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

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: chlorite dismutation; cryptic O2 cycling; dark oxygen production; hypoxia; nitric oxide dismutation; subsurface microbiome.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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 , 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 , Révész et al. , Wassenaar and Hendry , Smith et al. , Parker et al. , , Ruff et al. 2023). Nearly half of all measurements are best explained by in situ production of isotopically light O₂ (Supplementary Table 3).

Figure 6.

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 , 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. , Lösekann et al. , Aburto et al. , Mills et al. , Ruff et al. , , Stępniewska et al. , , Tavormina et al. , Pytlak et al. , Tiano et al. , Kalvelage et al. , Martinez-Cruz et al. , Padilla et al. , Bhattacharya et al. , He et al. , Mosley et al. , Almog et al. , 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. , Padilla et al. , Cheng et al. , Ruff et al. , , Elbon et al. , 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. , Lichtenberg et al. , Kraft et al. 2022). The figure and legend features select examples, details are provided in the respective section of this review.