Methanogenesis in the presence of oxygenic photosynthetic bacteria may contribute to global methane cycle

Domain: earth_science
Published: 2024-07-06
Editorial outcome: major_revision

Abstract
Accumulating evidences are challenging the paradigm that methane in surface water primarily stems from the anaerobic transformation of organic matters. Yet, the contribution of oxygenic photosynthetic bacteria, a dominant species in surface water, to methane production remains unclear. Here we show methanogenesis triggered by the interaction between oxygenic photosynthetic bacteria and anaerobic methanogenic archaea. By introducing cyanobacterium Synechocystis PCC6803 and methanogenic archaea Methanosarcina barkeri with the redox cycling of iron, CH4 production was induced in coculture biofilms through both syntrophic methanogenesis (under anoxic conditions in darkness) and abiotic methanogenesis (under oxic conditions in illumination) during the periodic dark-light cycles. We have further demonstrated CH4 production by other model oxygenic photosynthetic bacteria from various phyla, in conjunction with different anaerobic methanogenic archaea exhibiting diverse energy conservation modes, as well as various common Fe-species. These findings have revealed an unexpected link between oxygenic photosynthesis and methanogenesis and would advance our understanding of photosynthetic bacteria’s ecological role in the global CH4 cycle. Such light-driven methanogenesis may be widely present in nature.

Introduction
Atmospheric methane (CH4), one of the most important greenhouse gases, reached an exceptionally high concentration of 1912 part per billion in 20221,2. This calls for an immediate action to understand and address CH4 emission problems. Freshwater ecosystems such as rivers, streams, lakes, oceans, and wetlands, play a vital role in contributing to the global atmospheric CH4 budget3. It is widely recognized that CH4 in freshwater ecosystems is primarily produced via the transformation of organic matters in anoxic profundal and littoral sediments4,5. Nevertheless, despite the limited exchange between the oxic surface layers of freshwater ecosystems and sediments due to the deep water columns, a prevalent CH4 supersaturation was observed6. This unexpected phenomenon, also known as the methane paradox wherein methane concentrations exceed atmospheric equilibrium values, suggests the existence of a significant CH4 production process that has yet to be defined.

Photosynthetic bacteria hold a dominant presence in the surface layers of freshwater ecosystems and exhibit excellent phototactic motility and versatile metabolic patterns7. Their interaction with other coexisting microorganisms significantly influences the biogeochemical cycle of elements via harnessing solar light as an energy source. The correlation between photosynthetic bacteria and CH4 production under illumination has been reported previously8,9, but the underlying mechanisms are yet to be elucidated. It is likely that an oxygenic photosynthetic bacteria act as photosensitizers, driving the CO2-to-CH4 conversion with anaerobic methanogenic archaea when being cocultured in an anoxic layer10. The role of oxygenic photosynthetic bacteria in the context of CH4 supersaturation is largely unknown. This oversight arises from the traditional belief that methanogenic archaea are highly sensitive to oxygen exposure and can only thrive in highly reduced, anoxic environments11. However, the coexistence of oxygenic photosynthetic bacteria and anaerobic methanogenic archaea occurs in various natural habitats, such as microbial mats, soil crusts, and aerobic epilimnion of an oligotrophic lake12–14. The in situ detection of the close attachment between methanogenic archaea and photosynthetic bacteria in these oxygenated and methane-rich environment, along with the finding that methanogens can survive oxygen exposure15, suggested their potential interactions through direct nutrient exchange or signal transduction16,17. Thus, a comprehensive understanding of photosynthetically regulated CH4 production is of ecological and biogeochemical importance, and will offer valuable insights into global CH4 cycle with implications for climate change.

Here, we demonstrated the methanogenesis involved in the coculture of Cyanobacterium Synechocystis sp. strain PCC6803 (hereafter PCC6803) and Methanosarcina barkeri (hereafter M. b). PCC6803 is a model oxygenic photosynthetic bacterium that can perform solar energy conversion of water and CO2 to carbohydrates and oxygen. In the absence of light, the produced carbohydrates are metabolized to generate CO2 and ATP through a respiratory system, creating an anoxic microenvironment suitable for microbial methanogenesis18. Meanwhile, M. b as a model methanogen was chosen owing to its widespread environmental presence with physiological and metabolic diversity19. It is reported that iron exists in many open water systems and is quantitatively the most important trace metal in photosynthetic bacteria20. Over 99% of the dissolved Fe pool is complexed by organic ligands21. Therefore, Fe-ethylenediaminetetraacetic acid (Fe-EDTA) was selected as a typical iron species in this study due to its stability and solubility in aqueous solutions. The results showed that CH4 production by the interaction of oxygenic photosynthetic bacteria and anaerobic methanogenic archaea was significantly enhanced through the redox cycling of Fe-ethylenediaminetetraacetic acid (Fe-EDTA), involving both syntrophic methanogenesis and abiotic methanogenesis during the periodic dark-light cycles (Fig. 1). Specifically, in darkness, the organics and H2 produced by PCC6803 during dark fermentation were utilized as carbon sources and reducing equivalents by M. b for syntrophic methanogenesis under anoxic conditions. The significantly lowered hydrogen pressure by M. b, in turn, created more thermodynamically favorable conditions for PCC6803. In contrast, in illumination, the photosynthesized organic compounds and intermediate products by PCC6803 served as potential methyl donors (-CH3). Meanwhile, the simultaneously produced O2 stimulated reactive oxygen species (ROS) production by M. b. Along with the Fenton reaction with Fe-EDTA, various methyl donors were oxidized by ROS to form methyl radicals (•CH3) as intermediates that eventually resulted in abiotic methanogenesis under oxic conditions. Further studies indicated that other model oxygenic photosynthetic bacteria and anaerobic methanogenic archaea were also able to conduct this light-driven methanogenesis process. These findings not only unveil an unexpected link between oxygenic photosynthesis and methanogenesis, but also advance our understanding of the ecological role of photosynthetic bacteria in the global CH4 cycle.

Results
Light-driven methanogenesis with PCC6803 and M. b
PCC6803 and M. b were cocultured in a defined medium (Supplementary Table 1), where CO2 served as the only electron acceptor. The visible light LEDs (12±0.6 W m-2) over the wavelength range of 380–800 nm was used as a simulated sunlight source (Supplementary Fig. 1). The experiment was conducted under a light-dark cycle of 4 hours of light and 20 hours of darkness at 35 oC but some tests were performed with a light-dark cycle of 12 h-12 h that simulates a full day. Compared to the pure M. b control that had a negligible CH4 production under the same condition, the coculture of PCC6803 and M. b achieved a higher CH4 yield of 1.0±0.1 µmol. The addition of Fe-EDTA (hereafter PCC6803-M. b-Fe-EDTA) further enhanced the yield to 2.5±0.5 µmol (Fig. 2a), with a linear correlation between the CH4 yield and the Fe-EDTA concentration (Supplementary Fig. 2). The single-factor experiments showed that the removal of any following components: PCC6803, M. b, Fe-EDTA or light, would result in a significant declination of methanogenesis performance, demonstrating the importance of each component in this process (Fig. 2a, Supplementary Fig. 3). Notably, the CH4 yield with PCC6803-M. b-Fe-EDTA increased both under illumination and in darkness (Fig. 2b), different from the light-dependent CH4 production with anoxygenic photosynthetic bacteria and methanogenic archaea that was only activated by light but inhibited in darkness10. This difference might be attributed to the production of O2 by PCC6803 with H2O as electron donors under illumination, thereby stimulating the generation of ROS by M. b. These ROS, in turn, could oxidize organic matters to create potential carbon sources for biotic methanogenesis with M. b in the dark (see detailed discussion below). The CH4 production rate with PCC6803-M. b-Fe-EDTA continuously increased during the three successive cycles (Fig. 2c), contributing to the rapid growth and formation of a stable syntrophic coculture for methanogenesis (Supplementary Fig. 4).

To understand the source of CH4 produced by PCC6803-M. b-Fe-EDTA, we performed isotopic labeling experiments. The selected m/z=45 (13CO2) and 17 (13CH4) were detected when 13C-labeled NaHCO3 was used as the sole carbon source (Fig. 2d). This provided direct evidence that the produced CH4 was derived from the CO2 conversion. Unexpectedly, the relatively weak signals at m/z=44 (12CO2) and 16 (12CH4) were also detected, indicating the existence of other CH4 production pathways that might utilize the carbon sources synthesized during the initial cultivation process, such as biomass and oxidation intermediates (see detailed discussion below). A noticeable periodic variation in the dissolved oxygen (DO) concentration was observed in the coculture during the light-dark cycles (Fig. 2e), likely attributed to the alternate photosynthetic oxygen evolution and respiratory oxygen consumption22. The addition of Fe-EDTA would reduce the oxygen concentration under illumination and expedite the oxygen consumption in darkness, through the oxygen oxidation with Fe(II) chelated by EDTA, resulting in an anaerobic microenvironment for the growth and metabolism of M. b. This hypothesis was confirmed by the increased copy number of mcrA gene, which is ubiquitous in M. b during the methanogenesis with PCC6803-M. b-Fe-EDTA (Fig. 2f), along with a simultaneous rise in the copy number of cpcG that referred to as rod-core linker genes in PCC6803. However, the growth of PCC6803 led to an enhanced photosynthetic oxygen evolution, elevating DO accumulation in the light and extending the time required to establish an anaerobic microenvironment for biotic methanogenesis in darkness (Fig. 2e). As a result, the rate of CH4 production with PCC6803-M. b-Fe-EDTA slowed down somewhat after day 2 but persisted (Fig. 2b).

