Methane formation driven by light and heat prior to the origin of life and beyond

Ernst, Leonard; Barayeu, Uladzimir; Hädeler, Jonas; Dick, Tobias P.; Klatt, Judith M.; Keppler, Frank; Rebelein, Johannes G.

doi:10.1038/s41467-023-39917-0

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Published: 01 August 2023

Methane formation driven by light and heat prior to the origin of life and beyond

Nature Communications volume 14, Article number: 4364 (2023) Cite this article

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Carbon cycle

Abstract

Methane is a potent greenhouse gas, which likely enabled the evolution of life by keeping the early Earth warm. Here, we demonstrate routes towards abiotic methane and ethane formation under early-earth conditions from methylated sulfur and nitrogen compounds with prebiotic origin. These compounds are demethylated in Fenton reactions governed by ferrous iron and reactive oxygen species (ROS) produced by light and heat in aqueous environments. After the emergence of life, this phenomenon would have greatly intensified in the anoxic Archean by providing methylated sulfur and nitrogen substrates. This ROS-driven Fenton chemistry can occur delocalized from serpentinization across Earth’s humid realm and thereby substantially differs from previously suggested methane formation routes that are spatially restricted. Here, we report that Fenton reactions driven by light and heat release methane and ethane and might have shaped the chemical evolution of the atmosphere prior to the origin of life and beyond.

Introduction

Methane (CH₄) is a potent greenhouse gas which has in the past and is still today contributing to climate change¹. Atmospherically accumulated CH₄ and ethane (C₂H₆) might also explain the “faint young sun paradox”, which describes the apparent contradiction of a fainter sun (70 – 83% of the current solar energy output) but a climate that was at least as warm as today during early Earth (4.5–2.5 Ga ago)^2,3,4. Although these CH₄ levels would be essential to keep the Earth a liquid hydrosphere to allow the evolution of life during the Archean (4.0–2.5 Ga), the source of CH₄ prior to the origin of life is still under debate⁵. While CH₄ was released by submarine volcanism, most CH₄ is suggested to be formed as side product of serpentinization⁵. After the evolution of microbial methanogenesis latest by 3.5 Ga⁶, methanogenesis could have been responsible for a CH₄ flux comparable to today⁷. Thus, methanogenesis is expected to be the main source of CH₄ during the Archean, supported by light carbon isotope values in sedimentary deposits⁸. However, isotope signals can only manifest upon reoxidation and CH₄ itself does not leave much of a signature in the geological record. Thus, the actual CH₄ concentrations and the potential abiotic sources during early Earth remain elusive. Based on mass-independent fractionation of sulfur, at least 20 ppmv CH₄ was present around 2.4 Ga ago⁹. A more recent study analyzing the fractionation of xenon isotopes suggests CH₄ levels of >5000 ppmv around 3.5 Ga ago¹⁰. Catling et al. expect even higher CH₄ levels at the beginning of the Archean (4 Ga)³ before methanogenesis evolved. Yet, the processes responsible for these high CH₄ levels and their relative contributions remain controversial.

Recently, we discovered a non-enzymatic CH₄ formation mechanism expected to occur in all living organisms¹¹. The mechanism has been demonstrated to be active in over 30 very diverse organisms¹¹ and suggested to explain previously observed CH₄ formation by cyanobacteria¹², freshwater and marine algae^13,14, saprotrophic fungi¹⁵ and plants¹⁶. The CH₄ formation is driven by a cascade of radical reactions, governed by the interplay of reactive oxygen species (ROS) and ferrous iron (Fe²⁺), methylated sulfur (S)- and nitrogen (N)-compounds are oxidatively demethylated by hydroxyl radicals (∙OH) and oxo-iron(IV) complexes ([Fe^IV=O]²⁺) to yield methyl radicals (·CH₃)¹¹.

Here we show that this abiotic mechanism occurs also outside living cells and might have contributed to CH₄ levels before life emerged. All needed components: (i) methylated S- and N-compounds, (ii) Fe²⁺ and (iii) ROS are found under early-earth conditions. (i) In a prebiotic world, methylated S-compounds like methanethiol, dimethyl sulfide (DMS) or dimethyl sulfoxide (DMSO) were formed abiotically under the reducing conditions of hydrothermal vents^17,18,19 or transported to Earth by carbonaceous meteorites during early Earth meteorite bombardment^20,21. Upon the emergence of life, more methylated S-/N-compounds were produced by cells and organisms, i.e. methionine, dimethylsulfoniopropionate or trimethylamine²². (ii) Under the anoxic conditions of the early Earth, oceans were rather ferruginous, i.e. rich in Fe²⁺ required for Fenton chemistry^23,24, nonetheless ferric iron (Fe³⁺) also occurred in Archean seawater²⁵. Additionally, the mechanism driven by Fe²⁺ can be enhanced by Fenton-promoting Fe²⁺-chelators, e.g. ATP or citrate²⁶. Under anoxic conditions, Fe(III)-carboxylate complexes are photochemically reduced via ligand-to-metal charge transfer (LMCT)²⁷, resulting in Fe²⁺ and organic radicals²⁸. (iii) Under ambient temperatures, low ROS levels exist in water that increase with heat²⁹, or can be generated by photolysis or radiolysis^30,31,32,33. Under acidic conditions, i.e. in volcanic lakes³⁴, illumination of Fe(III)-aqua complexes ([Fe(H₂O)₆]³⁺) forms Fe²⁺ and ROS^35,36. Thus, we hypothesized that the Fenton reaction of Fe²⁺ with H₂O₂, generated by heat and light, could have driven the formation of CH₄ from methylated S-/N-compounds independent of temperatures and pressures occurring at hydrothermal vents but at ambient conditions as early as the prebiotic world of the Hadean (4.5–4.0 Ga, Fig. 1a). To identify critical components of such a mechanism, we used aqueous model systems to determine the influence of heat, light, and (bio)molecules on CH₄ formation in abiotic and biotic environments.

