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. 2024 Sep 26;100(11):fiae132. doi: 10.1093/femsec/fiae132

Widespread occurrence of dissolved oxygen anomalies, aerobic microbes, and oxygen-producing metabolic pathways in apparently anoxic environments

S Emil Ruff 1,✉, Laura Schwab 2, Emeline Vidal 3, Jordon D Hemingway 4, Beate Kraft 5, Ranjani Murali 6
PMCID: PMC11549561  PMID: 39327011

Abstract

Nearly all molecular oxygen (O2) on Earth is produced via oxygenic photosynthesis by plants or photosynthetically active microorganisms. Light-independent O2 production, which occurs both abiotically, e.g. through water radiolysis, or biotically, e.g. through the dismutation of nitric oxide or chlorite, has been thought to be negligible to the Earth system. However, recent work indicates that O2 is produced and consumed in dark and apparently anoxic environments at a much larger scale than assumed. Studies have shown that isotopically light O2 can accumulate in old groundwaters, that strictly aerobic microorganisms are present in many apparently anoxic habitats, and that microbes and metabolisms that can produce O2 without light are widespread and abundant in diverse ecosystems. Analysis of published metagenomic data reveals that the enzyme putatively capable of nitric oxide dismutation forms four major phylogenetic clusters and occurs in at least 16 bacterial phyla, most notably the Bacteroidota. Similarly, a re-analysis of published isotopic signatures of dissolved O2 in groundwater suggests in situ production in up to half of the studied environments. Geochemical and microbiological data support the conclusion that “dark oxygen production" is an important and widespread yet overlooked process in apparently anoxic environments with far-reaching implications for subsurface biogeochemistry and ecology.

Keywords: dark oxygen production, chlorite dismutation, nitric oxide dismutation, subsurface microbiome, hypoxia, cryptic O2 cycling

Geochemical and microbiological data suggest that molecular oxygen is produced by microbes in dark ecosystems, revealing a widespread yet overlooked process with important implications for subsurface biogeochemistry and ecology.

Principles of light-independent oxygen production and consumption

By far the most important source of molecular oxygen (O2) on Earth is photosynthesis, a biotic process that generates O2 as a byproduct through the lysis of water molecules (Nelson and Ben-Shem 2004, Fischer et al. 2016). Despite the quantitative importance of photosynthesis, O2 is additionally produced by light-independent abiotic and biotic reactions. Here, we refer to all light-independent production pathways, whether biotic or abiotic, as “dark oxygen production" (DOP; Fig. 1).

Figure 1.

Figure 1.

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Processes involved in the production, recycling, and consumption of O2 in modern environments. Reactions responsible for net O2 production (reactions 1–4) are shown above, and those that recycle or consume O2 (reactions 5–9) are below the dotted line. Abiotic reactions are shown to the left and biotic reactions to the right of the dashed lines. Note: intermediate reactions and electrons are not shown. This overview highlights the reactions reviewed here and is not exhaustive, e.g. most abiotic and biotic O2-consuming redox reactions are not shown.

Abiotic DOP specifically proceeds via the radiolysis of water (Gutsalo 1971, Das 2013), which is driven in dark geological systems such as aquifers and bedrock by the decay of radioactive elements present in surrounding rock, and via the consumption of surface-bound radicals on Si-bearing minerals such as quartz (He et al. 2021, 2023, Stone et al. 2022). Although direct O2 formation is likely negligible (Le Caër 2011), both pathways produce abundant reactive oxygen species (ROS) such as hydroxyl radical (OH•), superoxide (O2•−), and hydrogen peroxide (H2O2). ROS can be subsequently disproportionated biotically by the enzymes superoxide dismutase and catalase—or abiotically by ferrous iron and other reduced metals—to form O2 and H2O (Xu et al. 2013, Sutherland et al. 2022). However, surface-bound radical generation requires either intense mechanical abrasion (He et al. 2021, 2023) or hydrothermal temperatures (Stone et al. 2022). Thus, while possibly important earlier in Earth’s history, this mechanism is likely insignificant in most low-temperature, anoxic systems today. In contrast, water radiolysis followed by ROS disproportionation can act as a net source of O2 to groundwater systems over geologic timescales (i.e. the timescale of radioactive decay). It was recently proposed that molecular oxygen may also be produced via the electrolysis of seawater at polymetallic nodules in the deep ocean, yet the details of this reaction are so far unknown (Sweetman et al. 2024).

Biotic DOP is carried out by microorganisms belonging to several different and generally well-known lineages within the Archaea and Bacteria. Microbial DOP proceeds via three fundamentally different microbial processes: chlorite (ClO2−) dismutation (Xu and Logan 2003), nitric oxide (NO) dismutation (Ettwig et al. 2012), and the lysis of water via methanobactins (Dershwitz et al. 2021). Despite this variety of microbial metabolisms producing O2, these processes were all reported relatively recently: chlorite dismutation in the mid-1990s (van Ginkel et al. 1996), NO dismutation in 2010 (Ettwig et al. 2010), and water lysis by methanobactins only three years ago (Dershwitz et al. 2021). In both dismutation pathways, the crucial O2-producing step is the electron-neutral dismutation of ClO2− or NO into O2 and chloride (Cl−) or O2 and dinitrogen gas (N2) or nitrous oxide (N2O). Chlorite dismutase (CLD) and proposed nitric oxide dismutase (NOD) enzymes are both heme-containing oxidoreductases (Hofbauer et al. 2014, Murali et al. 2022), yet they are unrelated belonging to distant protein families. In contrast, the molecular mechanism for methanobactin-dependent lysis of water remains unclear to date (Dershwitz et al. 2021), and, unlike for both dismutation pathways, the boundary conditions for the energetic feasibility of methanobactin-dependent water lysis do not suggest a substantial occurrence in natural ecosystems.