Syntrophic methanogenesis by organic degradation and CO2 reduction
The close contact between different microorganisms can enhance the interspecies exchange of matter and energy to shape specific communities16. As shown in Fig. 3a, PCC6803 and M. b were connected to form dense PCC6803-M. b biofilm at the bottom of the culture bottles after 10 days of coculturing. Both optical microscopy images (Fig. 3b) and fluorescence in situ hybridization (FISH) images (Fig. 3c) showed the aggregation of PCC6803 and M. b cells in the biofilm. An increase in the biofilm thickness from ∼30 μm on day 0 to ∼45 μm on day 10 was observed by confocal laser scanning microscopy (CLSM) (Supplementary Fig. 5), demonstrating the growth of both microbes in the coculture biofilms.

PCC6803 can synthesize carbohydrates via photosynthesis, and then degrade these carbohydrates through a respiration process in darkness, excreting a variety of organic matters23. These organic matters, along with their intermediates produced via the oxidative degradation by the concomitant reactive oxygen species (ROS), would serve as the potential carbon sources for biotic methanogenesis with M. b (Fig. 3d). Lactate, pyruvate and acetate were detected by the 1H nuclear magnetic resonance (NMR) spectra (Fig. 3e). These findings were further confirmed by two-dimensional NMR spectra, which provide the crucial through-bond correlations existing between the coupled protons [two-dimensional gradient-selected homonuclear correlation spectroscopy (gCOSY) in Fig. 3e] and between protons and carbons via multiple-bond correlations [gradient-selected heteronuclear multiple bond correlation (gHMBC) in Fig. 3f] for each compound24. The 1H and 13C chemical shifts of lactate, pyruvate, methanol, and acetate correspond well to values from known library spectra (PubChem Database), such as the chemical shifts of 1H (1.96 ppm) and 13C (176.5 ppm) in acetate, confirming the presence of these compounds in aqueous solution during syntrophic methanogenesis. Although pyruvate was reported to be the sole carbon and energy source for the growth of M. b25, this species is more efficient in converting acetate to CH4 (CH3COOH→CH4+CO2) due to less energy requirement for acetate activation26, resulting in a lower acetate yield with PCC6803-M. b-Fe-EDTA than that with pure PCC6803 (Fig. 3h).

In addition to organic substances, H2 was also detected in the headspace with PCC6803-M. b-Fe-EDTA (Supplementary Fig. 6). These H2 molecules could originate from the direct (proton reduction with hydrogenase) and indirect (hydrogen release from the carbohydrate degradation) biophotolysis, and serve as electron donors for CH4 production via a hydrogenotrophic pathway (CO2+4H2→CH4+2H2O)27. Compared with pure PCC6803, a lower H2 yield was detected with PCC6803-M. b-Fe-EDTA after 18 days of coculture (Supplementary Fig. 6), which is probably because that H2 produced by PCC6803 was used as electron donors for methanogenesis. To confirm this argument, sodium 2-bromoethanesulfonate (SBES) was added to the coculture medium to inhibit hydrogenases in methanogenic archaea. More H2 residue and a lower CH4 yield indicated that the hydrogenotrophic pathway was existed but inhibited by SBES in PCC6803-M. b-Fe-EDTA (Supplementary Fig. 7). However, it should be noted that PCC6803 contains a single [NiFe]-hydrogenase, HoxEFUYH, which is involved in fermentative hydrogen production as well as working as an electron valve when photosynthesis resumes under anaerobic conditions28. Notably, HoxEFUYH works bidirectionally with a bias to proton reduction rather than hydrogen oxidation29. Therefore, an increase in hydrogen pressure due to hydrogen accumulation during dark fermentation might result in a significant decrease in hydrogenase activity, thereby influencing the growth and metabolism of PCC680330. This inference was confirmed by the lower chlorophyll concentration, quantum yield of PSII primary photochemical reactions (Fv/Fm), and copy number of cpcG in PCC6803, along with a higher H2 concentration compared with syntrophic methanogenesis (Supplementary Fig. 8). The superior activity of PCC6803 during syntrophic methanogenesis were attributed to the versatile metabolic pathways of M. b, which significantly lowered hydrogen pressure via CO2/H2 methanogenesis (Supplementary Fig. 7), and created more thermodynamically favorable conditions for the dark fermentation of PCC6803.

A distinct photocurrent was also observed under illumination during syntrophic methanogenesis (Supplementary Fig. 9a), indicating that PCC6803 can release electrons extracellularly. Although M. b served as electron acceptors capable of accepting photoelectrons from PCC6803 for CO2 reduction (CO2+8e-+8H+→CH4+2H2O)31,32, the syntrophic methanogenesis via a direct interspecies electron transfer (DIET) pathway was less likely to occur with PCC6803-M. b-Fe-EDTA under illumination. This was because that the growth and metabolism of M. b is highly sensitive to the oxygen exposure during the photosynthetic oxygen evolution. Besides the extracellular electron transfer for Fe(III) reduction, the light-induced electrons by PCC6803 would form excited triplet state of chlorophyll in the photosystem II reaction center, which then interacts with molecular oxygen for singlet oxygen (1O2) production33, eventually leading to the production of other ROS for abiotic methanogenesis (see detailed discussion below). In contrast, previous studies have shown that bare photosynthetic microorganisms can also generate an electrical current exclusively in darkness, using illumination as a recharge stage34,35. To validate this, two-chamber H-cells were constructed, with PCC6803 and M. b separately inoculated into each chamber and then electrically connected by an external circuit, to mitigate the influence of direct electron exchange between M. b and PCC6803 on the photoelectron measurement. Similar results were observed that a continuous current was recorded during its dark fermentation period, albeit with lesser intensity compared to that in the light (Supplementary Fig. 9b). This finding indicates the potential DIET between PCC6803 and M. b via an electric syntrophic coculture in darkness.

Electron flow and energy metabolisms at the genetic level
Syntrophic methanogenesis with PCC6803-M. b-Fe-EDTA was further confirmed by transcriptomic analyses (Fig. 4). The transcript levels of PSII and cytochrome b6f complex (cytb6f) exhibited similar oscillation patterns that were highly upregulated during the light period. With plastocyanin and cytc553 in the thylakoid lumen as alternated donors, the produced electrons would further transfer from cytb6f to PSI, resulting a higher expression of genes encoding PSI. Meanwhile, ATP synthase (ATPase) and ferredoxin-NADP+ reductase (FNR) were upregulated during the light period and downregulated during the dark period. The upregulation during the light period was expected, as all transcripts associated with photosynthetic electron transport and ATP synthesis were upregulated during this phase. In addition, most of the CO2 fixation machinery was highly active in the light period. For example, the key genes for the Calvin cycle, such as RubisCO (rbc), phosphoribulokinase (prk) and glyceraldehyde-3-phosphate dehydrogenase (gap)36, were significantly upregulated to ensure the efficient CO2 fixation for producing carbohydrates (Fig. 4, Supplementary Fig. 10). The accumulated carbohydrates, acting as storage molecules, then serve as carbon sources for microbial metabolism. The oxidation of carbohydrates was further confirmed by the upregulated transcript level of genes encoding the tricarboxylic acid (TCA) cycle, such as citrate synthase (glt), isocitrate dehydrogenase (icd), synthetase (suc) and malate dehydrogenase (mdh) (Fig. 4, Supplementary Fig. 11). Likewise, genes related to the biosynthesis of lactate and acetate were significantly upregulated, which could be oxidized by ROS and used as substrates by M. b. Remarkably, the iron uptake and transport (ferric uptake (Fut) and ferrous iron transport (Feo) systems) were also enhanced with higher gene expression, particularly in the light period, thereby facilitating the Fe redox process during syntrophic methanogenesis.

In darkness, genes responsible for the complete pathways for oxidation of organic compounds (i.e., acetate and pyruvate) and CO2 reduction were also highly expressed, suggesting the existence of multiple CH4 production pathways. For instance, the significantly increased activity of pyruvate-ferredoxin oxidoreductase (Por) could oxidize pyruvate to AcCoA and CO2, and the subsequent conversion of AcCoA to CH4 would occur via the native aceticlastic pathway37. Notably, the transcriptional levels of genes encoding functional hydrogenases for H2 production and consumption (e.g., energy-converting [NiFe]-hydrogenase (Ech), methanophenazine-reducing [NiFe]-hydrogenase (Vht) and F420H2 dehydrogenase (Fpo))38 were upregulated, implying that the CO2 reduction was conducted via H2 transfer during the dark period.