**Fig. 1: Heat and light drive CH₄ formation under abiotic conditions.**

Results

Methane is formed under abiotic conditions

To investigate CH₄ formation under abiotic conditions (Fig. 1a), we designed a chemical model system consisting of a nitrogen atmosphere, a potassium phosphate-buffered solution (pH 7, expected during the Archean at 4.0 Ga³⁷) supplemented with Fe²⁺ and the abiotically formed DMSO which serves as methyl donor for ROS-driven CH₄ formation. Over the course of the experiments, no pH change was observed, while low amounts of Fe(OH)₂ precipitated. In this model system, CH₄ was consistently formed from DMSO in the dark (Fig. 1b). CH₄ formation rates increased with rising temperatures from 30 to 97 °C, consistent with the previously reported temperature-dependency of ROS levels in water²⁹. While only marginal CH₄ formation rates derived from DMSO were observed at 30 °C (~0.02 μM h⁻¹), rates increased 41-fold to ~0.82 μM h⁻¹ at 97 °C. In addition, low C₂H₆ amounts were formed (Supplementary Fig. 1), most likely resulting from the recombination of two methyl radicals²³. At 37 °C, the CH₄:C₂H₆ ratio was ~110, with an increasing trend towards higher temperatures. As the ROS-driven CH₄:C₂H₆ ratios are substantially lower than those observed for archaeal methanogenesis³⁸, the CH₄:C₂H₆ ratios could serve as indicator to distinguish microbial from abiotic processes.

Light enhanced the abiotic CH₄ formation rates (Fig. 1c) by photolysis of water and generation of H₂O₂ at 30 °C (Supplementary Fig. 2). Notably, CH₄ amounts increased ~4-fold from ~0.02 μM h⁻¹ to ~0.08 μM h⁻¹ upon broad-spectrum illumination (~ 350 nm < λ < ~1010 nm at 82 ± 4 µmol photons m⁻²s⁻¹, Supplementary Fig. 3). This data provides evidence that light-driven CH₄ formation from methylated S-compounds can occur even in the absence of biomolecules. The addition of oxygen to the samples stopped the formation of CH₄ in this pH-neutral model system supplemented with Fe³⁺ (Fig. 1c). In contrast, under acidic (pH 3), illuminated conditions CH₄ formation rates increased ~5-fold upon Fe³⁺-supplementation in comparison to Fe²⁺-addition, indicating light-driven ROS and Fe²⁺ formation from [Fe(H₂O)₆]³⁺ complexes (Fig. 1d)³⁵. Upon supplementation of 1 mM Fe³⁺ and 1 mM Fe²⁺, keeping the overall iron concentration unchanged at 2 mM, CH₄ formation rates increased to ~0.33 μM h⁻¹. This 5-fold rate increase is driven by both ROS-inducing Fe³⁺ and Fenton-driving Fe²⁺. Under pH-neutral conditions, mixing Fe²⁺ and Fe³⁺ only increased CH₄ formation rates by ~1.3-fold in comparison to Fe²⁺-supplemented samples, while only trace amounts of CH₄ were obtained from Fe³⁺-supplemented samples (Supplementary Fig. 4). Thus, illuminated [Fe(H₂O)₆]³⁺ complexes generate both Fe²⁺ and ROS, thereby contributing to the ROS-driven CH₄ formation under acidic conditions.

Taken together, we demonstrated that heat and light drive the formation of CH₄ and C₂H₆ in an anoxic, abiotic environment under ambient temperatures and pressures. These results establish a ROS-driven mechanism based on Fenton chemistry that can occur delocalized from serpentinization across Earth’s humid realm and thereby substantially differs from previously suggested mechanisms that are spatially restricted. Thus, this non-enzymatic hydrocarbon formation mechanism could have released CH₄ and C₂H₆ into the atmosphere of the Hadean and Archean. Besides CH₄, C₂H₆ is considered an important factor in keeping the early Earth warm, since C₂H₆ absorbs from 11 to 13 µm in an atmospheric window (roughly 8–13 µm) where H₂O and CO₂ do not absorb strongly². Together, the hydrocarbons produced by these pathways might offer a solution to the “faint young sun paradox”^3,4.

(Bio)molecules enhance the heat-driven CH₄ formation

Even before life emerged, several metabolites, e.g. citrate and malate, could have been formed via an ancient, non-enzymatic TCA cycle predecessor driven by ROS^39,40. Catalyzed by iron particles, the formation of pyruvate from CO₂ was recently reported⁴¹. Intriguingly, citrate and malate, as well as other primordial (bio)molecules with a putative prebiotic origin, including ATP⁴² or serine⁴³, have been reported to act as Fenton-promoting Fe²⁺-chelators²⁶. We therefore investigated if these hydroxylated and carboxylated (bio)molecules enhance the ROS-driven CH₄ formation rates (Fig. 2a).