Balancing the O2 sources, the most important sink for O2 is respiration. The reduction of O2 provides the largest free energy release per electron transfer, with the exception of fluorine and chlorine, and is thus a powerful electron acceptor (Catling et al. 2005, Jørgensen 2006). Aerobic respiration is therefore widespread in the tree of life. Many single-celled eukaryotes (Zimorski et al. 2019), ∼70% of bacteria (Morris and Schmidt 2013), and essentially all macroscopic life forms respire O2 (Hedges et al. 2004). However, in recent years, it is becoming increasingly recognized that many aerobes occur in ecosystems in which O2 does not seem to be available or at least does not accumulate. For example, strictly aerobic methanotrophs are reported to be active in apparently anoxic sediments of hydrocarbon seeps (Ruff 2020) and wetlands (Reis et al. 2024). The presence of aerobes in such environments has historically been explained either by (i) their capability to exist in a facultative anaerobic lifestyle, (ii) their capability to remain dormant during unfavorable conditions, or (iii) using small amounts of oxygen that become available through advection, storage, or diffusion, or that are produced via photosynthesis. Indeed, recently developed dissolved O2 sensors reveal that nanomolar (i.e. ∼10−9 mol l−1) concentrations of O2 are common in nature, leading to the presence of nanaerobic microbes in many apparently anoxic environments (Berg et al. 2022). In this review, we compile and provide evidence for a fourth, largely overlooked mechanism by which aerobic microbes can survive in apparently anoxic environments even when sunlight is not available, advection is not present, and diffusion from oxic systems is too slow: in situ DOP.

Biochemistry and physiology of microbial light-independent oxygen production

Of the enzymes involved in microbial DOP, CLD is the best understood. CLD is a relatively small heme-dependent enzyme (∼120 Da) that consists of two (Celis et al. 2015) or four identical subunits (van Ginkel et al. 1996, Mehboob et al. 2009). It is highly specific for the dismutation of chlorite to chloride and O2 with no other side products (Lee et al. 2008). The organisms capable of chlorite dismutation are specialized on the reductive dissimilation of (per)chlorate into chloride, with chlorite being a strongly oxidative and toxic intermediate (Xu and Logan 2003, Coates and Achenbach 2004). Although being facultative anaerobes, some of these organisms can use the generated O2 for aerobic degradation of organics (Carlström et al. 2015). The details concerning the biochemical properties of CLD and the physiology of (per)chlorate reducers are thoroughly discussed and reviewed elsewhere (Coates and Achenbach 2004, Mlynek et al. 2011, Hofbauer et al. 2014, Schaffner et al. 2015, 2017) and are not the focus of this review.

Less is known about the inner workings of the enzymes involved in NO dismutation because the putative NOD has not been biochemically or structurally characterized. A putative NOD was first postulated as a key enzyme in the nitrite-driven anaerobic oxidation of methane performed by Candidatus Methylomirabilis oxyfera (Ca. M. oxyfera; Ettwig et al. 2010). While living in anoxic ecosystems, these organisms generate oxygen internally from nitrite—a process that involves NOD—then use the generated O2 to oxidize methane via an aerobic metabolic pathway (Wu et al. 2011). It seems clear that the putative NOD evolved from the closely related quinol-dependent nitric oxide reductase (qNOR). Both enzymes belong to the heme-copper oxidoreductase (HCO) superfamily and differ in only few amino acid residues (Ettwig et al. 2012, Murali et al. 2022). NOD seems to have lost a quinol-binding site and thus cannot take up external electrons (Ettwig et al. 2012), indicating its adaptation for a purpose different from its ancestral scaffold.

In pure cultures, O2 generation coupled to N2 or N2O production from nitrite is indicative of NO dismutation. As O2 production and consumption may be tightly coupled, inhibitors of the O2-consuming processes (e.g. respiration and ammonia or methane oxidation) may be required to observe oxygen accumulation (Ettwig et al. 2010, Kraft et al. 2022). In most cases, the ability of microorganisms—including the ammonia-oxidizing archaeon Nitrosopumilus maritimus (Kraft et al. 2022) and Pseudomonas aeruginosa (Lichtenberg et al. 2021)—to produce O2 from nitrite or NO was shown via 15N-isotope labeling combined with low-concentration O2 measurements. However, neither the genome of N. maritimus nor of P. aeruginosa encodes a nod gene, and the enzyme that is responsible for catalyzing NO dismutation in these organisms remains to be discovered. The presence of two distinct pathways to dismutate NO, which involve different key enzymes, indicates that the capability of NO-dismutation evolved independently at least twice.

Several key differences exist in the physiologies of the NO dismutation pathways of Ca. M. oxyfera, N. maritimus, and P. aeruginosa. Firstly, Ca. M. oxyfera consumes all O2 that it produces directly for its own metabolism, whereas O2 accumulates up to concentrations of several hundred nanomoles per liter in N. maritimus cultures (Kraft et al. 2022). Secondly, although both pathways reduce nitrite to NO that is then dismutated, Ca. M. oxyfera dismutates NO to O2 and N2 directly, whereas N. maritimus dismutates NO to O2 and N2O that is subsequently reduced to N2 (Fig. 2). Thirdly, P. aeruginosa cultures produce a short-lived O2 peak that occurs immediately after O2 is depleted through respiration. This peak transiently exceeds O2 concentrations accumulated by N. maritimus. The products, stoichiometry, and responsible enzyme remain to be resolved.

Figure 2.

Figure 2.

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Overview of the different microbial DOP metabolisms. Reported Gibbs free energies (∆G) only refer to the O2-generating step of each process. (1) Chlorite dismutation (Mehboob et al. 2009). (2a) NO dismutation to N2 and O2 (Ettwig et al. 2010). (2b) NO dismutation to N2O and O2 (Kraft et al. 2022). Pcr: perchlorate reductase, Clr: chlorate reductase, Cld: chlorite dismutase, NirS: Fe-nitrite reductase, Nod: NO-dismutase, NirK: Cu-nitrite reductase, unk: unknown.