Abiotic methanogenesis via the oxidation of methyl donors by ROS
Substantial CH4 production during the light period was observed with PCC6803-M. b-Fe-EDTA (Fig. 2b). However, deletional control experiment revealed that PCC6803 produced almost no CH4 under illumination without the presence of other factors (Supplementary Fig. 3), suggesting the potential existence of an abiotic CH4 production process. ROS are vital cellular metabolic products found in all living organisms. They are responsible for oxidizing the methyl groups (-CH3) of methyl donors and can play a crucial role in modulating chemical CH4 formation (Fig. 5a). Electron paramagnetic resonance (EPR) spectra revealed clear signals of •CH3, •OH, and •O2- from PCC6803-M. b-Fe-EDTA (Fig. 5b, Supplementary Fig. 12). In addition, H2O2 was produced as shown in the ultraviolet–visible (UV–Vis) absorption spectra (Fig. 5c). The concentrations of these ROS, particularly •OH and H2O2, significantly increased under light illumination (Fig. 5d), likely attributed to the produced O2 by PCC6803 during the photosynthetic oxygen evolution that significantly stimulated the ROS production by M. b. This inference was confirmed through the ROS production experiments with M. b under varying O2 concentrations. It was found that higher O2 concentrations led to the production of more ROS, such as •OH and H2O2 (Supplementary Fig. 13). Stable isotope analysis with 18O further confirmed that the produced ROS stemmed from O2 reduction, evidenced by the observed 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-18OH (m/z=132.09, Supplementary Fig. 14). Previous studies have shown that when anaerobic cells were exposed to oxygen-rich environments, molecular O2 adventitiously abstracted electrons from the reduced flavins or metal centers of some redox enzymes, resulting in the ROS formation (O2→•O2-→H2O2→•OH)39–41. As these events relied on collision frequency, the ROS production rate was directly proportional to the O2 concentration42. Subsequently, the produced ROS were inadvertently released extracellularly43. Meanwhile, ROS could also be produced by PCC6803 during the photochemical energy conversion for bioenergetic production44. These ROS could be further used for -CH3 and Fe(II) oxidization. Particularly, the light-driven H2O2 may also interact with EDTA chelated-Fe(II) for •OH production via Fenton reaction (Fe(II)+H2O2→Fe(III)+•OH), leading to an increasing Fe(III) concentration under illumination in the first light-dark cycle (Fig. 5e). The produced Fe(III) through the oxidation of ROS and O2 could be reduced by PCC6803 (Supplementary Fig. 15), either through intracellular metabolism or extracellular electron transfer, and finally established an effective Fe(III)/Fe(II) redox cycle.

EPR spectra also revealed the prominent signals of DMPO/•CH3 (Fig. 5b). The potential sources of methyl donors for •CH3 production with PCC6803-M. b-Fe-EDTA were diverse. On one hand, PCC6803 could synthesized organic compounds containing sulfur-bonded methyl groups, such as pyruvate and ethanol (Fig. 3g). Meanwhile, the existence of DMS was confirmed by gas chromatography-mass spectrometry (GC-MS) with ion signal at m/z=62, along with the retention time of 8.7 min in 400 MHz 1H nuclear magnetic resonance (NMR) spectra (Fig. 5f). The DMS production might be partly attributed to the ROS oxidation of DMSP, because the DMSP was confirmed by the increasing intensity of DMS after alkali treatment for 12 h via the DMSP-to-DMS conversion45. On the other hand, the possible release of the intracellular metabolites by M. b, such as 2-(methylthio)ethanesulfonic acid (CH3-S-CoM), might also serve as potential sources of methyl donors. These methyl donors could be oxidized by ROS for •CH3 formation, ultimately leading to abiotic CH4 production46,47. The role of ROS was further examined by the scavenger trapping tests, which revealed that the methanogenesis process was significantly inhibited after the addition of ROS quenching reagents (Supplementary Fig. 16). The CH4 yield with PCC6803-M. b-Fe-EDTA was decreased from 2.5 µmol to 0.5 µmol with the •OH scavenger of tert-butyl alcohol (TBA, 10 mmol-1). The results were confirmed by previous studies in which abiotic CH4 production occurred with highly reactive •OH generated not only through Fenton reaction48,49 but also through ubiquitous non-Fenton chemistry reactions driven by diversified external fields50. Because •OH is involved in both the organic degradation and -CH3 oxidization, the difference in the CH4 yield with and without the •OH scavenger suggested that the contribution of abiotic methanogenesis (i.e., methyl donors-to-CH4 conversion) to total CH4 production would be around 65.4%, and the remaining 34.6% of CH4 came from syntrophic methanogenesis (i.e., organics/CO2-to-CH4 conversion). In addition, the ROS-induced •CH3 could also combine with O2 under illumination47, contributing to the formation of CH3OH as a carbon source for biotic methanogenesis in darkness.

Notably, excessive ROS accumulation has been reported to induce oxidative stress, causing damage to cellular components such as DNA, proteins, and lipids, ultimately inhibiting microbial growth and survival51. However, ROS were effectively consumed during the light-driven methanogenesis for the oxidation of -CH3 and organic compounds, as well as Fe(II) oxidation, thereby suppressing their accumulation, particularly in the dark (as shown in Fig. 5d). Additionally, the detoxification systems in both microorganisms, including superoxide dismutase, catalases, and peroxidases, were found to be significantly upregulated (Supplementary Fig. 17), effectively alleviating the potential oxidative stress and toxicity for M. b and PCC6803 during syntrophic methanogenesis.

Universality of the light-driven methanogenesis in nature
The investigation of the light-driven methanogenesis was conducted to other oxygenic photosynthetic bacteria and anaerobic methanogenic archaea (Fig. 6). In addition to PCC6803 (Cyanophyta), Tribonema minus (Xanthophyta), Euglena gracilis (Euglenophyta) and Chlorella sp. (Chlorophyta) from various phyla also produced CH4 with M. b. Furthermore, similar methanogenesis was observed when M. b was replaced with Methanobacterium bryantii (hydrogenotrophic methanogen), Methanococcoides orientis (methylotrophic methanogen), or Methanosphaera stadtmaniae (aceticlastic methanogen). These results suggest that the light-driven methanogenesis process with cocultures is not dependent on the energy conservation types of methanogenic archaea. We also found that various common Fe-species, such as zero-valent iron, ferric citrate and ferric oxalate, could enhance the performance of light-driven methanogenesis. Therefore, these results have provided strong evidence that the methanogenesis by oxygenic photosynthetic bacteria and anaerobic methanogenic archaea may be a prevalent occurrence in nature.

Methods
Light-driven methanogenesis experiments
PCC6803 was purchased from the China General Microbiological Culture Collection Center, and cultured in BG11 medium under illumination by visible light LEDs (12±0.6 W m-2). M. b MS (DSM 800) was purchased from the German Collection of Microorganisms and Cell Cultures, and cultured in heterotrophic medium modified from DSM 311b medium31,32. When PCC6803 and M. b grew to their late exponential stages, cells were collected and washed with 0.9% NaCl solution for three times by centrifuging at 5000×g for 20 min at 4 °C (Eppendorf AG 5811, Hamburg, Germany). Then, the washed cells were used to initiate coculture by inoculating PCC6803 and M. b cells in 200 mL of autotrophic medium with different concentrations of sterile Fe-EDTA. The suspension was sparged with sterile N2/CO2 (80/20, vol/vol), creating an initial anaerobic environment with CO2 as the sole carbon source, and incubated without illumination for 24 h to promote heterotrophic respiration with the residual carbon sources from the initial microbial cultivation. After that, the PCC6803-M. b-Fe-EDTA coculture was cultivated for CH4 production under a light-dark cycle of 4 h-20 h at 35 oC using visible light LEDs (12±0.6 W m-2) as light sources. For comparison, a series of single-factor experiments were conducted, by removing M. b, PCC6803, Fe-EDTA or light. The coculture in suspension were harvested after 18 days of light-driven methanogenesis, and the feedstocks were then replaced with the fresh sterilized autotrophic medium to start a new cycle (for a total of three cycles). To evaluate the origin of the produced CH4 with PCC6803-M. b-Fe-EDTA, isotopic labeling experiments were conducted with 13C-labeled NaHCO3 as a sole carbon source. Sodium 2-bromoethanesulfonate (SBES) as a hydrogenase inhibitor was added to evaluate the contribution of H2 to the light-driven methanogenesis. ROS production experiments with M. b under varying O2 concentrations (0.0%, 0.5% and 1.0%) were conducted to evaluate the role of M. b in abiotic CH4 production under illumination. Meanwhile, stable isotope experiments using 18O-labeled O2 were carried out to further determine the origin of the produced •OH. The DMPO-18OH, formed through the interaction between DMPO and •18OH, was identified using an ultrahigh-performance liquid chromatography-triple quadrupole mass spectrometer (LC‒MS, TSQ Endura, Thermo Fisher, USA)58.

