Methane is a highly potent greenhouse gas that affects Earth’s climate. Around 70 % of all emissions to the atmosphere derive from biogenic sources10. Biological CH4 formation has long been considered to occur only under strictly anoxic conditions in organisms belonging to the domain Archaea. To generate the cellular fuel ATP, methanogenic archaea convert simple compounds, such as CO2, H2 or acetate, into CH4. This process of methanogenesis depends on reactions that are catalysed by unique sets of enzymes and co-enzymes9. In addition, small amounts of CH4 can be formed via ‘mini-methanogenesis’ in several sulphate-reducing bacteria which contain the enzyme carbon monoxide dehydrogenase11,12. However, during the past 15 years, evidence has been accumulating that other organisms produce CH4 under aerobic conditions. These include both multicellular organisms, such as plants2–4 and saprotrophic fungi5, and unicellular organisms, including marine and freshwater algae6,13 and cyanobacteria8. These organisms generate energy via photosynthesis and/or respiration, and it is unclear why and how they release CH4. Multiple marine and freshwater bacteria harbouring the C-P lyase pathway have been reported to generate CH4 from methylphosphonate14–18. Several bacteria and archaea have also been shown to possess alternative nitrogenases19,20 or nitrogenase-like reductases21, which can produce CH4 and other hydrocarbons. In addition – as we will demonstrate here – living systems can form CH4 without the need for specific enzymes, and such pathways could drive CH4 formation in all cells.
In plants, CH4 formation is enhanced by UV irradiation3,22,23, inhibition of cytochrome c oxidase24 and environmental stressors4,25. This raises the question whether reactive oxygen species (ROS, e.g., hydrogen peroxide H2O2, hydroxyl radicals ·OH and superoxide radicals O2-) might be involved in the formation of CH4. In highly oxidizing environments generated in vitro by a chemical model system containing iron (II/III), hydrogen peroxide (H2O2) and the radical scavenger ascorbic acid, CH4 is readily formed from organosulphur compounds at ambient temperatures26. Under Fenton-type conditions, non-heme oxo-iron(IV) ([FeIV=O]2+) oxidizes methyl sulphides to sulphoxides, which then results in selective formation of methyl radicals by sulphoxide demethylation, and ultimately leads to CH4 and methanol formation depending on oxygen availability26,27. Moreover, ROS such as ·OH and H2O2 react with methyl sulphoxides to produce methyl radicals or peroxomethyl radicals in the presence of oxygen28, subsequently resulting in generation of CH4 or oxidized C1 species.
Fenton chemistry takes place in living cells, as iron is an essential trace element29 and H2O2 is produced during metabolism30. Hydrogen peroxide and Fe2+ react to either ferric iron (Fe3+), OH- and ·OH radicals, or alternatively [FeIV=O]2+ and water31. This therefore raises the possibility that non-enzymatic CH4 formation occurs in cells under oxic conditions at ambient temperatures. Suitable methyl donors for CH4 formation could, in principle, be derived from a wide spectrum of molecules containing sulphur- and/or nitrogen-bonded methyl groups, which are endogenously produced during cellular metabolism and exogenously secreted during natural product formation. Such methylated sulphur compounds include methionine, dimethylsulphoniopropionate (DMSP), dimethyl sulphide (DMS) and dimethyl sulphoxide (DMSO), which are ubiquitous in the environment32. Thus, we tested the hypothesis that a non-enzymatic pathway of CH4 formation exists in all cells, which is based on interactions between ROS, iron and methyl donors (Fig. 1A).
Formation of methane by Bacillus subtilis
We first explored CH4 formation in the Gram-positive model bacterium Bacillus subtilis which, to the best of our knowledge, is not known to release CH4. To detect authentic CH4 formation, we performed stable carbon isotope labelling experiments, using sterile media as baseline controls, and measured CH4 in the headspace of atmospherically sealed bacterial cultures (Fig. 1B). We first investigated whether CH4 could be derived from glucose, an abundant carbon source that is readily taken up and metabolized by B. subtilis. Indeed, small but significant amounts of CH4 were formed in bacterial cultures from [13C6]-glucose (0.21 nmol, p < 0.001). Moreover, the corresponding 13C-isotope values (Δδ13C; difference between culture and sterile media) were highly enriched (79 ± 1 ‰). This indicates that glucose metabolism in B. subtilis results in at least one precursor compound that contributes to the formation of CH4. We next investigated whether B. subtilis could utilize methyl sulphide and methyl sulphoxide as exogenous precursor carbon compounds for CH4 formation. In fact, addition of either 13C-labelled DMS or DMSO to bacterial cultures resulted in a clear shift in the isotopic value, demonstrating that the methyl groups of each sulphur compound were converted into CH4 by reduction. Addition of DMSO increased CH4 formation by 8-fold (1.85 nmol, p < 0.001) relative to glucose. Formation of CH4 containing 13C-labelled carbon derived from DMS was also observed. Although in the latter case CH4 amounts were low, and varied from experiment to experiment, Δδ13C values were correlated with the amount of CH4 produced. Taken together, these experiments provide unambiguous evidence for CH4 formation from both exogenous and endogenous carbon compounds by B. subtilis.