In summary, while O2 is produced in all NO dismutation pathways, the byproducts differ. One implication is that ammonia-oxidizing archaea (AOA) produce half the amount of O2 per nitrite consumed as compared to Ca. M. oxyfera. In environments in which nitrite is limiting and NO-dismutating microbes compete with other nitrite-consuming microbes, such as denitrifiers, this could constitute a substantial disadvantage. Finally, recent research indicates that the number of potential dismutation substrates discovered to date may not be exhausted. For example, it was suggested that novel Methylomirabilota methanotrophs potentially couple methane oxidation to iodate reduction via the production of O2 from hypoiodous acid (Zhu et al. 2022). Future work is therefore needed to explore additional possible dismutating metabolisms.

Diversity and phylogeny of nod genes and of organisms potentially mediating DOP

Organisms that have the metabolic capabilities to dismutate chlorite or NO are found across the tree of life. For example, it has long been known that CLD is widespread in Pseudomonas sp. (Mehboob et al. 2009), Nitrospira sp. (Maixner et al. 2008), Nitrobacter sp. (Mlynek et al. 2011), Klebsiella sp. (Celis et al. 2015), as well as in all (per)chlorate respiring organisms such as Dechloromonas sp. and Dechlorosoma sp. (Coates et al. 1999, Achenbach et al. 2001). Furthermore, a recent publication showed the presence of cld genes in organisms affiliated with >60 genera within 13 phyla, mostly among Proteobacteria (Barnum and Coates 2023).

The list of organisms that are known to contain a putative nod gene is not quite as extensive as that for cld yet has rapidly expanded in recent years. In particular, there are at least five Candidatus species within the Methylomirabilota that contain nod genes: Ca. M. oxyfera, Ca. M. lanthaniphila, Ca. M. sinica, Ca. M. limnetica, and Ca. M. iodofontis (Ettwig et al. 2010, He et al. 2016, Graf et al. 2018, Versantvoort et al. 2018, Zhu et al. 2022). In addition to Methylomirabilota, nod genes were detected in Sediminibacterium sp., Algoriphagus sp., and Muricauda sp.—all within the phylum Bacteroidota (Ettwig et al. 2012, Murali et al. 2022, Ruff et al. 2023)—as well as in Planctomycetota and Proteobacteria (Hanke et al. 2016, Murali et al. 2022, Elbon et al. 2024, Lv et al. 2024). The alkane-oxidizing Gammaproteobacteria HdN1 contains a putative nod and may use oxygen for the activation of alkanes, although the process and activity of this NOD remain unclear (Zedelius et al. 2011). An even larger diversity of putative nod genes has been found through PCR-based studies and environmental surveys (Zhu et al. 2017, 2019, 2020, Zhang et al. 2018, Hu et al. 2019). Furthermore, nod-like genes have been reported in viral genomes in oceanic oxygen-deficient zones (Gazitúa et al. 2021) as well as in oxygen-depleted sediments colonized by foraminifera (Gomaa et al. 2021).

To expand upon the diversity of microorganisms that contain nod genes, we identified all nod gene sequences within the Genome Taxonomy Database (GTDB; Parks et al. 2022) using a recently published nod-specific hidden Markov Model (HMM; Murali et al. 2024). In doing so, we show that at least 16 phyla and 162 genera contain NOD (Supplementary Table 1). Remarkably, more than half of the identified putative NODs are affiliated with lineages within the phylum Bacteroidota. Our phylogenetic analysis of NOD sequences shows that this protein family—belonging to the larger HCO superfamily—diverges into four subclades, two of which almost exclusively comprise sequences affiliating with Bacteroidota (Fig. 3). All four of these NOD subclades retain amino acids that are conserved across the HCO scaffold—the putative low-spin heme ligand (H358, numbering according to NOD in Ca. M. oxyfera, CBE69502.1), the high-spin heme ligand (H662), and two out of three histidine ligands to the non-heme metal in the active site. In all NOD, one of the histidines that bind a non-heme active site metal in other HCO is replaced by an asparagine (N564) residue (Murali et al. 2022). Despite these similarities, these divergent subclades appear to be robust, sharing no more than 40%–50% sequence similarity between the groups. To assess the biochemical differences that drove diversification of NODs, characterization of homologs from each of these groups will be necessary.

Figure 3.

Figure 3.

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Phylogenetic diversity and taxonomic distribution of NOD. NOD sequences were retrieved from release 214 of the GTDB (Parks et al. 2022) using an HMM, built with curated NOD sequences (Murali et al. 2022), available on GitHub (https://github.com/ranjani-m/HCO). These sequences were aligned using MUSCLE (Edgar 2004) ), and a phylogenetic tree was inferred using IQ-TREE (Minh et al. 2020) with qNOR sequences as outgroup. A substitution model was identified with IQ-TREE’s ModelFinder, and the tree was validated with 1000 ultrafast bootstraps. The tree was visualized using iTOL, and the label background of each leaf on the tree was colored according to the phylum of the bacteria or archaea that contained the NOD (Supplementary Table 1). The phylum-level classification was made according to GTDB taxonomy. The tree appeared to diverge into four clades, each of which is color-coded here. The protein accession ID of each sequence in the tree according to GTDB is available in Supplementary Table 1.

We also supplemented environmental surveys from PCR-based studies (Fig. 4A; Supplementary Table 2) by categorizing the environments in which we identified nod-containing metagenome-assembled genomes (MAGs) in our GTDB analysis (Fig. 4B; Supplementary Table 2). Our analysis shows that nod genes were found in metagenomes from both natural environments such as freshwater and marine sediments, groundwaters, soils, hydrothermal vents, and methane seeps, as well as in man-made environments such as wastewater treatment plants and bioreactors. This supports earlier marker gene-based studies in which nod genes were found in lakes , wetlands, aquifers, soils, oil reservoirs, and wastewater treatment plants (Zhu et al. 2017, 2020, Zhang et al. 2018, Hu et al. 2019). Interestingly, the nod genes occurred in such artificial environments at higher abundances than in natural environments, and there existed no trend between the phylogenetic affiliation of nod genes and their environmental distribution. This most likely reflects the diverse lifestyles of microorganisms capable of nod-mediated DOP. For example, microbes affiliating with Bacteroidota thrive in many anoxic environments ranging from marine sediments to bioreactors. Their presence in these environments is largely consistent with biogeochemical evidence for microbial DOP in these environments.