**Fig. 2: (Bio)molecules enhance heat-driven CH₄ formation.**

Indeed, the addition of pyruvate, glucose, serine, ATP, malate or citrate to the heat-driven (97 °C) model system increased the abiotic CH₄ formation rate, e.g. more than 11-fold for citrate (Fig. 2b). Corresponding C₂H₆ rates significantly increased for glucose, serine, malate and citrate, resulting in CH₄:C₂H₆ ratios between ~190 (glucose) and ~1100 (citrate, Fig. 2b). To test if these enhancing effects were indeed driven by Fe²⁺ chelation, we supplemented the assays with the Fe²⁺-competitor Ca²⁺ (Fig. 2c). Since (bio)molecules like citrate can alternatively chelate Ca²⁺ ions, we expected that increasing Ca²⁺ concentrations result in decreasing CH₄ formation rates by replacing Fenton-promoting Fe²⁺-citrate complexes with Ca²⁺-citrate complexes. Upon addition of 10 mM and 500 mM Ca²⁺, CH₄ formation rates significantly decreased from ~6.32 μM h⁻¹ to ~4.86 μM h⁻¹ and ~1.29 μM h⁻¹, respectively. Thus, 500 mM Ca²⁺ suppressed ~90% of the Fenton-promoting effect of citrate supplementation. The Ca²⁺ concentration-dependent decrease of the heat-driven CH₄ formation rate supports the role of citrate as a Fenton-promoting Fe²⁺-chelator, which is further indicated by citrate dissolving any ferruginous precipitate.

Together, ROS generated by heat interact with iron and thereby drive the formation of methyl radicals from S-/N-methylated compounds, resulting in CH₄ and C₂H₆. Moreover, several hydroxylated or carboxylated (bio)molecules with a putative prebiotic origin were shown to act as Fenton-promoting Fe²⁺-chelators, indicating that ROS-driven CH₄ formation may have already been widespread within the timeframe of the transition from prebiotic chemistry to the origin of life. The rise of life would have fostered the abiotic, non-enzymatic CH₄ formation due to the consequential formation and release of biomolecules serving as chelators and substrates.

A light-driven iron redox cycle sustains CH₄ formation

During Fenton chemistry, Fe²⁺ is either oxidized to [Fe^IV= O]²⁺ or ferric iron (Fe³⁺). As Fe³⁺ cannot drive Fenton reactions^23,24, CH₄ formation rates decrease with increasing reaction time and increasing concentrations of Fe³⁺. While this effect may have been minor in the ferruginous Archean oceans, Fe³⁺ likely dominated the iron pool in the photic zone of the oceans latest by the rise of photoferrotrophy and was also prevalent in several ecological niches, e.g. volcanic lakes³⁴. The evolution of photosynthesis and the subsequent biological production of O₂ oxidized the majority of the available Fe²⁺ to Fe³⁺. Thus, abiotic ROS-driven CH₄ formation would have been hindered in the sunlit realm by the late Archean in the absence of an iron redox cycle at neutral pH. Intriguingly, besides acting as Fenton-promoting Fe²⁺-chelators²⁶, (bio)molecules like citrate were reported to reduce Fe³⁺ to Fe²⁺ via LMCT under oxic and anoxic conditions²⁷. Therefore, (bio)molecules may have facilitated widespread iron redox cycling, e.g. by forming Fe(III)-carboxylate complexes. Furthermore, previous studies showed that, upon illumination of water hydroxyl radicals (·OH) and hydrogen atoms are generated, forming H₂O₂ and H₂^30,31,32,33. Thus, we hypothesized that light could drive CH₄ formation in the absence of Fe²⁺ by simultaneously (i) generating ROS from water and (ii) reducing Fe³⁺ to Fe²⁺ via LMCT, thereby recycling Fe³⁺ and keeping the Fenton reaction running (Fig. 3).

**Fig. 3: A light-driven iron redox cycle drives and enhances CH₄ formation.**

To verify our hypothesis, we first confirmed light-dependent ROS production in our model system in the absence of substrate, iron and organic ligands by measuring final reaction products of photolysis: H₂ and H₂O₂ (Fig. 3a, b). We measured H₂ production at a rate of ~1.3 nM h⁻¹ in anoxic samples under broad-spectrum illumination but not in samples kept in the dark (Fig. 3a). A continuous formation of H₂O₂ was measured online using microsensors, which confirmed light-dependent production dynamics in pure buffer (Fig. 3b). Via fluorescence-based H₂O₂ endpoint measurements, we found that both iron and DMSO reduced the H₂O₂ concentrations. The decrease in H₂O₂ levels can be attributed to Fenton reactions between H₂O₂, Fe²⁺ and the radical scavenger DMSO (Supplementary Fig. 5).

Building on this, we closely investigated the interplay of LMCT and iron photochemistry on CH₄ formation. For this purpose, we analyzed our chemical model system containing a buffered solution (pH 7), Fe²⁺ or Fe³⁺, DMSO, in the presence or absence of citrate for the formation of CH₄ and the concentration of available Fe²⁺ (Fig. 3c). The influence of the following parameters on the formation of CH₄ was tested: (i) O₂ ( ~ 21% in air), (ii) oxidation state of the supplemented iron species (Fe²⁺ vs. Fe³), (iii) light and (iv) presence/absence of citrate. (i) CH₄ formation rates under anoxic conditions always exceeded rates under oxic conditions. (ii) Without citrate, initial Fe²⁺-supplementation was required to form significant CH₄ levels. (iii) CH₄ formation always increased with light. (iv) Upon citrate addition, CH₄ formation was enhanced in illuminated and anoxic samples containing DMSO and Fe²⁺ or Fe³⁺. Besides elevated CH₄ formation rates, citrate addition also increased the final Fe²⁺ concentrations, e.g. from ~0 mM Fe²⁺ to ~1.5 mM Fe²⁺ in illuminated and anoxic samples.

After determining the influence of the four parameters (i) O₂, (ii) iron (iii) light and (iv) (bio)molecules, we further investigated them individually to gain a better understanding of their contribution and role in the light-driven CH₄ formation.