Free iron and oxidative stress promote CH4 formation
We further tested our hypothesis by varying the availability of suitable methyl donors, free iron and the level of oxidative stress, respectively (Fig. 1C). To minimize the effects of biomass variation on CH4 formation, we used the same stationary-phase culture in a minimal medium and systematically varied the reaction conditions for CH4 formation by supplying the substrate DMSO, additional free iron in the form of Fe2+ (as FeSO4) and hypochlorous acid (HOCl) to induce oxidative stress in all possible combinations. The optical density (OD600nm) of stationary cultures changes only marginally if at all (±1%), which implies that the observed variation in CH4 can be attributed to increased CH4 formation by B. subtilis. Under these conditions, external supply of DMSO (S) was required to stimulate significant CH4 formation, as the amounts of CH4 produced in its absence were close to atmospheric levels (Fig. 1C). In low-iron environments, addition of DMSO resulted in a small but significant quantity of CH4 (0.45 nmol ± 0.03 nmol). Supplementation with HOCl to stimulate oxidative stress (S+O) increased this yield by ~40%. Moreover, an iron-rich environment (S+F) boosted CH4 formation from DMSO by 17-fold, while the additional imposition of HOCl-induced oxidative stress (S+F+O) resulted in a 35-fold increase in CH4 levels relative to substrate alone (S). In contrast, heat-inactivated B. subtilis released only marginal amounts of CH4 (0.038 ± 0.006 nmol, p = 0.007) in conditioned media containing DMSO, Fe2+ and HOCl, suggesting that viable but not dead biomass promotes formation of CH4. In accordance with our hypothesis, these data show that CH4 formation from DMSO in stationary-phase B. subtilis cells is enhanced by both free iron and oxidative stress.
CH4 formation is restricted to metabolically active life-cycle stages
According to our model, the generation of H2O2/ROS by living organisms is a prerequisite for CH4 formation in the presence of suitable methyl donors. B. subtilis is a spore-forming organism that cycles between a dormant endospore state and a vegetative cell that grows and divides. Dormant spores are considered to be metabolically inactive, but resume metabolism upon germination33. DMSO readily enters dormant Bacillus spores and does not interfere with spore revival34. We thus asked whether the ability of B. subtilis to generate CH4 from DMSO is restricted to its metabolically active life-cycle stages (Fig. 2A). To investigate this, we performed stable isotope labelling experiments on dormant and germinated B. subtilis spores using 2H-labelled DMSO (Fig. 2B). The use of 2H-labelled compounds and measurements of δ2H values increases the detectability of CH4 formation by orders of magnitude in comparison to 13C labelling. Despite the exquisite sensitivity of the assay, CH4 release from dormant spores after 10 h of incubation with DMSO was within the margin of error (0.02 nmol, p = 0.066), as were the stable hydrogen isotope signatures (Δδ2H = 10 ± 9 ‰). In contrast, when spores were germinated by supplying the nutrient germinant mixture AGFK, CH4 formation was clearly detected (0.1 nmol, P = 0.019). Moreover, the stable hydrogen isotope signatures increased by three orders of magnitude, unambiguously confirming the conversion of the 2H-labelled methyl group of 2H-DMSO into CH4 (Fig. 2B). Note that, under our experimental conditions, AGFK-induced germination is evidenced by the loss of spore refractivity; however, these spores did not grow out (Fig. 2B, insets). These data therefore suggest that germinated and thus metabolically active – but not dormant – spores can generate CH4 from DMSO, in accordance with our hypothesis.
CH4 accumulates during growth under oxic conditions
According to our model, CH4 is mainly formed under oxic conditions. Since B. subtilis is a facultative anaerobe35, we followed the dynamics of CH4 accumulation during growth in complex media while monitoring the development of oxygen levels in the sample headspace (Fig. 2C). Methane levels increased during the exponential growth phase when oxygen was at atmospheric levels. Coincident with oxygen depletion, population growth slowed down rapidly, while CH4 levels continued to increase for another ~ 2.5 h into stationary phase. Little additional CH4 was formed during later cultivation stages when oxygen levels were low and the OD600nm of the culture gradually declined. This indicates that B. subtilis forms CH4 under oxic conditions, in agreement with our model.
Methane can be generated by organisms from all three domains of life
Finally, we investigated CH4 formation by other cells from different domains of life that were previously not known to release CH4. Escherichia coli DH5α served as a model organism for Gram-negative bacteria and Corynebacterium glutamicum ATCC 13032 for Gram-positive bacteria. The yeast Saccharomyces cerevisiae S288C and the mold Aspergillus niger DSM 821 served as fungal models. Human HEK293T cells were used as the model system for mammalian cells in particular and animals in general (Fig. 3A).
All microorganisms formed CH4, and levels were enhanced under oxidative stress induced by the addition of HOCl (Fig. 3B). Cellular CH4 formation by bacteria and fungi exceeded the respective (sterile) media controls by factors from ~1.1 minimum (non-stressed S. cerevisiae) to ~423 maximum (HOCl-stressed A. niger). We also observed CH4 formation in HEK293T cells (Fig. 3C). Comparing culture and corresponding sterile media, CH4 was formed by cultures supplemented with unlabelled DMSO (0.65 nmol, P = 0.064) or 2H-DMSO (1.07 nmol, P = 0.004). Furthermore, the stable hydrogen isotope signatures of 2H-labelled cultures clearly indicated that CH4 is also formed from DMSO by mammalian HEK293T cells. Together with the well-established role of Archaea in producing CH4, we conclude that organisms from all three domains of life release CH4.