To evaluate the universality of such light-driven methanogenesis in nature, several other oxygenic photosynthetic bacteria and anaerobic methanogenic archaea were selected. Oxygenic photosynthetic bacteria Tribonema minus (FACHB-2214, medium BG-11), Euglena sp. (FACHB-1862, Medium HUT) and Chlorella sp. (FACHB-5, medium BG-11) were obtained from the collection of Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB), China. Anaerobic methanogenic archaea Methanobacterium bryantii (ATCC 33272, DSM medium 1523) and Methanosphaera stadtmaniae (CCAM 456, DSM medium 322) were purchase from Biogas Institute of Ministry of Agriculture and Rural Affairs, China. The initial inoculum of Methanococcoides orientis (PRJNA718391, DSM medium 141c) was graciously obtained from the laboratory of Prof. Guangyu Li in Third Institute of Oceanography, Ministry of Natural Resources, China. All culture and sampling manipulations were performed using the sterile technique.

Microscopy and fluorescent in situ hybridization
An optical microscope (Nikon Eclipse E200, Japan) was used to examine the cocultured cells grown at their logarithmic phase. Fluorescent in situ hybridization was conducted with PCC6803-M. b cocultured cells that were first fixed in premixed paraformaldehyde (1%) and glutaraldehyde (0.5%), and then dehydrated in a graded ethanol series (30%, 50%, 70%, 80% and 100% for 3 min at 4 °C). Next, the samples were incubated in a UVPHL-2000 HybriLinker hybridization oven at room temperature for 2 h with PCC6803 (red, autofluorescence) and green-fluorescing probes specific for M. b (5’-(FAM) GTGCTCCCCCGCCAATTCCT-3’). Ultimately, the obtained samples were washed for 30 min in washing buffer, rinsed with Milli-Q water, and then visualized with a Carl Zeiss LSM 880 confocal laser scanning microscope. In addition, to investigate the variation of biofilm thickness with PCC6803-M. b-Fe-EDTA during light-driven methanogenesis, a graphite plate was positioned on the bottom of the culture bottle. Then the formed biofilm was stained by a LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, CA), and examined with a Carl Zeiss LSM 880 confocal laser scanning microscope.

Quantitative analysis of mcrA and cpcG genes
RT-PCR was performed for the quantitative analysis of mcrA gene in M. b and cpcG gene in PCC6803 via a Roche LightCycler 480 System (Roche Applied Science, Penzberg, Germany). mcrA gene was amplified using the primer pair mcrAFor (5ʹ-GGYGGTGTMGGDTTCACM-CARTA-3ʹ) and mcrARev (5ʹ- CGTTCATBGCGTAGTTVGGRTAGT-3ʹ). cpcG gene was amplified using the primer pair cpcGFor (5ʹ-GTCGGGAAGCGGGTGA-3ʹ) and cpcGRev (5ʹ-TTGGCGGCAGGGTTGA-3ʹ). The detailed procedures for RNA collection and quantitative RT-PCR quantification could be found in the Supporting Information59.

Transcriptomic analysis
PCC6803-M. b cocultured cells from triplicate experiments were collected by centrifugation at 12,000×g for 2 min at 4 °C (Eppendorf AG 5811, Hamburg, Germany). Total RNA was extracted RNAprep pure cell/bacteria Kit (TIANGEN, Beijing, China), and the RNA integrity was assessed using the Agilent RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system. Library preparation for strand-specific transcriptome sequencing was generated using NEBNext Ultra II Directional RNA Library Prep Kit following the manufacturer’s recommendations. All the raw sequencing data were quality-checked, and the clean reads were obtained by removing sequencing adapters, trimmed ambiguous bases (N) from the start and end, and other low-quality reads. The remaining reads were subsequently used to map against the published genome of PCC6803 (GCF_018845095.1) and M. b (GCF_000970025.1).

Analytical techniques
The concentrations of CH4 and H2 were determined using a Shimadzu Gas Chromatograph (GC-2014, Shimadzu, Japan) equipped with both flame ionization detector (FID) and thermal conductivity detector (TCD), as well as a Porapak Q column (3.00 mm ID, 5.0 m long). Nitrogen gas (purity >99.995%) with a flow rate of 30 mL min-1 was used as the carrier gas. The injector port and detector temperatures were set at 100 °C and 250 °C, respectively. The injection volume was 100 μL, with the detection limits of 0.1 ppm for CH4 and 5 ppm for H2. The gas products during the isotopic labeling experiments were determined by a Shimadzu GC-2010 equipped with a Shimadzu AOC-20i autosampler system, and interfaced with a Shimadzu QP2010S mass spectrometer (Shimadzu, Japan) (DB-5 capillary column (30 m×0.25 mm×0.25 μm), Helium (99.999%) as carrier gas with a flow rate of 1.2 mL min-1). The DO concentration was measured using UNISENSE OX-NP oxygen needle sensor with a detection limit of 0.3 μM, which was calibrated according to the manufacturer’s instructions.

The photocurrents (I-t) were initially measured in a single-chamber microbial fuel cell (MFC) by applying an external potential bias of -0.5 V. Carbon cloth with a size of 3 cm×3 cm was used as the working electrodes, and autotrophic medium with EDTA-Fe was employed as electrolyte. The cell had a liquid volume of 200 mL and a headspace volume of 150 mL. Data were recorded every 1 min by a data acquisition system (model 2700, Keithley Instruments, Ohio, USA). In addition, photocurrent measurement was also conducted under identical condition, but using two-chamber H-cells instead of the single-chamber MFC10,60. A proton exchange membrane (Nafion 117, DuPont Co., USA) was used to separate the anodic and cathodic chambers, along with autotrophic medium as electrolyte. To examine the compositions of organic substances produced with PCC6803-M. b, the coculture cells were centrifuged at 15,000×g for 20 min at 4 °C (Eppendorf AG 5811, Hamburg, Germany), and then the supernatant was characterized by the Varian INOVA 600-MHz NMR spectroscopy via 1H NMR, gCOSY and gHMBC. The concentration of acetate was monitored by a Shimadzu Nexis GC-2030 equipped with an FID detector and a 10 m×0.53 mm HP-FFAP fused-silica capillary column. Nitrogen was used as the carrier gas with a flow rate of 1.0 mL min-1. The reactive species were characterized using a Bruker A300-10/12 EPR spectrometer, in which •CH3 and •OH were captured with DMPO while •O2- was captured with TEMPO. The concentrations of •OH and H2O2 were measured by a terephthalic acid method and the Shimadzu UV-2600 UV–Vis spectroscopy by adding o-tolidine as the peroxide indicator, respectively. Aqueous Fe(II) concentration was determined using the ferrozine method at a wavelength of 562 nm with the Shimadzu UV-2600 UV–Vis spectroscopy46. Total iron (Fetotal) was measured after the reduction of Fe(III) to Fe(II) with hydroxylamine-HCl. The Fe(III) concentration was subsequently calculated as the difference between the Fetotal and Fe(II) concentrations61. DMSP measurement was made by cleaving DMSP into DMS with strong alkali and quantifying DMS by gas chromatography-mass spectrometry (GCMS-QP2020NX, Shimadzu, Japan)62.

Cells were harvested by centrifugation at 5000×g for 10 min (Eppendorf AG 5404, Hamburg, Germany). The pellet was resuspended in 1 mL of 90% (v/v) acetone and remained in the dark and at 4 °C for 1 h or until no green pigment was visible. Subsequently, the samples were centrifuged again at 5000×g for 10 min to separate the water soluble phase and the cell fragments (pellet) from the acetone extract (supernatant).

The quantification of Chl a was conducted with a Varian Cary Eclipse (now Agilent, USA) spectrofluorometer with the following equation63:

Chla (μg mL-1) = (11.93×A664) - (1.93×A647) (1)

Where A is the absorbance at different wavelengths (nm).

Cells were collected in a 1.5 mL tube and kept in the dark for 20 min. Then the photosynthetic efficiency was measured with an AquaPen-C AP-C 100 (Photon Systems Instruments, Brno, Czech Republic). The maximum quantum yield at PSII was measured in dark-adapted cells by providing a blue light (455 nm) to excite chlorophyll. Fv/Fm was calculated according to the following equation63:

Fv/Fm = (Fm - F0) / Fm (2)

Where Fm is the maximal fluorescence yield observed in dark-adapted cells after stimulation with a saturating light pulse (3000 μmol photons m-2 s-1); F0 is the fluorescence yield measured in dark-adapted sample when all PSII reaction centers are open and it was measured with a measuring light (0.045 μmol photons m-2 s-1).