Figure 4.

Figure 4.

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Environmental distribution of NOD. (A) Map of NOD presence and abundance reported in the literature (Supplementary Table 2). (B) Phylogenetic tree showing NOD sequences colored by the environments in which they were identified as described in the metadata accompanying the BioSample of each MAG from which NOD was recovered (Supplementary Table 2). The four clades identified in the NOD phylogenetic tree in Fig. 3 were colored in the same shades to correlate the distribution of Nod clades in different environments. BTEX: benzene, toluene, and xylenes; WWTP: wastewater treatment plant.

Occurrence of O2-dependent enzymes, metabolisms, and microbes in anoxic environments

In addition to the widespread occurrence of microorganisms that have the metabolic capabilities to produce O2 in the absence of light, there are many records of the presence of O2-consuming, strictly aerobic organisms and metabolic pathways inapparently anoxic environments. Despite not being detectable using traditional methods, O2 is likely produced and consumed in these environments as indicated, e.g. by the occurrence and expression of O2-dependent oxygenases. Below, we outline evidence for active O2 cycling in a non-exhaustive list of select environments.

Marine environments

In the marine environment, gene transcripts of oxygenases and the presence of strict aerobes were reported from oxygen-deficient water bodies. In particular, O2-dependent monooxygenases (Hayashi et al. 2007, Tavormina et al. 2013, Padilla et al. 2017) and aerobic respiration were found in the oxygen-deficient waters off the coast of Namibia, Mexico, and Peru (Tiano et al. 2014, Kalvelage et al. 2015). The presence of O2 in oxygen minimum zones (OMZs) is often attributed to laterally or vertically advected “whiffs,” which is certainly a possible explanation for the prevalence of aerobic microbes. However, the presence of oxygenic phototrophs (Garcia-Robledo et al. 2017) and of microbes expressing nod genes (Padilla et al. 2016, Elbon et al. 2024) in OMZs also indicates the potential for in situ production—with or without light—as an O2 source. Despite this possible importance, such production is difficult to detect directly because trace levels of produced O2 in OMZs would not be expected to accumulate to detectable levels but would likely be consumed immediately (Canfield and Kraft 2022).

Evidence for the activity of aerobes was also found several meters below the seafloor of the permanently anoxic Arabian Sea OMZ (Bhattacharya et al. 2020). These sediments contained obligate aerobes that, upon isolation, only grew with oxygen. Also, Nitrosopumilus sp. and nod genes were present, and oxidase genes were transcribed (Sarkar et al. 2024). In marine seeps and mud volcanoes, aerobic methanotrophs are abundant in methane-rich sediments well below the measurable oxygen penetration depth (Lösekann et al. 2007, Ruff et al. 2013, 2015, 2019). It was speculated that these sediments may be actively oxygenated, e.g. by bioturbation due to seep-associated fauna. Nitrosopumilaceae as well as Methylomirabilota are also widespread and often abundant in deep subseafloor sediments indicating a genetic potential for DOP in environments that have been disconnected from surface processes on geologic time scales (Ruff et al. 2024).

Lake and wetland environments

Aerobic methanotrophs have been detected in anoxic sediments of Lake Constance, Germany (Pester et al. 2004), and of thermokarst Lake Vault, Alaska (Martinez-Cruz et al. 2017), as well as in sediments (up to 70 cm depth) at an active methane seep in Lake Qalluuraq, Alaska (He et al. 2022). Anoxic mesocosms using sediments from these Alaskan sites contained aerobic gammaproteobacterial methanotrophs that dominated methane assimilation, as shown by DNA-based stable isotope probing (Martinez-Cruz et al. 2017, He et al. 2022). In the seep site, the authors speculate that methanotrophy was coupled to iron reduction, yet it is unlikely that the first step of methane oxidation, catalyzed by methane monooxygenases (encoded by e.g. pmoA gene) could occur without O2. Evidence of aerobic methanotrophy has also been detected in the suboxic/upper anoxic zones of Lake Untersee, Antarctica (Brady et al. 2023). Similar observations were made in Lake Zug, Switzerland, where aerobic alpha- and gammaproteobacterial methanotrophs were found to be more abundant in anoxic waters than in the oxycline and oxic waters (Oswald et al. 2016). In a follow-up study, the methane-oxidizing Methylococcales were shown to consume up to 0.2 µM methane per day, under both hypoxic and anoxic conditions (Schorn et al. 2024). These findings are supported by incubation experiments using methanogenic sediments of Lake Kinneret, Israel (Almog et al. 2024). Here, the authors found that Methylococcales made up one-third of the microbial community under hypoxic conditions. Both studies describe potential adaptations of Methylococcales to hypoxia or apparent anoxia, e.g. the metabolic potential for fermentation-based methanotrophy to overcome oxygen limitation (Kalyuzhnaya et al. 2013). However, the initial step in methane oxidation still requires O2 even in this process. In Lake Zug, the anoxic layer also comprises denitrifying methanotrophs affiliating with Methylomirabilota (Graf et al. 2018, Schorn et al. 2024). These organisms that are capable of producing O2, yet have not been shown to leak dissolved oxygen into the environment.