(i) O₂: The influence of O₂ on LMCT and CH₄ formation was studied in citrate-supplemented samples by adding various amounts of air. Fe²⁺ concentrations and CH₄ formation rates decreased with increasing O₂ levels (Supplementary Fig. 6). In comparison to 0 % O₂, the Fe²⁺ concentration dropped drastically already at 0.2 % O₂ and was ~96 % lower at 2 % O₂, while CH₄ formation rates decreased approximately linearly with the O₂ level. This indicates the presence of a Fe-cycle, in which most LMCT-formed Fe²⁺ is instantly re-oxidized, either by O₂ or Fenton reactions. The balance between these Fe²⁺ sinks depend on O₂ availability and governs CH₄ formation rates. In the presence of O₂, we also detected methanol (CH₃OH) formation rates ranging from ~0.003 μM h⁻¹ (0.2 % O₂) to ~0.07 μM h⁻¹ (21 % O₂). CH₃OH is preferentially formed through the reaction of ·CH₃ with O₂^23,44. Without the addition of O₂, no CH₃OH was detected, indicating anoxic conditions in our standard assays.

(ii) Iron: The role of the LMCT-rate and the corresponding Fe²⁺ availability for CH₄ formation was tested by supplementing the assays with various Fe³⁺ concentrations (Supplementary Fig. 7). At lower Fe³⁺ concentrations, CH₄ formation rates increased steeper than the measured Fe²⁺ concentrations. At high Fe³⁺ concentrations, CH₄ formation rates leveled off, while Fe²⁺ concentrations continued to increase. This indicates that Fe²⁺ is limiting the demethylation rates at low iron concentrations, because it is immediately re-oxidized, while light-dependent ROS production is limiting CH₄ formation at high iron concentrations. Most importantly, these data highlight that a light- and ROS-driven iron cycle can facilitate high rates of CH₄ formation, even in the presence of O₂ and the absence of detectable Fe²⁺, which opens the possibility of widespread abiotic CH₄ production after the great oxidation event as well as in diverse modern habitats. Next, we investigated the role of the alkali metal magnesium (Mg²⁺) due to its high environmental abundance and found that Mg²⁺ does not facilitate CH₄ formation in illuminated buffer containing DMSO and citrate (Supplementary Fig. 8). Upon additional Fe³⁺ supplementation, Mg²⁺ also decreased CH₄ formation rates by replacing Fenton-promoting Fe³⁺-citrate complexes by Mg²⁺-citrate complexes, thereby acting similar to Ca²⁺ that was demonstrated to decrease heat-driven CH₄ formation (Fig. 2c). Besides iron, the transition metals copper, cerium, cobalt, nickel and manganese were reported to drive Fenton chemistry^45,46, resulting in the release of CH₄. Thus, we tested different transition metals in our chemical model system, containing DMSO as substrate and ascorbate as a strong metal reductant^47,48. We observed that copper, cobalt and cerium also enhanced CH₄ formation rates (Fig. 4a). However, the activity of copper, cobalt and cerium was lower than iron. The high activity of iron combined with its ubiquitous abundance in the Precambrian highlights the global distribution and importance of this mechanism.

**Fig. 4: Transition metals, wavelengths and methylated sulfur- and nitrogen compounds mediate light-driven CH₄ formation.**

(iii) Light: It is established that light quality has an important influence on photolysis. Short wavelength light in the ultraviolet spectrum was reported to drive water photolysis and LMCT more efficiently than longer wavelengths⁴⁹. We expected that shorter wavelength light would increase both CH₄ formation rates and Fe²⁺ levels. Indeed, CH₄ formation rates surged from ~0.3 μM h⁻¹ (λ_max = 534 nm) to ~1.23 μM h⁻¹ (λ_max = 388 nm, Fig. 4b) and Fe²⁺ concentrations almost tripled from ~1.3 mM (λ_max = 534 nm) to ~4.2 mM (λ_max = 388 nm). Although the broad-spectrum light had a 1.5-fold higher energy flux (57 ± 2 kJ m⁻² h⁻¹) compared to the 388 nm-LED light (37 ± 2 kJ m⁻² h⁻¹), the CH₄ formation rate under the broad-spectrum light was only half (0.7 μM h⁻¹). Given that the stratospheric ozone layer was absent during the Hadean and Archaean, higher fluxes of short wavelength light (i.e. ultraviolet light), reached aqueous environments and may have further enhanced the ROS-driven CH₄ formation.

(iv) (Bio)molecules: After illumination of Fe³⁺-ligand complexes, one electron is transferred via LMCT from a carboxylated ligand (L¹) to Fe³⁺, an organic radical (·L¹), i.e. citrate radical, is generated. As described in the literature²⁸, we observed the subsequent CO₂ disassembly from citrate radicals (Supplementary Fig. 9). We speculated that the remaining organic radical (·L²) could react with DMSO, resulting in ·CH₃ and the formation of CH₄ (Fig. 3). Since we cannot directly detect organic radicals, we mimicked the proposed reaction in an anoxic model system only containing DMSO and the radical-generating 2,2’-azobis(2-amidinopropane) dihydrochloride (APPH) that readily decomposes into carbon-centered organic radicals at 40 °C (Supplementary Fig. 10). Indeed, we observed CH₄ formation in a mixture of DMSO and APPH, while only trace amounts of CH₄ were observed from either DMSO or AAPH alone, suggesting an organic radical-driven CH₄ formation mechanism. In short, carboxylates like citrate facilitate LMCT, thereby reducing Fe³⁺ to Fe²⁺ and forming organic radicals. Both resulting compounds drive CH₄ formation. Overall, CH₄ can be formed under anoxic conditions via (i) water thermolysis, (ii) water photolysis, (iii) [Fe(H₂O)₆]³⁺ photolysis and (iv) LMCT-induced carbon-centered radicals. Apart from serving as chelators, some (bio)molecules could also serve as substrates for Fenton reactions. Thus, we investigated four S-/N-methylated compounds in the presence of the chelator citrate. Upon illumination, CH₄ was formed from dimethyl sulfide, methionine, 2-methylthioethanol and trimethylamine (Fig. 4c). These observations indicate that ROS-driven CH₄ formation significantly increased after the origin of life by providing biomolecules as chelators and substrates.