The conversion efficiency of photosynthate to methane (i.e., percentage of gross primary production diverted to CH4 formation) was calculated based on the reduced concentration of inorganic carbon and CH4 yield.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I am honored to review this paper, which is a research direction that I am interested in and currently\nengaged in. In this paper, Ye and colleagues conducted an extensive study that uncovered\npreviously overlooked connections between oxidative photosynthesis and metagenesis. This has a\nbeneficial impact on advancing our comprehension of the microbial and abiotic processes involved\nin the natural methane cycles. This research has a meticulously crafted methodology, a substantial\namount of data, and a uniform writing style. Frankly speaking, I am cautiously inclined to publish\nthis study in Nature Communications. Nevertheless, considering the current discussion, the author\nmust clarify and revise the subsequent key issues.\nLine 16-18. In the abstract, the author needs to provide a more in-depth introduction to the external\nbiotic and abiotic factors that are responsible for the occurrence of syntrophic methanogenesis in\nnatural environments.\nLine 244-289. The generation of a significant quantity of reactive oxygen species (ROS) chemicals\ncan potentially reach a level of toxicity for symbiotic microbe, particularly in situations when these\ncompounds are not consumed and progressively accumulate. There is a need for additional\ndiscussions to be included here.\nLine 327-348. It is advisable to take into account the metabolic diversity of the potential\nmicroorganisms and, if feasible, compare them with established methane production pathways in\norder to approximate the proportionate contribution of this claimed methane generation pathway in\nparticular habitats of the natural environment.\nLine 327-348. The authors should include a thorough overview of the most recent comparable\naccomplishments in the relevant domain, along with offering specific recommendations for future\nresearches to validate and investigate this methane production process in specific natural\nenvironments.", "reviewer_2": "Syntrophic methanogenesis has received much attention in the scientific community in recent\nyears, which provides a potentially important pathway for microbial metabolism. Particular\nemphasis has been placed the electron transfer and utilization by methanogens in strictly\nanaerobic conditions. However, it must be admitted that the detailed information about alternative\nsyntrophic methanogenesis by two microorganisms with different tolerance levels of oxygen for\nsurvival and reproduction has not been reported.\nIn this study, Zhou et al. present a novel syntrophic methanogenesis triggered by the interaction\nbetween oxygenic photosynthetic bacteria and anaerobic methanogenic archaea with the redox\ncycling of iron. Such syntrophic methanogenesis may be widely present in nature, and contribute to\nglobal methane cycle, therefore with big geochemical impacts. Overall, the topic and scientific part\nare quite interesting and appropriate for Nature Communications. The experiments reported herein\nare well-designed, and the manuscript is well-written. However, the manuscript has several\nshortcomings that requires careful revisions. To further improve the work, I have made several\ncomments listed below:\nGeneral Comments:\n(1) Although the presented results about syntrophic methanogenesis by oxygenic photosynthetic\nbacteria and anaerobic methanogenic archaea are novel, some assumptions made in the\nmanuscript are based on prior work, particularly the abiotic methanogenesis part. Therefore, some\nrecent literatures should be cited and discussed in the manuscript to strength your conclusions,\nsuch as J. Am. Chem. Soc., 2023, 145, 24590-24602; Nat. Commun., 2023, 14, 4364.\n(2) Overall, although there is enough detail provided for most of the methods used in this study,\nsome ones are not sufficiently described. For instance, detection limits and method settings were\nnot included, though these are essential for data interpretation. Please provide more details on\nthese methods.\nSpecific comments:\n(1) Lines 28-29, More information about the prevalent CH4 supersaturation may be provided. under\noxic conditions?\n(2) Lines 44-45, If possible, more information about the coexistence of oxygenic photosynthetic\nbacteria and anaerobic methanogenic archaea should be provided. In addition, are there any\nevidences to support the statement about their potential interaction through nutrient exchange or\nsignal transduction?\n(3) Line 62, Why did you choose Fe-ethylenediaminetetraacetic acid (Fe-EDTA) as Fe species for\nexperiments? Please rationalize these choices.\n(4) Line 80, More information for the applied light source for illumination should be provided, such\nas the wavelength spectra for the visible light LEDs.\n(5) Lines 93-94, Why the methanogenesis behaviour was different between anoxygenic\nphotosynthetic bacteria and oxygenic photosynthetic bacteria under the light-dark conditions?\n(6) Lines 97-98, Is it possible to provide more evidences to support the statement about the rapid\ngrowth and formation of a stable syntrophic coculture for methanogenesis?\n(7) Lines 130-132, Can you show the change of biofilm during the coculture in a time scale with\ndifferent pictures, maybe in Supporting Information? This may give the readers with more intuitive\nimpression.\n(8) Lines 144-146, Maybe more discussion about the results of two-dimensional NMR should be\nconducted.\n(9) Lines 165-170, Is it possible for M. b to directly use the electrons from the respiration process of\nPCC6803 in darkness for methanogenesis? I can understand that it may be very difficult to provide\nthe direct evidences, but you can add the related discussion in the content.\n(10) Lines 274-275, More information and discussion should be replenished for Fig. 5f.\n(11) Based on the discussion in the manuscript, it is better to adjust the order of different\nparameters in Figure 6 (First oxygenic photosynthetic bacteria, then anaerobic methanogenic\narchaea, and finally Fe-species). Meanwhile, the related experimental conditions should be more\nclearly clarified. For instance, which kind of Fe-species did you use when you investigated the\ninfluence of different oxygenic photosynthetic bacteria on the performance of syntrophic\nmethanogenesis?\n(12) Line 335, The related information about the natural sunlight should be replenished.", "reviewer_3": "This is a very interesting and very complex article. Stepping back and taking a broad perspective on\nthe findings, the authors conclude from their experiments that a variety of methanogenic bacteria\nproduce methane in the light and in the dark when co-cultured with a variety of different oxygenic\nphototrophs. More methane is made in the light (Fig. 2b), and FeEDTA considerably enhanced\nmethane production. I suggest the authors choose among the good ideas they report here and build\nstronger experimental support for the ideas they want to prioritize. It would be a significant\ncontribution to prove a general mechanism that supports a partnership between methanogens and\noxygenic photoautotrophs. Presumably (but not explicitly clear in this manuscript), part of this\npartnership is the phototrophs benefit from the methanogens when they deplete oxygen by\nrespiration. In addition to this concept, also in this manuscript are the concepts of direct\ninterspecies electron transfer via Fe-EDTA, and abiotic methanogenesis via ROS. These three\nconcepts all have some support from the reported experiments but none has compelling\nexperimental support. In a manuscript with this many complex ideas and experiments, the\nsupporting documentation can be very important for explaining concepts when page length\nlimitations prevent digression. But, the supporting documents include interesting figures with very\nlittle explanation. Overall, I find the ideas in this manuscript interesting but the presentation of the\ndata made it difficult to fully evaluate the experimental support the author’s ideas. Although I think\nclarity and focus are a problem, I suspect with greater clarity and focus I would ask for more\nexperiments. At this time I don't want to ask for more experiments when I’m not sure which if the\nfindings are most central.\n1. The authors refer to the relationship between phototrophs and methanogen as a syntrophy but I\ndid not notice evidence supporting the idea that the phototrophs benefited. In fact, in Fig 2e we see\nthat the introduction of methanosarcina (M.b.) +Fe-EDTA seems to accelerate O2 decline (note\nthere is no PCC6308 control shown for comparison). My interpretation is that M.b. is consuming\noxygen, probably resulting in more rapid depletion of O2 by the cyanobacteria when they are\nrespiring in the dark, which likely will have negative consequences for the cyanobacteria. How does\nthe cyanobacterium benefit? The impact appears to be negative. I recommend to not use the term\n\"syntrophy\" unless you can show benefits to the cyanobacterial partner. I appreciate that it is\npossible that the methanogens are making energy-yielding anaerobic metabolism more favorable\nfor the cyanobacteria when O2 is exhausted, by lowering H2 partial pressures, but I found the\ndiscussion of that important mechanism too brief and unconvincing.\n2. A rather straightforward, conventional explanation for methanogenic activity would be that the\nmethanogens, in the absence of other cell types except the photoauxotrophs, are benefiting from\nphotosynthetic organic matter production, performing methanogenesis when oxygen is depleted by\ncyanobacterial respiration in the dark. While not surprising from a theoretical perspective, this\npotentially is an interesting finding. What percentage of gross primary production was diverted to\nmethane formation? Are your findings a manifestation of common microbiological activities, or is\nthere a co-evolved, specific interaction occurring? I found myself unable to decide which was the\ncase for the data provided.