The purple sulfur bacteria Chromatium okenii thriving in Lake Cadagno, Switzerland, aerobically oxidize sulfide in dark and apparently anoxic waters. It was hypothesized that in this case, O2 could derive from transport and convection processes occurring in the lake, allowing microbial populations at the oxic/anoxic interface to be active (Berg et al. 2019). In addition to convection of O2, the occurrence of local microbial DOP could be contemplated. In Lake Lugano, Switzerland, pmoA genes were found in the anoxic, but not in the oxic part of the water column. Here, most of the detected bacterial populations affiliated with Methylobacter sp. and showed maximum activity at the suboxic–anoxic interface and in the deeper anoxic layer (Blees et al. 2014). In the boreal lake Alinen-Mustajärvi, Finland, aerobic methanotrophic Methylobacter and newly described Ca. Methyloumidiphilus alinesnsis were active, and transcripts of pmoA were present in the anoxic hypolimnion and in dark and anaerobic incubations (Rissanen et al. 2018). The addition of nitrate to the incubations stimulated methane oxidation, and the authors speculated that trace amounts of O2 could have diffused through or out of the septa allowing the micro-aerobic methane activation and subsequent coupling to denitrification (Rissanen et al. 2018).

Similar to lakes, Methylobacter sequences have been found to be active in an Arctic wetland, with the highest activity detected in the anoxic and the transition zones (Graef et al. 2011). Further, Methylobacter and Methylococcus sequences were detected in the Zoige wetland, China, both in oxic and anoxic zones. While the abundance of aerobic methanotrophs was highest in the oxic zone, their diversity was highest in the anoxic zone, which indicates diverse aerobic niches that can support distinct populations of aerobes (Yun et al. 2010). Anaerobic continuous cultures inoculated with wetland sediments and fed with methane and nitrous oxide were dominated by aerobic methanotrophic Methylocaldum after 500 days; stable isotope analyses and an increase of methane monooxygenase suggested that aerobic methane oxidation was driven by nitrous oxide consumption (Cheng et al. 2021). The widespread occurrence, activity, and persistence of aerobic methanotrophs in anoxic freshwater habitats were suggested to be caused by diverse mechanisms, including direct electron transfer to metal oxides, alternative electron acceptors, and the presence of intracellular gas vesicles (Reis et al. 2024). This review adds DOP to the list of possible processes that can explain aerobic niches in anoxic wetland habitats.

Groundwater and subsurface environments

AOA, which require O2 for the oxidation of ammonia to hydroxylamine, have been found together with Methylomirabilota in dysoxic aquifers (Mosley et al. 2022). In that study, it was speculated that O2 is provided to AOA by Methylomirabilota via microbial cooperation. Such a “leakage” of dark O2 into the surrounding environment has indeed been observed in a laboratory setup (Kraft et al. 2022). However, the AOA N. maritimus itself was shown to produce and leak O2 in dark and anoxic conditions (Kraft et al. 2022), thus obviating the need to invoke Methylomirabilota as an O2 source. Microbial O2 leakage from chlorite or NO dismutation could also explain the presence of O2 and the observed co-occurrence of strictly aerobic methanotrophic Methylobacter with nod and cld gene-containing organisms in old groundwaters (Ruff et al. 2023). A laboratory experiment using sand-packed microcosms and groundwater as inoculum showed that methane oxidation in the Methylobacter-dominated microcosms was strictly dependent on O2, despite the presence of nitrate, which underlines that O2 is needed for the activation of methane even if the oxidation of methanol can be coupled to denitrification (Kuloyo et al. 2020). Deep aquifer systems can also contain viable aerobic heterotrophs that are indicative of relatively oxidizing environments (Hicks and Fredrickson 1989).

Groundwater environments are often contaminated with hydrocarbons from industrial or military facilities. Interestingly, benzene-contaminated aquifers have been shown to comprise aerobic and denitrifying communities at low- to below-detection-limit O2 concentrations (Aburto et al. 2009). The characterization of benzene-oxidizing and chlorate-reducing enrichment cultures indicated that anaerobic benzene degradation could be bypassed by DOP from chlorate-reducing communities (Weelink et al. 2007). In another study, O2 production was measured during benzene-degradation under nitrate-reducing conditions (Atashgahi et al. 2018). For the toluene-degrading microbe Georgfuchsia toluolica, it was shown that traces of air introduced during sampling are sufficient to supply O2 for the O2-dependent enzymatic steps of aerobic toluene degradation, while the main electron flux and energy generation occurred simultaneously via nitrate reduction (Atashgahi et al. 2021). This shows that very small amounts of O2, e.g. supplied by DOP, can suffice for the initial activation of hydrocarbons, and establish aerobic niches in anoxic environments.

Strictly aerobic methanotrophs have also been found in lignite and coal formations (Mills et al. 2010, Stępniewska et al. 2013, 2014, Pytlak et al. 2014). Similarly, cores from oil sands and coal beds in Alberta have been shown to contain unexpectedly high proportions of aerobic hydrocarbon-degrading bacteria as well as metagenomes with high proportions of genes for enzymes involved in aerobic hydrocarbon metabolisms (An et al. 2013, Ridley and Voordouw 2018). Deep crystalline bedrock was shown to contain strictly aerobic methanotrophs (Kalyuzhnaya et al. 1999, Hirayama et al. 2011, Kietäväinen and Purkamo 2015, Rajala et al. 2015) and water samples from rock fractures of the Deep Mine Microbial Observatory comprised methane monooxygenases and enzymes for oxygen respiration (Momper et al. 2023). Even ancient fluids of Canadian shield bedrock contained viable heterotrophic aerobes that apparently belonged to an autochthonous community (Song et al. 2024). The radiolysis of water is a source of O2 in certain subsurface systems and could explain, e.g. the presence of strict aerobes in sediment and rock layers near a uranium mine (Mills et al. 2010). However, the widespread occurrence of O2-dependent pathways and of dissolved O2 at detectable concentrations in deep subsurface ecosystems merits a closer investigation of other possible processes, e.g. using stable-isotope methods.