Finally, synergistic effects between light and heat were observed (Fig. 3d). For Fe²⁺-supplemented samples, CH₄ rates at 4 °C increased from ~0.056 μM h⁻¹ in the dark over ~0.19 μM h⁻¹ under light to ~0.65 μM h⁻¹ in illuminated samples at 30 °C. For Fe³⁺-supplemented samples, only CH₄ rates below 0.03 μM h⁻¹ were obtained in the dark, while CH₄ formation rates were slightly above Fe²⁺-supplemented samples in the light, again demonstrating the effects of LMCT and LMCT-induced carbon-centered radicals. Thus, the two factors heat and light synergistically combine for a stable and enhanced ROS and CH₄ formation.

Biomass-derived CH₄ with an abiotic isotope fractionation

Considering the impact of (bio)molecules on the LMCT-driven Fenton reaction, organic radical generation and the role of biomolecules as substrates, we expect the discussed mechanisms to have played and still play the most important role in the vicinity of decaying biomass. To demonstrate that CH₄ is indeed formed from dead biomass in the presence of a variety of biomolecules and not just in our well-defined model systems, we conducted deuterium labeling experiments. For this purpose, we grew the bacterium B. subtilis in Luria-Bertani medium supplemented with 10% D₂O and inactivated the cells by sonication and freezing (see Methods).

The obtained dead biomass was supplemented with Fe³⁺ and ascorbate and incubated under broad-spectrum light. Around 40 fmol CH₄ h⁻¹ mg⁻¹ dry weight was obtained from labeled and unlabeled biomass (Fig. 5a). In addition, stable hydrogen isotope values (δ²H) of CH₄ from D₂O-treated biomass showed strong enrichment in deuterium (~5900 ‰) in comparison to unlabeled biomass (~−225 ‰), demonstrating a direct conversion of isotopically labeled biomass to CH₄. This suggests that the availability of biomass, upon the emergence of life, has increased the CH₄ formation by delivering both (i) S-/N-methylated compounds and (ii) Fenton-promoting iron chelators. The presence of CH₄ has been suggested to be crucial for the evolution of life, since it could serve as life´s first carbon source via methanotrophy^50,51,52. Following this line of thought, we could demonstrate that methanotrophic Methylocystis hirsuta grew on CH₄ generated by our light-driven model system, transferred to the headspace of the M. hirsuta culture (Supplementary Fig. 11). In fact, the “last methane-metabolizing ancestor” had likely the genes to perform methanogenesis and anaerobic methane oxidation⁵³, suggesting that, under high CH₄ concentrations, methanotrophy could have emerged prior to methanogenesis.

**Fig. 5: Isotope labeling studies confirm dead biomass as substrate and show an abiotic isotope fractionation for ROS-driven CH₄ formation.**

Finally, we speculated that ROS-driven CH₄ formation leads to different stable carbon isotope values (δ¹³C) compared to biological processes, i.e., methanogenesis. The observed δ¹³C values for CH₄ generated by heat or light were less negative (~−54 ± 1.1‰) compared to the δ¹³C value of the methanogen Methanothermobacter marburgensis (~−59.2 ± 2.3‰, Fig. 5b). While the isotopic fractionation during abiotic ROS-driven CH₄ formation remains to be studied in depth, these results suggest a lower carbon isotope fractionation for ROS-driven CH₄ formation than for enzymatic methanogenesis. Together with the observed CH₄:C₂H₆ ratios, isotopic signatures may therefore serve to differentiate between CH₄ formed enzymatically or abiotically on Earth and extraterrestrial planets.

Discussion

In this work, we demonstrated that the interplay of Fe²⁺ and H₂O₂, generated by heat and light, drives CH₄ and C₂H₆ formation from methylated S-/N-compounds via Fenton chemistry under conditions that were globally prevalent in the Hadean and Archean. As we observed CH₄ formation under suboxic and oxic conditions, these mechanisms could, in principle, also contribute to extant CH₄ emissions from aqueous environments that were recently shown to correlate with light instead of specific enzymatic pathways⁵⁴. The here described pathways allow CH₄ and C₂H₆ formation in many aqueous environments including oceans, lakes, rivers, and ponds, delocalized from restricted hotspots for (bio)molecule formation such as hydrothermal vents or ultramafic rocks, in superficial water layers driven by light and throughout the entire water column driven by heat. After the emergence of life, this phenomenon would have greatly intensified in the anoxic Archaean and the subsequent “boring billion”^55,56. The increasing amounts of biomass provided methylated S-/N-substrates, Fe-chelating biomolecules reducing Fe³⁺ to Fe²⁺ and releasing organic radicals and thus enhance ROS-driven CH₄ formation. Possibly, these reactions facilitated elevated CH₄ and C₂H₆ levels during the Hadean and Archean. These hydrocarbons would have contributed to atmospheric temperatures on Earth and allowed the evolution of life in a liquid hydrosphere which could have influenced the evolution of metabolism by allowing the rise of methanotrophy prior to methanogenesis. This work lays the foundation to explore further the mechanism’s role in shaping the evolution of the atmosphere on Earth and other planets and its influence on the current climate change.