\nI recommendation that you estimate the percentage of gross primary production accounted for by\nmethane production. Is there evidence that anaerobic PCC6308 can use fermentative metabolism\nto produce energy in the absence of oxygen, which would indicate they might benefit from the\nmethanogens creating thermodynamically favorable conditions for fermentation?\n3. The manuscript indicates that Fe-EDTA supports methanogenesis, presumably by serving as a\ncarrier for electrons DIET. Can you prove that PCC6803 reduces Fe-EDTA and Mb. oxidizes it?\n4. Figure 4. “Mechanisms of biotic methanogenesis with PCC6803-M. b-Fe-EDTA as revealed by\ntranscriptomic analyses”. It’s confusing to show ROS in this figure but no role for Fe-EDTA, which is\npurportedly important for biological methane production.\n5. In Fig.2, oxygen production appears to continue for 5 days (2e) but methane production falls off\nsharply after day 2. Is this the correct interpretation of the data? Why does methane production\nstop?\n6. There is almost no explanation of the term “photocurrent”, and the description of these\nexperiments is too brief. If the photocurrent is through a graphite substrate, does this mean its\ncompeting with electron transfer to M.b.?\nLegend, Fig. 2. Indicate that grey means dark, if that is correct.\nThe legends for the supporting figures are too brief. Please add details."}, "author_response": "Re: NCOMMS-24-04041\nPoint-by-point responses to Referees' Comments\nReviewer #1 (Remarks to the Author):\nI am honored to review this paper, which is a research direction that I am interested in and currently\nengaged in. In this paper, Ye and colleagues conducted an extensive study that uncovered previously\noverlooked connections between oxidative photosynthesis and metagenesis. This has a beneficial\nimpact on advancing our comprehension of the microbial and abiotic processes involved in the natural\nmethane cycles. This research has a meticulously crafted methodology, a substantial amount of data,\nand a uniform writing style. Frankly speaking, I am cautiously inclined to publish this study in Nature\nCommunications. Nevertheless, considering the current discussion, the author must clarify and revise\nthe subsequent key issues.\nWe really appreciate the referee’s insightful suggestions to help us significantly improve the\nquality of our manuscript. We have carefully revised the manuscript, and sincerely hope that our\nrevisions have satisfactorily addressed the referee’s concerns.\n1) Line 16-18. In the abstract, the author needs to provide a more in-depth introduction to the external\nbiotic and abiotic factors that are responsible for the occurrence of syntrophic methanogenesis in\nnatural environments.\nResponse: We thank the referee for pointing out this issue. As suggested, a more in-depth\nintroduction to the external biotic and abiotic factors that are responsible for the occurrence of\nsyntrophic methanogenesis in natural environments has been replenished in the revised manuscript to\nthe strength the conclusion.\nRevised in text: “We further demonstrated CH production by other model oxygenic\n4\nphotosynthetic bacteria from various phyla, in conjunction with different anaerobic methanogenic\narchaea exhibiting diverse energy conservation modes, as well as various common Fe-species.\nTherefore, such syntrophic methanogenesis may be widely present in nature. These findings have\nrevealed an unexpected link between oxygenic photosynthesis and methanogenesis, and would\nadvance our understanding of photosynthetic bacteria’s ecological role in the global CH cycle.”.\n4\n2) Line 244-289. The generation of a significant quantity of reactive oxygen species (ROS) chemicals\ncan potentially reach a level of toxicity for symbiotic microbe, particularly in situations when these\n1\ncompounds are not consumed and progressively accumulate. There is a need for additional discussions\nto be included here.\nResponse: We thank the referee for pointing out this issue. As suggested, additional discussions\nabout the accumulation and consumption of produced ROS on the growth of symbiotic microbe have\nbeen included in the revised manuscript.\nRevised in text: “Notably, excessive ROS accumulation has been reported to induce oxidative\nstress, causing damage to cellular components such as DNA, proteins, and lipids, ultimately inhibiting\nmicrobial growth and survival.48 However, ROS were effectively consumed during syntrophic\nmethanogenesis for the oxidation of -CH and organic compounds, as well as Fe (II) oxidation, thereby\n3\nsuppressing their accumulation, particularly in the dark (as shown in Fig. 5d). Additionally, the\ndetoxification systems in both microorganisms, including superoxide dismutase, catalases, and\nperoxidases, were found to be significantly upregulated (Supplementary Fig. 15), effectively\nalleviating the potential oxidative stress and toxicity for M. b and PCC6803 during syntrophic\nmethanogenesis.”.\nSupplementary Fig. 15 Transcriptomic analyses of key antioxidant stress genes in M. b and PCC6803\nduring the syntrophic methanogenesis. Sod, superoxide dismutase; Kat, catalase; sll1621, type II\nperoxiredoxins; AhpC and AhpE, alkylhydroperoxidases; Htp and Hsp, heat shock proteins; Rbr,\nrubrerythrin; Dps, DNA-binding protein from starved cells.\nReference\nHong, Y., Zeng, J., Wang, X., Drlica, K., Zhao, X. Post-stress bacterial cell death mediated by\nreactive oxygen species P. Natl. Acad. Sci. 116, 10064-10071 (2019).\n3) Line 327-348. It is advisable to take into account the metabolic diversity of the potential\n2\nmicroorganisms and, if feasible, compare them with established methane production pathways in order\nto approximate the proportionate contribution of this claimed methane generation pathway in\nparticular habitats of the natural environment.\nResponse: We appreciate the reviewer's valuable suggestion. As suggested, the relevant\ndiscussion has been replenished in the revised manuscript.\nRevised in text: “Oxygenic photosynthesis has been recognized as the most important metabolic\ninnovation on Earth, enabling life to harness energy and reducing power directly from sunlight and\nwater, thus liberating it from the constraints of geochemically derived reductants.51 Consequently,\nthese diverse and intriguing oxygenic photosynthetic bacteria exist considerable metabolic flexibility,\nutilizing numerous unconventional central carbon metabolic pathways and novel enzymes for\nautotrophic, mixotrophic, and heterotrophic growth, tailored to their specific ecological niches.22,52\nConsidering the extensive coexistence and interaction of diverse microbial species in natural and\nengineered ecosystems,53 along with ferruginous environment on Earth (e.g., oceans with abundant\nFe(II) and Fe(III)-carboxylate complexes), syntrophic methanogenesis by oxygenic photosynthetic\nbacteria and anaerobic methanogenic archaea creates more thermodynamically favourable conditions\nfor both microorganisms. Thus, this syntrophic methanogenesis process surpasses the conventional\nmethane production pathways (i.e., acetoclastic methanogenesis and hydrogenotrophic\nmethanogenesis), and potentially making a more significant contribution to the global CH cycle.”.\n22.Saper, G., et al. Live cyanobacteria produce photocurrent and hydrogen using both the\nrespiratory and photosynthetic systems. Nat. Commun.9, 2168 (2018).\n51. Fischer, W. W., Hemp, J., Johnson, J. E. Evolution of oxygenic photosynthesis. Annu. Rev.\nEarth Pl. Sc. 44, 647-683 (2016).\n52.Tang, K.-H., Tang, Y. J., Blankenship, R. E. Carbon metabolic pathways in phototrophic\nbacteria and their broader evolutionary implications. Front. Microbiol. 2, 11061 (2011).\n53.Kato, S., Watanabe, K. Ecological and evolutionary interactions in syntrophic methanogenic\nconsortia. Microbes Environ. 25, 145-151 (2010).\n4) Line 327-348. The authors should include a thorough overview of the most recent comparable\naccomplishments in the relevant domain, along with offering specific recommendations for future\nresearches to validate and investigate this methane production process in specific natural\nenvironments.\nResponse: We appreciate the reviewer's valuable suggestion. As suggested, the relevant\ninformation has been replenished in the revised manuscript.\n4\nand photosynthesis.8,9,54 Various potential mechanisms for CH production by phototrophic\n4\nmicroorganisms having been extensively investigated, including the photosynthesis-driven\nmetabolism9,50 and ROS-driven demethylation of methyl donors.43-46 Our study innovatively\ndemonstrated the synergistic interaction between these two mechanisms, along with the Fe redox\ncycles. However, the existence of such syntrophic methanogenesis by oxygenic photosynthetic\nbacteria and anaerobic methanogenic archaea in the natural environments requires further validation\nwith multiple complementary approaches, including the evaluation of in situ CH profiles and\n4\nmicrobial composition, incubation experiments with freshwater microbial cultures using NaH13CO as\n3\na supplementation carbon source, and the assessment of the exact contribution of both abiotic and\nbiotic pathways. Meanwhile, recent studies have indicated the potential importance of various metal\nelements in the evolution of oxygenic photosynthesis, such as manganese.55 Therefore, the potential\ninvolvement of other metal elements in such syntrophic methanogenesis warrants further evaluation.”.\nReferences\n8. Fixen, K. R., et al. Light-driven carbon dioxide reduction to methane by nitrogenase in a\nphotosynthetic bacterium. P. Natl. Acad. Sci. 113, 10163-10167 (2016).