Detecting and quantifying DOP

Dissolved oxygen anomalies have been detected in several subsurface ecosystems, including shallow (Ronen et al. 1987) and deep oxygenated groundwater (Winograd and Robertson 1982, Ruff et al. 2023) and fluids trapped within ancient bedrock (Nisson et al. 2023). Despite the detection of O2 in subsurface ecosystems and the widespread occurrence of putative O2-producing enzymes and metabolisms in apparently anoxic environments, confidently quantifying the amount of O2 produced in situ remains challenging, particularly using traditional microbiological tools. Stable oxygen isotope analysis remains the most promising method to date to distinguish atmospherically derived from in situ produced dissolved oxygen, and to quantify their relative proportions. The utility of this approach largely relies on the so-called “Dole effect” (Dole 1936), in which isotope fractionation during oxic respiration leads to atmospheric O2 being enriched in 18O by several tens of permil relative to water (i.e. δ18O = 24‰ for atmospheric O2; δ18O ranging from ∼ −25 to 0‰ for typical meteoric waters and seawater (Sharp et al. 2018, Wostbrock et al. 2020). The major stable-oxygen isotope composition of any oxygen-bearing material is written as

graphic file with name TM0001.gif (1)

where 18R is the 18O/16O ratio and VSMOW is the Vienna Standard Mean Ocean Water international standard. Results are often reported in units of “permil” (‰) by multiplying Equation 1 by 1000. In contrast to respiration, photosynthesis—and possibly DOP—generates O2 with an isotopic composition lighter than that of the atmosphere. Specifically, the δ18O value of photosynthesis-derived O2 resembles that of the parent water from which it formed (Helman et al. 2005). The isotopic composition of DOP-derived O2 is currently unknown because DOP fractionation factors remain unconstrained. However, the detection of dissolved oxygen that is depleted in 18O relative to atmospheric O2 likely implies in situ production, the amount of which is quantitatively related to the difference in δ18O value between atmospheric O2 and measured dissolved oxygen.

Following these principles, several studies have analyzed δ18O values of dissolved oxygen in groundwaters to constrain O2 cycling dynamics (Aggarwal and Dillon 1998, Révész et al. 1999, Wassenaar and Hendry 2007, Smith et al. 2011, Parker et al. 2012, 2014, Ruff et al. 2023); we compiled these results here (Fig. 5A). Authors typically assume that groundwater initially contains dissolved O2 in equilibrium with the atmosphere. If this dissolved O2 subsequently undergoes closed-system consumption either by microbial respiration or by abiotic processes (e.g. Fe(II) or H2S oxidation; Oba and Poulson 2009a, 2009b), then residual dissolved oxygen δ18O values will become progressively higher due to isotopic fractionation (so-called “Rayleigh fractionation”). Such mechanisms successfully explain ≈35% of all groundwater observations compiled here (i.e. values that plot close to the green and blue/orange shaded regions in Fig. 5A).

Figure 5.

Figure 5.

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Groundwater dissolved oxygen isotope plots showing (A) δ18O as a function of dissolved oxygen concentration [DO] and (B) ∆′17O as a function of δ18O (so-called “triple-oxygen isotope plot”). Modern-day atmospheric O2 isotopic composition (Wostbrock et al. 2020) is shown in panel B as an open white circle. Dissolved O2 is assumed to begin in equilibrium with the atmosphere at standard temperature and pressure in fluids between 5°C and 15°C with salinity between 0 and 10 psu (thick black line in panel A; black circle in panel B; Garcia and Gordon 1992, Li et al. 2019). Groundwater dissolved O2 can then decrease in concentration and become isotopically fractionated via consumption by microbial respiration (blue shaded region) or abiotic processes (e.g. Fe(II) or H2S oxidation; orange shaded region). Mixing between inward-diffusing O2 and water that has undergone dissolved O2 consumption will result in isotope values between the green and blue/orange shaded regions. In contrast, mixing with in situ-produced O2 shifts isotopic compositions toward an end member resembling source water (modified by fractionation during the O2 production process). For example, the red arrow indicates in situ production starting from an arbitrary initial point within the respiration array (red diamond) assuming that in situ O2 forms from groundwater with δ18O = −17.5‰ and ∆′17O = 84 ppm, i.e. the average of all measured values compiled here, assuming they fall on the meteoric water line (Sharp et al. 2018) and assuming that O2/H2O fractionation is described by the temperature-dependent equilibrium fractionation factor (Hemingway et al. 2022). Regardless of the exact fractionation factors, in situ production is the only process within this framework that can explain high concentrations of 18O-depleted dissolved O2 (i.e. to the lower-right of the diffusion array). Gray circles in panel A represent 338 measured values from globally distributed groundwaters (Aggarwal and Dillon 1998, Révész et al. 1999, Wassenaar and Hendry 2007, Smith et al. 2011, Parker et al. 2012, 2014, Ruff et al. 2023). Nearly half of all measurements are best explained by in situ production of isotopically light O2 (Supplementary Table 3).

However, many studies also observe 18O-depleted dissolved oxygen in groundwater. This cannot be explained by consumption mechanisms alone, all of which are expected to lead to 18O enrichment (Mader et al. 2017). Rather, these authors have mostly invoked the downward diffusion of atmospheric O2 through the vadose zone to explain 18O-depleted values in shallow aquifers (Smith et al. 2011, Parker et al. 2012, 2014). Diffusion is known to induce isotopic fractionation due to differences in isotopologue-specific diffusion coefficients. Diffusion can thus lead to 18O-depleted dissolved oxygen despite being derived from atmospheric O2, but only in low concentrations (Knox et al. 1992, Li et al. 2019, Cao 2022). As diffusion-derived dissolved oxygen concentrations increase, isotopic compositions approach their equilibrium value of δ18O ≈ 24‰ (i.e. following the green shaded regions in Fig. 5). By considering diffusion in addition to consumption, the percentage of groundwater observations compiled here that can be explained by traditional mechanisms increases to ≈53%. This analysis thus suggests that nearly half of all groundwater dissolved oxygen δ18O values compiled here can only be explained by invoking an additional source of 18O-depleted O2, e.g. by in situ DOP. Such processes—regardless of specific biotic versus abiotic mechanism—will drive groundwater dissolved oxygen to higher concentrations and lower δ18O values (red arrows in Fig. 5).