Methods

General assay conditions

Unless otherwise indicated, 4 mL samples were incubated in closed 20 mL glass vials at 30 °C under a pure nitrogen (N₂) atmosphere and subsequently analyzed via gas chromatography (GC).

Heat assays

In total, 500 mM DMSO and 10 mM FeSO₄ were added to 20 mM degassed potassium phosphate buffer (pH 7) in an anaerobic tent. The headspace of the closed vials was then cycled three times with vacuum and N₂. Samples were incubated at 37 °C, 57 °C, 77 °C and 97 °C for 6 h in an incubator in the dark. Optionally, 20 mM citrate, malate, ATP, serine, glucose or pyruvate were also supplemented. Ca²⁺ was added in the form of CaCl₂. Samples were measured within the linear range of CH₄ formation rate via gas chromatography.

Light assays

In total, 500 mM DMSO, 2 mM of either FeCl₃ or FeSO₄ and, optionally, 10 mM citrate were added to 20 mM degassed potassium phosphate buffer (pH 7). Anoxic conditions were generated by drawing vacuum eight times for 1 min and a subsequent filling with N₂. For experiments investigating [Fe(H₂O)₆]³⁺ complexes, samples were incubated under anoxic, acidic conditions (20 mM Tris · HCl buffer, pH 3) and supplemented with 500 mM DMSO and either 2 mM FeCl₃, 2 mM FeSO₄ or 1 mM FeCl₃ and 1 mM FeSO₄, each. For the investigation of transition metals (Fig. 4a), 2 mM cerium (CeNH₄SO₄), manganese (MnSO₄), cobalt (CoNO₃), nickel (NiSO₄), copper (CuCl₂) or iron (FeCl₃) and 10 mM pH-neutral ascorbate were added to 500 mM DMSO and 20 mM potassium phosphate buffer with an incubation for 1 day. The effect of different wavelengths on CH₄ formation (Fig. 4b) was investigated by adding 5 mM FeCl₃, 10 mM citrate and 500 mM DMSO to 20 mM potassium phosphate buffer with an incubation for 1 day. For the determination of substrates for CH₄ formation (Fig. 4c), 500 mM DMS, methionine, 2-methylthioethanol, Trimethylamine N-oxide or DMSO were added to 10 mM FeCl₃ and 100 mM citrate in 20 mM potassium phosphate buffer with an incubation for 3 days. Samples were incubated under air or N₂ in the dark or under constant broad-spectrum illumination from light bulbs (Osram, Superlux, Super E SIL 60; Φ = 82 ± 4 µmol photons m⁻² s⁻¹, H = 52 ± 2 kJ m⁻² h⁻¹; Supplementary Fig. 3) for 1 day. Samples were measured within the linear range of the CH₄ formation via gas chromatography. Specific wavelengths were provided by diodes (H2A1 series, Roithner Lasertechnik, Austria) emitting UV-A, blue, cyan, green, red or near-infrared light (λ_max = 388 nm, Φ = 35 ± 1 µmol photons m⁻² s⁻¹, H = 36 ± 2 kJ m⁻² h⁻¹; λ_max = 436 nm, Φ = 45 ± 1 µmol photons m⁻² s⁻¹, H = 45 ± 1 kJ m⁻² h⁻¹; λ_max = 500 nm, Φ = 64 ± 4 µmol photons m⁻² s⁻¹, H = 55 ± 3 kJ m⁻² h⁻¹; λ_max = 534 nm, Φ = 63 ± 1 µmol photons m⁻² s⁻¹, H = 50 ± 1 kJ m⁻² h⁻¹; λ_max = 675 nm, Φ = 45 ± 4 µmol photons m⁻² s⁻¹, H = 29 ± 3 kJ m⁻² h⁻¹; or λ_max = 868 nm, Φ = 69 ± 7 µmol photons m⁻² s⁻¹, H = 35 ± 3 kJ m⁻² h⁻¹) Light intensity was determined using a fiber optic scalar irradiance microsensor⁵⁷ connected to a spectrometer (USB4000; Ocean Optics, USA) placed in the center of the incubation vials and calibrated using a spherical light probe (Walz) connected to a LI-250A light meter (Li-Cor Biosciences GmbH, Germany)⁵⁸. Concentration of Fe²⁺ was quantified with the colorimetric ferrozine method⁵⁹.

Bacillus subtilis biomass assays

B. subtilis was grown in 500 mL LB media, supplemented with 10 % H₂O or D₂O, grown for 36 h at 37 °C and 180 rpm. The obtained culture was collected by three cycles of centrifugation (10 min, 4743 × g) and resuspended in 35 mL 20 mM potassium phosphate buffer (pH 7) in order to remove the excess D₂O. Biomass was then generated by sonication (4-times, 1 min) and freezing of the samples. Subsequently, 80 mL buffer was supplemented with 10 mL biomass, 20 mM FeCl₃ and 50 mM ascorbic acid, saturated with N₂ for 30 min and incubated in 100 mL closed glass vials under N₂ and constant broad-spectrum illumination for 3 days. The gas headspace was extracted with a syringe and analyzed with regard to CH₄ content and δ²H values.