\n9. Perez-Coronel, E., Michael Beman, J. Multiple sources of aerobic methane production in\naquatic ecosystems include bacterial photosynthesis. Nat. Commun.13, 6454 (2022).\n43. Ernst, L., et al. Methane formation driven by reactive oxygen species across all living\norganisms. Nature603, 482-487 (2022).\n44.Althoff, F., et al. Abiotic methanogenesis from organosulphur compounds under ambient\nconditions. Nat. Commun.5, 4205 (2014).\n45.Hädeler, J., Velmurugan, G., Lauer, R., Radhamani, R., Keppler, F., Comba, P. Natural abiotic\niron-oxido-mediated formation of C1 and C2 compounds from environmentally important methyl-\nsubstituted substrates. J. Am. Chem. Soc. 145, 24590-24602 (2023).\n46.Ernst, L., et al. Methane formation driven by light and heat prior to the origin of life and\nbeyond. Nat. Commun.14, 4364 (2023).\n50.Bižić, M., et al. Aquatic and terrestrial cyanobacteria produce methane. Sci. Adv. 6, 5343\n(2020).\n54.Deines, P., Hammerschmidt, K., Bosch, T. C. Microbial species coexistence depends on the\nhost environment. mBio 11, 10-1128 (2020).\n55.Huang, L., Liu, X., Rensing, C., Yuan, Y., Zhou, S., Nealson, K. H. Light-independent\nanaerobic microbial oxidation of manganese driven by an electrosyntrophic coculture. ISME J. 17,\n163-171 (2022).\nSyntrophic methanogenesis has received much attention in the scientific community in recent\nyears, which provides a potentially important pathway for microbial metabolism. Particular emphasis\nhas been placed the electron transfer and utilization by methanogens in strictly anaerobic conditions.\nHowever, it must be admitted that the detailed information about alternative syntrophic\nmethanogenesis by two microorganisms with different tolerance levels of oxygen for survival and\nreproduction has not been reported.\nIn this study, Zhou et al. present a novel syntrophic methanogenesis triggered by the interaction\nbetween oxygenic photosynthetic bacteria and anaerobic methanogenic archaea with the redox cycling\nof iron. Such syntrophic methanogenesis may be widely present in nature, and contribute to global\nmethane cycle, therefore with big geochemical impacts. Overall, the topic and scientific part are quite\ninteresting and appropriate for Nature Communications. The experiments reported herein are well-\ndesigned, and the manuscript is well-written. However, the manuscript has several shortcomings that\nrequires careful revisions. To further improve the work, I have made several comments listed below:\nWe really appreciate the referee’s kind suggestions to help us further improve the quality of our\nmanuscript. We have carefully revised the manuscript, and sincerely hope that our revisions have\nsatisfactorily addressed the referee’s concerns.\nGeneral Comments:\n(1) Although the presented results about syntrophic methanogenesis by oxygenic photosynthetic\nbacteria and anaerobic methanogenic archaea are novel, some assumptions made in the manuscript\nare based on prior work, particularly the abiotic methanogenesis part. Therefore, some recent\nliteratures should be cited and discussed in the manuscript to strength your conclusions, such as J. Am.\nChem. Soc., 2023, 145, 24590-24602; Nat. Commun., 2023, 14, 4364.\nResponse: We appreciate the reviewer's valuable suggestion. As suggested, the recent literatures\nhave been cited and discussed in the revised manuscript to the strength the conclusion.\nRevised in text: “These methyl donors could be oxidized by ROS for •CH formation, ultimately\n3\nleading to abiotic CH production.43,44”.\n4\n“In addition, the ROS-induced •CH could also combine with O under illumination,44\n3 2\ncontributing to the formation of CH OH as a carbon source for biotic methanogenesis in darkness.”.\n3\n4\noccurred with highly reactive •OH generated not only through Fenton reaction45,46 but also through\nubiquitous non-Fenton chemistry reactions driven by diversified external fields.47”.\nReferences\n43.Ernst, L., et al. Methane formation driven by reactive oxygen species across all living\norganisms. Nature603, 482-487 (2022).\n44.Althoff, F., et al. Abiotic methanogenesis from organosulphur compounds under ambient\nconditions. Nat. Commun.5, 4205 (2014).\n45.Hädeler, J., Velmurugan, G., Lauer, R., Radhamani, R., Keppler, F., Comba, P. Natural abiotic\niron-oxido-mediated formation of C1 and C2 compounds from environmentally important methyl-\nsubstituted substrates. J. Am. Chem. Soc. 145, 24590-24602 (2023).\n46.Ernst, L., et al. Methane formation driven by light and heat prior to the origin of life and\nbeyond. Nat. Commun.14, 4364 (2023).\n47.Jie, Y., et al. Abiotic methane production driven by ubiquitous non-Fenton-type reactive\noxygen species. Angew. Chem. Int. Ed. e202403884 (2024).\n(2) Overall, although there is enough detail provided for most of the methods used in this study, some\nones are not sufficiently described. For instance, detection limits and method settings were not\nincluded, though these are essential for data interpretation. Please provide more details on these\nmethods.\nResponse: We thank the referee for pointing out this issue. As suggested, more detailed\ninformation has been replenished in the revised manuscript.\nRevised in text: “The concentrations of H and CH were determined using a Shimadzu Gas\n2 4\nChromatograph (GC-2014, Shimadzu, Japan) equipped with both flame ionization detector (FID) and\nthermal conductivity detector (TCD), as well as a Porapak Q column (3.00 mm ID, 5.0 m long).\nNitrogen gas (purity >99.995%) with a flow rate of 30 mL min-1 was used as the carrier gas. The\ninjector port and detector temperatures were set at 100 °C and 250 °C, respectively. The injection\nvolume was 100 μL, with the detection limits of 0.1 ppm for CH and 5 ppm for H .”.\n4 2\n“The gas products during the isotopic labelling experiments were determined by a Shimadzu GC-\n2010 equipped with a Shimadzu AOC-20i auto sampler system, and interfaced with a Shimadzu QP\n2010S mass spectrometer (Shimadzu, Japan) (DB-5 capillary column (30 m × 0.25 mm × 0.25 μm),\nHelium (99.999%) as carrier gas with a flow rate of 1.2 mL min-1).”.\n“The DO concentration was measured using UNISEN SE OX NP oxygen needle sensor with a\n6\ndetection limit of 0.3 μM, which was calibrated according to the manufacturer’s instructions.”.\n“The photocurrents (I-t) were initially measured in a single-chamber microbial fuel cell (MFC)\nby applying an external potential bias of -0.5 V. Carbon cloth with a size of 3 cm × 3 cm was used as\nthe working electrodes, and autotrophic medium with EDTA-Fe was employed as electrolyte. The cell\nhad a liquid volume of 200 mL and a headspace volume of 150 mL. Data were recorded every 1 min\nby a data acquisition system (model 2700, Keithley Instruments, Ohio, USA). In addition, photocurrent\nmeasurement was also conducted under identical condition, but using two-chamber H-cells instead of\nthe single-chamber MFC.10 A proton exchange membrane (Nafion 117, DuPont Co., USA) was used\nto separate the anodic and cathodic chambers, along with autotrophic medium as electrolyte.”.\n“The concentration of acetate was monitored by a Shimadzu Nexis GC-2030 equipped with an\nFID detector and a 10 m × 0.53 mm HP-FFAP fused-silica capillary column. Nitrogen was used as the\ncarrier gas with a flow rate of 1.0 mL min-1.”.\n“Aqueous Fe(II) concentration was determined using the ferrozine method at a wavelength of 562\nnm with the Shimadzu UV-2600 UV-Vis spectroscopy.43 Total iron (Fe ) was measured after the\ntotal\nreduction of Fe(III) to Fe(II) with hydroxylamine-HCl. The Fe(III) concentration was subsequently\ncalculated as the difference between the Fe and Fe(II) concentrations.57”.\ntotal\n“The syntrophic methanogenesis experiments was also conducted on the roof of the Research\nCenter for Water Resources and Security Building at Fujian Agriculture and Forestry University in\nFuzhou, China (latitude: 26.05 oN"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have thoroughly addressed the comments of the three reviewers by providing\ncomprehensive responses and making the necessary modifications. As a result, the quality of the\ncurrent paper has substantially improved. Hence, I propose accepting it.", "reviewer_2": "I am satisfied with the revised manuscript. The authors carefullly addressed all comments.", "reviewer_3": "This manuscript reports a diverse collection of experiments that collectively support the\nconclusion that methanogens and cyanobacteria may interact synergistically, benefitting both cell\ntypes in dense co-cultures on light dark cycles by providing methanogens with a source of\nelectrons for methanogenic energy production and cyanobacteria with an electron sink that\nincreases the energy yield of fermentative metabolism of stored carbohydrates during dark periods.\nTheoretically, such an association makes sense, and, because of the prevalence of cyanobacterial\nmats, it could potentially have a broad impact by widespread methane production. However, most\nof the methane production by the culture occurs in the light (Fig. 2b), which points to the proposed\nabiotic mechanisms of methane production. But, that does not entirely make sense because it’s\nnot clear why methanogenic bacteria would be required for that process. Does PCC6803 make\nmethane in the light without the other factors? This is an important control that I did not find. If not,\ndoes this not diminish the argument that production of methane in the light is due to abiotic\nmechanisms?\nThe syntrophy idea is better supported in this version of the manuscript, but the role of\nmethanogenic bacteria in abiotic methane production needs further support. The authors modified\ntheir manuscript to address one of my foremost criticisms, which was the lack of clarity in the\noriginal manuscript about the mechanisms by which methanogenesis benefits the cyanobacteria.