In addition to canonical 18O measurements, recent analytical advancements have led to increased use of so-called “triple-oxygen isotopes” (written as ∆′17O) to track O2 cycling. The triple-oxygen isotope composition of any oxygen-bearing material is typically written as

graphic file with name TM0002.gif (2)

where 17R is the 17O/16O ratio and θRL is a reference line slope. Here, we let θRL = 0.5305 (Bao et al. 2016). Results are often reported in units of “parts per million” (ppm) by multiplying Equation 2 by 106. Of relevance here is the fact that atmospheric O2 is anomalously depleted in 17O due to mass-independent isotope exchange processes in the stratosphere (Hemingway and Claire 2025). This leads to atmospheric O2 with a ∆′17O value of ≈ −500 ppm. In contrast, seawater and all meteoric fluids fall along a mass-dependent meteoric water line, with ∆′17O ≥ 0 ppm (Sharp et al. 2018). Like in δ18O versus dissolved oxygen concentration space, several processes of interest here will lead to unique fractionation trajectories in a triple-oxygen isotope plot (Fig. 5B). Importantly, only by combining all three measurements—concentration, δ18O, and ∆′17O—can the source and cycling of dissolved oxygen be uniquely constrained. For example, partial closed-system respiration followed by DOP could be falsely interpreted as reflecting diffusion in a concentration versus δ18O plot (intersection of red arrow and green shaded region in Fig. 5A), but these processes can be uniquely separated when including ∆′17O (no intersection of red arrow and green shaded region in Fig. 5B).

Astrobiological relevance of DOP

Atmospheric production of (per)chlorate occurs in Earth’s stratosphere from oxidation of Cl−, ClO−x, or ClOx through ozone or light (Sturchio et al. 2009, Jackson et al. 2010), whereas nitrate is formed through oxidation of nitrogen dioxide gas to nitric acid (Smith et al. 2014). Accumulation of these compounds after deposition on the Earth’s surface correlates with aridity, thus explaining the elevated levels of (per)chlorate in dry conditions such as those of the Atacama Desert (Catling et al. 2010). Evidence exists that both nitrate and (per)chlorate also occur on the lunar surface, on Mars, and within Martian meteorites (Kounaves et al. 2014, Stern et al. 2015, Martin et al. 2020), with detected nitrate levels in Martian sediments (up to 1000 ppm; Stern et al. 2015) reaching those found in the Atacama Desert (Walvoord et al. 2003). Microbial enrichment cultures from this nitrate-rich arid environment have shown microbial growth possibility (Shen et al. 2019), and NO3−/ClO4− reducing microbial populations were identified from desert soil samples that serve as sites analogous to the Martian environment (Cortés et al. 2024). Due to highly conserved ratio of NO3−/ClO4− in non-biologically active areas on Earth, it may be possible to use alterations of this ratio as a biomarker of past biological activity on Mars (Jackson et al. 2015).

Subsurface nitrates on Mars could potentially provide a source of available nitrogen and O2 to support past or present life, which is most likely found in the planet’s subsurface ecosystems (Michalski et al. 2018). Aerobic niches caused by DOP could in theory be present also in dark environments on ocean worlds, as biologically available nitrogen was detected in Enceladus’ plume. Furthermore, Enceladus’ global ocean is suspected to harbor hydrothermal activity (Hsu et al. 2015, Waite et al. 2017). On Earth, hydrothermal systems present available forms of nitrogen and are colonized by a variety of microorganisms involved in the nitrogen cycle (Zeng et al. 2021). Hydrothermal systems additionally drive abiotic O2 generation via the surface-bound radical mechanism (Stone et al. 2022). DOP—whether abiotic or biotic—could therefore be a source of O2 in hydrothermally active, nitrate-rich ocean worlds even if light is not available.

Concluding remarks and outlook

Following a quote from the 1982 paper “Deep Oxygenated Groundwater: Anomaly or Common Occurrence?” (Winograd and Robertson 1982) in which the authors write: “We hope that this report will stimulate a systematic appraisal of DO [dissolved oxygen] in future geochemical studies of shallow and deep ground water,” we would like to conclude this review on similar hopes. Oxygen anomalies have been reported from numerous ecosystems, aerobic organisms are widespread in anoxic environments, and the metabolic capabilities to produce O2 via dismutation of chlorite or NO are nearly ubiquitous (Fig. 6). In contrast to the early 1980s, we now have advanced molecular tools to detect putative nod genes, e.g. through specific oligonucleotide primers for gene amplification (Bhattacharjee et al. 2016, Zhu et al. 2017, Hu et al. 2019), and achieve high-quality long-read whole genome sequencing data even from low-biomass samples and environments (Simon et al. 2023). The widespread availability and utilization of improved sequence-similarity tools that include curated HMMs to detect target proteins will improve the characterization of genes and pathways involved in DOP and allow us to understand their global diversity, abundance, and evolution. Indeed, our analysis of published sequencing data demonstrates the unexpected and striking occurrence of nod genes in the phylum Bacteroidota and suggests a role for Bacteroidota in NOD-associated DOP. Putting together the environmental co-occurrence between hydrocarbon degradation and DOP and the genomic potential in many Bacteroidota to perform hydrocarbon oxidation, we could infer that aerobic hydrocarbon degradation in anoxic environments is one of the major driving forces for the evolutionary selection of DOP well beyond the phylum Methylomirabilota. Future studies looking at the co-expression of DOP and hydrocarbon degradation pathways within apparently anoxic environments by gene transcript sequencing, and protein mass spectrometry will provide insight into potential fluxes of O2 from DOP into various aerobic pathways.

Figure 6.

Figure 6.