Methylocystis hirsuta and Methanothermobacter marburgensis cultivation

M. hirsuta growth media contained 0.5 g Na₂HPO₄ · 2H₂O, 0.22 g KH₂PO₄, 1 g KNO₃, 0.4 mg CaCl₂ · 2H₂O, 2 mg MgSO₄ · 7H₂O per liter, supplemented with 5 mg Na₂EDTA, 0.06 mg CuCl₂ · 5H₂O, 2 mg FeSO₄ · 7H₂O, 0.1 mg ZnSO₄ · 7H₂O, 0.03 mg MnCl₄ · 4H₂O, 0.05 mg H₃BO₃, 0.2 mg CoCl₂ · 6H₂O, 0.02 mg NiCl₂ · 6H₂O and 0.03 mg Na₂MoO₄ · 2H₂O per liter. M. hirsuta was cultivated in 100 mL closed glass vials containing 30 mL culture and was incubated at 25 °C and 150 rpm under an air atmosphere. Methane was produced by supplementing 2 L degassed 20 mM potassium phosphate buffer with 1 M DMSO, 25 mM FeSO₄ and 50 mM ascorbic acid, incubating the solution under constant illumination in 1 L flasks and collecting the formed CH₄ with syringes. M. hirsuta cultures were either supplemented with 25 mL light-generated CH₄ or 25 mL pure N₂. M. marburgensis was cultivated as previously described⁶⁰.

Continuous H₂O₂ measurements using microsensors

To visualize H₂O₂ production in the illuminated anoxic model system, an H₂O₂ microsensor was positioned in the solution. The H₂O₂ microsensors were built, calibrated and used as described previously⁶¹. We sealed the vial opening with self-adhesive tape, rigorously bubbled the liquid with N₂ and then adjusted a gentle flow of N₂ through the headspace to minimize oxygen input from the atmosphere. Light was provided from halogen lamps (KL2500, Schott) at an intensity of 1027 µmol photons m⁻² s⁻¹. We did not attempt to calculate light-dependent H₂O₂ production rates due to the open design of the system, which allowed for the exchange of H₂O₂ with the headspace across the water interface.

End-point H₂O₂ measurements

After illumination, 290 µL sample was mixed anaerobically with 9 µL Amplex Ultrared (Thermofisher, A36006, 30 µM final concentration) and 1 µL recombinant APEX2 (0.23 µM final concentration). Fluorescence was then measured with a plate reader (BMG ClarioStar™) at 568 nm excitation / 581 nm emission. A calibration curve was established with H₂O₂ following the same procedure. To prevent O₂-driven H₂O₂ generation while sample preparation, all buffers were saturated with N₂ and the plate reader was kept at a partial oxygen pressure of 0.1% with an atmospheric control unit (Clariostar, BMG). Before sample preparation, all sample components (20 mM potassium phosphate buffer, DMSO, 1 M citrate and 100 mM FeCl₃) were degassed and kept in an anoxic tent overnight.

Quantification of CH₄, C₂H_6, CO₂, and H₂ (GC-FID)

Amounts of formed CH₄, C₂H₆, CO₂ and H₂ were determined via headspace analysis using a PerkinElmer® Clarus®690 GC system (GC–‍FID/TCD) with a custom-made column circuit (ARNL6743). The headspace samples were injected by a TurboMatrixX110 (PerkinElmer Inc, Waltham, USA) autosampler, heating the samples to 45 °C for 15 min prior to injection. The samples were then separated on a HayeSep column (7’ HayeSep N 1/8” Sf; PerkinElmer®), followed by molecular sieve (9’ Molecular Sieve 13×1/8” Sf; PerkinElmer®) kept at 60 °C. Subsequently, the gases were detected with a flame ionization detector (FID, at 250 °C) and a thermal conductivity detector (TCD, at 200 °C). The quantification of CH₄, C₂H₆, CO₂ and H₂ was based on linear standard curves that were derived from measuring varying amounts of these gases.

CH₃OH measurements (GC-FID)

CH₃OH was quantified with a GC-FID (Shimadzu GC-2010 Plus, FID-2010 Plus, 280 °C) containing an AOC 20i autosampler and a ZB-WAXplus (Zebron) column (30 m x ⌀ = 0.25 mm, df, 0.25 µm). A H₂O sample (1 μL) was injected in the split liner (250 °C, split 5,15,50). The temperature program was kept at 35 °C for 5 min and then increased by 50 °C min⁻¹ until 200 °C which was kept for 3 min. Helium served as carrier gas (flow rate: 1.95 ml min⁻¹) and the FID was operated with 400 ml min⁻¹ synthetic air, 40 ml min⁻¹ H₂ and 30 ml min⁻¹ N₂, serving as a makeup gas. For Split 5, a calibration curve (R² = 0.9931) was generated by diluting CH₃OH (99.9% purity), while an R² = 0.9981 for split 15 and an R² = 0.9997 for split 50 was determined.

δ ¹³C stable isotope measurements (GC-C-IRMS)

δ¹³C values of CH₄ were determined by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Aliquots of headspace gas were transferred to an evacuated sample loop (40 mL) and a cryogenic pre-concentration unit to trap CH₄. CH₄ was trapped on HayeSep D, separated from interfering compounds by GC and transferred to the GC-C-IRMS. The system consists of a cryogenic pre-concentration unit directly connected to an HP 6890 N GC (He flow rate: 1.8 mL min⁻¹; Agilent Technologies, Santa Clara, USA) fitted with a GS-Carbonplot capillary column (30 m * 0.32 mm i.d., d_f 1.5 µm; Agilent Technologies) and a PoraPlot capillary column (25 m * 0.25 mm (i.d.), d_f 8 µm; Varian, Lake Forest, USA). The GC flow was coupled using a press-fit connector to a combustion reactor comprised of an oxidation reactor (ceramic tube (Al₂O₃), length 320 mm, inner diameter 0.5 mm, with oxygen-activated Cu/Ni/Pt wires inside; reactor temperature 960 °C) and a GC Combustion III Interface (ThermoQuest Finnigan) to decompose CH₄ into CO₂. ¹³C/¹²C ratios were determined with a Delta^PLUSXL mass spectrometer (ThermoQuest Finnigan, Bremen, Germany). High-purity CO₂ (Messer Griesheim, Frankfurt, Germany) was used as the working monitoring gas. ¹³C/¹²C ratios (δ¹³C values) are expressed in the conventional δ notation in per mil versus VPDB, calculated as:

$$\,\delta {}^{13}C_{{VPDB}}=\left(\frac{{\left(\frac{13C}{12C}\right)}_{{Sample}}}{{\left(\frac{13C}{12C}\right)}_{{Standard}}}\right)-1$$

