined in a sub-set of experiments (designated by a dash [-]) because the chambers failed to seal sediments at the end of the experiment, which meant that the volume of water in the
chamber could not be determined (designated NA) once the lander arrived back on deck. DOP flux rates were determined from the change in O2 concentration from the point when the
optodes started logging to t = 47-hr. The determination of net production/ respiration dominated experiments was based on the O2 optode profiles seen in Fig. 1.




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Brief Communication                                                                                                                 https://doi.org/10.1038/s41561-024-01480-8


Extended Data Table 3 | Theoretical diffusion times for thin versus thick-walled bubbles at the seafloor




Theoretical diffusion times (seconds) for thin versus thick-walled bubbles at the seafloor. The theoretical time for O2 to diffuse from a trapped air bubble at the seafloor was estimated
by calculating the size of air bubble that would be required at the surface to produce the O2 increase seen in each chamber. This was estimated from the difference between the max O2
concentration value recorded by the optode and the initial O2 optode reading in the chamber, the volume (L) of the water phase in the chamber and assuming air was comprised of 21% O2.
The size of the bubble at the seafloor was then computed using Boyle’s law and the in-situ pressure calculated from the deployment depth. The O2 concentration gradient (dC) between the
bubble and the water phase of the chamber was determined from the concentration of O2 in the theoretical bubble and the initial concentration of O2 in the chamber. The diffusion distance
(dZ) was set at 10 and 10,000 nm to calculate the diffusion times across a thin versus thick-walled bubble. The diffusion coefficient for O2 (1.064 x 10−5 cm−2 s−1) was calculated from the O2
diffusion coefficient based on an in-situ salinity of 35 and temperature of 1.6oC. Fick’s first law of diffusion was then used to calculate the diffusion time based on the diffusion coefficient,
dZ and dC.




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Brief Communication                                                                                                            https://doi.org/10.1038/s41561-024-01480-8


Extended Data Table 4 | Minimum and maximum voltage potentials (mV) measured on the surface of polymetallic nodules




Minimum and maximum voltage potentials (mV) measured on the surface of the polymetallic nodules (nod.) at 21oC (nodules 1 – 12) and 5oC (nodules 6 and 7 cold), and a control (cont.) rock.
The voltages have not been corrected for the background voltages measured using only instant water.




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