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Overview of hypoxic or apparently anoxic ecosystems that have been reported to contain isotopically light dissolved oxygen, comprise strictly aerobic microorganisms, or O2-producing or -consuming metabolic genes and pathways. Traces of O2 were found in groundwaters (e.g. Winograd and Robertson 1982, Ruff et al. 2023) and fracture fluids (e.g. Nisson et al. 2023), while aerobic microorganisms and O2-dependent enzymes were reported from a variety of anoxic systems, such as bedrock, groundwater, marine and freshwater sediments, and hydrocarbon reservoirs. These microbes were associated with oxidation of methane, other hydrocarbons, and ammonia (e.g. Hayashi et al. 2007, Lösekann et al. 2007, Aburto et al. 2009, Mills et al. 2010, Ruff et al. 2013, 2019, Stępniewska et al. 2013, 2014, Tavormina et al. 2013, Pytlak et al. 2014, Tiano et al. 2014, Kalvelage et al. 2015, Martinez-Cruz et al. 2017, Padilla et al. 2017, Bhattacharya et al. 2020, He et al. 2022, Mosley et al. 2022, Almog et al. 2024, Schorn et al. 2024). Expression of nod and cld genes was detected in O2-deficient waters, sediments, groundwater, and wastewater treatment plants (e.g. Bhattacharjee et al. 2016, Padilla et al. 2016, Cheng et al. 2021, Ruff et al. 2023, 2024, Elbon et al. 2024, Sarkar et al. 2024), and DOP was confirmed in P. aeruginosa, Ca. M. oxyfera, and N. maritimus using stable-isotope labeling approaches (Ettwig et al. 2010, Lichtenberg et al. 2021, Kraft et al. 2022). The figure and legend features select examples, details are provided in the respective section of this review.

New observational and experimental tools are becoming routinely available to further constrain DOP. For example, the detection limit for bulk dissolved oxygen concentrations has decreased by several orders of magnitude in the last decade (Tiano et al. 2014, Lehner et al. 2015, Larsen et al. 2016). The measurement of dissolved O2—including its (triple)-oxygen isotopic composition—should become a standard procedure when measuring and monitoring environmental parameters in subsurface ecosystems, along with nitrate, nitrite, (per)chlorate, and chlorite concentration measurements. If shown to be isotopically unique and utilized in biosynthetic pathways, it may even be possible to track DOP-derived oxygen incorporation into biomolecules such as long-chain alcohols (Johnson and Galy 2022). Furthermore, experiments and incubations using isotopically labeled substrates, e.g. 15N18O, can yield valuable insights into dismutation processes, the involved organisms, and metabolic rates. Finally, AI-based protein structure prediction tools (Varadi et al. 2024) and heterologous expression of nod genes and pathways into a recipient cell such as Escherichia coli may shed additional light on the diversity and biochemistry of the enzymes of interest. Such experiments—including those targeting the responsible enzymes—will additionally benefit from continued efforts to culture and isolate the organisms capable of DOP.

Despite the promise of stable oxygen isotopes as a method to track dark-oxygen production, several key uncertainties remain. In particular, the (triple-)oxygen isotope fractionation factors for many processes of interest are unknown. First, only 18O fractionation for abiotic O2 consumption has been determined to date (Oba and Poulson 2009a, 2009b), thus leading to large uncertainty in the triple-oxygen isotope effect of this process. Second, to our knowledge, no fractionation factor measurements currently exist for any in situ DOP mechanism, neither biological metabolisms nor abiotic processes such as radiolysis. Third, O2 produced via DOP may not accumulate to high enough levels to assess isotope fractionation due to consumption by nanaerobic microbes. Future work should aim to directly quantify these fractionation factors using laboratory experiments and culturing studies. Once the fractionation factors have been accurately quantified, then (triple-)oxygen isotope analysis of dissolved oxygen will provide a powerful tool to quantitatively track O2 cycling, including the effect of DOP.

Overall, in this review, we show that DOP is likely an overlooked process of global relevance not only in subsurface ecosystems, but also in many apparently anoxic ecosystems on Earth’s surface. DOP can explain many of the O2-related anomalies and enigmatic occurrences of strict aerobes that have been reported for decades. The production of molecular oxygen in the dark may be crucial for the biogeochemistry, ecology, and evolution of many globally distributed ecosystems.

Supplementary Material

fiae132_Supplemental_Files
fiae132_supplemental_files.zip (85.8KB, zip)

Acknowledgments

We would like to thank the participants of the 2nd Joint Symposium of the International. Societies for Environmental Biogeochemistry & Subsurface Microbiology 2023 for their lively discussions and feedback.

Contributor Information

S Emil Ruff, Marine Biological Laboratory, Ecosystems Center and J Bay Paul Center for Comparative Molecular Biology and Evolution, Woods Hole, MA 02543, United States.

Laura Schwab, Institute of Biodiversity, Aquatic Geomicrobiology, Friedrich Schiller University, 07743 Jena, Germany.

Emeline Vidal, Marine Biological Laboratory, Ecosystems Center and J Bay Paul Center for Comparative Molecular Biology and Evolution, Woods Hole, MA 02543, United States.

Jordon D Hemingway, Geological Institute, Department of Earth and Planetary Sciences, ETH Zurich, Sonneggstrasse 5, 8092 Zurich, Switzerland.

Beate Kraft, Nordcee, Department of Biology, University of Southern Denmark, 5230 Odense, Denmark.

Ranjani Murali, School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV 89119, United States.

Author contributions

S. Emil Ruff (Conceptualization, Investigation, Supervision, Visualization, Writing – original draft, Writing – review & editing), Laura Schwab (Investigation, Visualization, Writing – original draft, Writing – review & editing), Emeline Vidal (Investigation, Visualization, Writing – original draft, Writing – review & editing), Jordon D. Hemingway (Conceptualization, Investigation, Visualization, Writing – original draft, Writing – review & editing), Beate Kraft (Conceptualization, Investigation, Visualization, Writing – original draft, Writing – review & editing), and Ranjani Murali (Conceptualization, Investigation, Visualization, Writing – original draft, Writing – review & editing)

Conflict of interest

None declared.

Funding

S.E.R., J.D.H., and B.K. received funds from the Human Frontier Science Program (RGEC34/2023). This work was supported by a grant from the Simons Foundation [824763, S.E.R.]. This work is part of a project that has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program [946150, J.D.H.].

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