(1)

δ¹³C values were corrected using three reference standards of high-purity CH₄ with δ¹³C values of –54.5 ± 0.2 ‰ (Isometric Instruments, Victoria, Canada), –66.5 ± 0.2 ‰ (Isometric Instruments) and –42.3 ± 0.2 ‰ (in-house), calibrated against International Atomic Energy Agency and NIST reference substances.

δ²H stable isotope measurements (GC-TC-IRMS)

δ²H values for CH₄ were determined using GC-temperature conversion-isotope ratio mass spectrometry (GC-TC-IRMS). The analytical set-up was the same as the one used for δ¹³C stable isotope measurements except that the He flow rate was changed to 0.6 ml min⁻¹ and, instead of combustion to CO₂ and H₂O, CH₄ was thermolytically converted (at 1450 °C) to hydrogen and carbon. After IRMS measurements, the obtained δ²H values were corrected by using two reference standards of high-purity CH₄ with δ²H values of –149.9‰ ± 0.2‰ (T-iso2, Isometric Instruments) and –190.6‰ ± 0.2‰ (in house). All δ²H values are expressed in the conventional δ notation in per mil versus Vienna Standard Mean Ocean Water (VSMOW), calculated as

$$\,{{{{{\rm{\delta }}}}}}{}^{2}H_{{VSMOW}}=\left(\frac{{\left(\frac{2H}{1H}\right)}_{{Sample}}}{{\left(\frac{2H}{1H}\right)}_{{Standard}}}\right)-1$$

(2)

Statistics

Unless indicated otherwise, all experiments were performed with N = 3 replicates (3 biological replicates). To test for significant differences in CH₄ formation between two samples, single-factor analysis (two-tailed students t test) of variance (ANOVA) was used.

Data availability

All data are available in the main text or the supplementary information. The data generated in this study have been deposited on the Edmond database⁶², the open repository of the Max Planck Society, under https://doi.org/10.17617/3.6X6JXR.

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Acknowledgements

We thank N. Oehlmann, F. Schmidt, H. Addison, A. Lago Maciel, G. Marijan, A. Goldman, K. Guo, F. Arriaza-Gallardo, M. Schneider, C. Hoyer, M. Schroll and M. Greule for practical and theoretical support; I. Bischofs, S. Shima and W. Liesack for providing strains; G. Hochberg, M. Preiner and T. Erb for providing critical comments and feedback. Figures 1A, 2A and 3A, B were created with BioRender.com. This work was supported by German Research Foundation (DFG) grant 446841743 (JGR), SPP2306 (UB, TPD) and KE884 19-1 (FK). J.G.R., L.E. and J.M.K are grateful for generous support from the Max Planck Society. L.E thanks the Friedrich Naumann Foundation for support. J.M.K. is grateful for the support from the state of Hesse and the UMR.

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Authors and Affiliations

Max Planck Institute for Terrestrial Microbiology, 35043, Marburg, Germany
Leonard Ernst & Johannes G. Rebelein
Center for Synthetic Microbiology (SYNMIKRO), 35032, Marburg, Germany
Leonard Ernst, Judith M. Klatt & Johannes G. Rebelein
Division of Redox Regulation, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, 69120, Heidelberg, Germany
Uladzimir Barayeu & Tobias P. Dick
Faculty of Biosciences, Heidelberg University, 69120, Heidelberg, Germany
Uladzimir Barayeu & Tobias P. Dick
Institute of Earth Sciences, Heidelberg University, 69120, Heidelberg, Germany
Jonas Hädeler & Frank Keppler
Microcosm Earth Center, Max Planck Institute for Terrestrial Microbiology & Philipps University Marburg, 35032, Marburg, Germany
Judith M. Klatt
Biogeochemistry Group, Department for Chemistry, Philipps University Marburg, 35032, Marburg, Germany
Judith M. Klatt
Heidelberg Center for the Environment HCE, Heidelberg University, 69120, Heidelberg, Germany
Frank Keppler

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Leonard Ernst
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Contributions

J.G.R. and L.E. conceived the project. J.G.R. supervised and administered the project. J.G.R., F.K., J.M.K., T.D. and L.E. acquired funding. L.E. and J.G.R designed and analyzed the experiments. L.E. performed the experiments. J.M.K. was involved in H₂O₂ microsensor and LED experiments (Figs. 3B, 4B, Supplementary Fig. 3). U.B. measured H₂O₂ formation (Supplementary Figs. 2, 5 and 10). J.H. measured methanol formation (Supplementary Fig. 6). L.E. and J.G.R. conceptualized, visualized and wrote the original draft. J.G.R., LE, FK and JMK edited the draft. All authors read and reviewed the manuscript.

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Ernst, L., Barayeu, U., Hädeler, J. et al. Methane formation driven by light and heat prior to the origin of life and beyond. Nat Commun 14, 4364 (2023). https://doi.org/10.1038/s41467-023-39917-0

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Received: 05 May 2023
Accepted: 03 July 2023
Published: 01 August 2023
DOI: https://doi.org/10.1038/s41467-023-39917-0

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