\nThe mechanisms reported are multiple and complex, which makes the narrative difficult to follow.\nAlthough this is a challenge throughout the manuscript, figure 1, a summary figure, does not help\nand should be altered (and perhaps the legend needs to change), for clarity. The figure shows\noxygenic photosynthetic bacteria in the light and anaerobic methanogenic bacteria in the dark, but\nthey are both together all the time in the light and the dark, so I think this is not a good way to\nillustrate the concepts. Rather, I suggest that you show them both together in the dark and the light,\nand use the figure to illustrate how the proposed mechanisms of methane production and\ninteractions of these two cell types change between light and dark.\nThe manuscript give the impression that across aquatic environments these processes are\noccurring. The proposed syntrophy only occurs in micro environments that periodically become\nanoxic. At some positions in the manuscript this is clear, but in the text explaining the methane\nparadox this point needs clarification.\nMy overall impression is that this manuscript is not yet ready to publish. Very complex ideas are\nadvanced to explain the observations. This makes the manuscript a challenge to write, because a\nvariety of different processes that are proposed must be proven. It's also a challenge to read,\nbecause of the multiple interpretations for methane production."}, "author_response": "Re: NCOMMS-24-04041A\nPoint-by-point responses to Referees' Comments\nReviewer #1 (Remarks to the Author):\nThe authors have thoroughly addressed the comments of the three reviewers by providing\ncomprehensive responses and making the necessary modifications. As a result, the quality of the\ncurrent paper has substantially improved. Hence, I propose accepting it.\nResponse: We thank the reviewer very much for the kind comments.\nReviewer #2 (Remarks to the Author):\nI am satisfied with the revised manuscript. The authors carefully addressed all comments.\nResponse: We thank the reviewer very much for the kind comments.\n1) This manuscript reports a diverse collection of experiments that collectively support the conclusion\nthat methanogens and cyanobacteria may interact synergistically, benefitting both cell types in dense\nco-cultures on light-dark cycles by providing methanogens with a source of electrons for methanogenic\nenergy production and cyanobacteria with an electron sink that increases the energy yield of\nfermentative metabolism of stored carbohydrates during dark periods. Theoretically, such an\nassociation makes sense, and, because of the prevalence of cyanobacterial mats, it could potentially\nhave a broad impact by widespread methane production. However, most of the methane production by\nthe culture occurs in the light (Fig. 2b), which points to the proposed abiotic mechanisms of methane\nproduction. But, that does not entirely make sense because it’s not clear why methanogenic bacteria\nwould be required for that process. Does PCC6803 make methane in the light without the other factors?\nThis is an important control that I did not find. If not, does this not diminish the argument that\nproduction of methane in the light is due to abiotic mechanisms?\nResponse: We thank the reviewer for pointing out this issue. The relevant results indicated that the\nO produced by PCC6803 during the photosynthetic oxygen evolution significantly stimulated the\n4\nproduction process (as detailed in the next comment). Moreover, additional experiments showed that\nPCC6803 produced almost no CH under illumination without the presence of other factors (Fig. 2b),\n4\nsuggesting the potential existence of an abiotic CH production process. The relevant information has\n4\nbeen replenished in the revised manuscript.\nRevised in text: “Substantial CH production during the light period was observed with PCC6803-\nalmost no CH under illumination without the presence of other factors (Fig. 2b), suggesting the\n4\npotential existence of an abiotic CH production process.”.\n4\n4\nthe first 4 days.\n2) The syntrophy idea is better supported in this version of the manuscript, but the role of methanogenic\nbacteria in abiotic methane production needs further support. The authors modified their manuscript\nto address one of my foremost criticisms, which was the lack of clarity in the original manuscript about\nthe mechanisms by which methanogenesis benefits the cyanobacteria.\nResponse: The relevant experiments indicated that the O produced by PCC6803 during the\n2\nphotosynthetic oxygen evolution significantly stimulated the ROS production by methanogenic\nbacteria (M. b) in illumination, thereby enhancing the abiotic CH production process. This inference\n4\nwas confirmed through the ROS production experiments with M. b under varying O concentrations.\n2 2 2\n(Supplementary Fig. 13). Stable isotope analysis with 18O further confirmed that the produced ROS\n2\nstemmed from O reduction, evidenced by the observed 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-\n2\n18OH (m/z = 132.09, Supplementary Fig. 14). Previous studies have shown that when anaerobic cells\nwere exposed to oxygen-rich environments, molecular O adventitiously abstracted electrons from the\n2\nreduced flavins or metal centers of some redox enzymes, resulting in the ROS formation (mBio 8(1),\ne01873-16 (2017); Mol. Microbiol. 75(6), 1389-1401 (2010); J. Biol. Chem. 266(11), 6957-6965\n(1991)). As these events relied on collision frequency, the ROS production rate was directly\nproportional to the O concentration (Nat. Rev. Microbiol. 19(12), 774-785 (2021)). Subsequently, the\n2\nproduced ROS were inadvertently released extracellularly (Biochem. Bioph. Res. Co. 36(6), 891-897\n3\n(1969)). Along with the potentially produced ROS by Fenton reaction with Fe-EDTA and/or PCC6803\nduring the photochemical energy conversion for bioenergetic production (P. Natl. Acad. Sci. 102, 6502-\n6507 (2005)), various methyl donors were oxidized by ROS to form methyl radicals (•CH ) as\n3\nintermediates that eventually resulted in abiotic methanogenesis under oxic conditions. The relevant\ninformation has been replenished in the revised manuscript.\nRevised in text: “The concentrations of these ROS, particularly •OH and H O , significantly\n2 2\nincreased under illumination (Fig. 5d), likely attributed to the produced O by PCC6803 during the\n2\nphotosynthetic oxygen evolution that significantly stimulated the ROS production by M. b. This\ninference was confirmed through the ROS production experiments with M. b under varying O\n2\nconcentrations. It was found that higher O concentrations led to the production of more ROS, such as\n2\n•OH and H O (Supplementary Fig. 13). Stable isotope analysis with 18O further confirmed that the\n2 2 2\nproduced ROS stemmed from O reduction, evidenced by the observed 5,5-dimethyl-1-pyrroline-N-\n2\noxide (DMPO)-18OH (m/z = 132.09, Supplementary Fig. 14). Previous studies have shown that when\nanaerobic cells were exposed to oxygen-rich environments, molecular O adventitiously abstracted\n2\nelectrons from the reduced flavins or metal centers of some redox enzymes, resulting in the ROS\nformation (O → •O - → H O → •OH).40-42 As these events relied on collision frequency, the ROS\n2 2 2 2\nproduction rate was directly proportional to the O concentration.43 Subsequently, the produced ROS\n2\nwere inadvertently released extracellularly.44”.\n“ROS production experiments with M. b under varying O concentrations (0.0%, 0.5% and 1.0%)\n2\nwere conducted to evaluate the role of M. b in abiotic CH production under illumination. Meanwhile,\n4\nstable isotope experiments using 18O-labeled O were carried out to further determine the origin of the\n2\nproduced •OH. The DMPO-18OH, formed through the interaction between DMPO and •18OH, was\nidentified using an ultrahigh-performance liquid chromatography-triple quadrupole mass spectrometer\n(LC‒MS, TSQ Endura, Thermo Fisher, USA).59”.\n2 2 2\nbars represent the standard deviation of n = 3 analytical replicates from one experiment. Different\nletters represent statistically significant difference (p < 0.05) in different groups.\nSupplementary Fig. 14 Mass spectra of DMPO-•OH with 18O as reaction species during ROS\n2\nproduction by M. b. The mass-to-charge ratios (m/z) of 130.09 and 132.09 represent DMPO-16OH and\nDMPO-18OH, respectively.\n40. Lu, Z., Imlay, J. A. The fumarate reductase of Bacteroides thetaiotaomicron, unlike that of\nEscherichia coli, is configured so that it does not generate reactive oxygen species.mBio8(1),\ne01873-16 (2017).\n41. Korshunov, S., Imlay, J. A. (2010). Two sources of endogenous hydrogen peroxide in Escherichia\ncoli.Mol. Microbiol. 75(6), 1389-1401 (2010).\n42. Imlay, J. A., Fridovich, I. Assay of metabolic superoxide production in Escherichia coli.J. Biol.\nChem.266(11), 6957-6965 (1991).\n43. Lu, Z., Imlay, J. A. When anaerobes encounter oxygen: mechanisms of oxygen toxicity, tolerance\nand defence. Nat. Rev. Microbiol. 19(12), 774-785 (2021).\n44. Massey, V., et al. The production of superoxide anion radicals in the reaction of reduced flavins\nand flavoproteins with molecular oxygen. Biochem. Bioph. Res. Co.36(6), 891-897 (1969).\n59. Huang, L., Liu, X., Rensing, C., Yuan, Y., Zhou, S., Nealson, K. H. Light-independent anaerobic\nmicrobial oxidation of manganese driven by an electrosyntrophic coculture. ISME J.17, 163-171\n(2022).\n3) The mechanisms reported are multiple and complex, which makes the narrative difficult to follow.\nAlthough this is a challenge throughout the manuscript, figure 1, a summary figure, does not help and\nshould be altered (and perhaps the legend needs to change), for clarity. The figure shows oxygenic\nphotosynthetic bacteria in the light and anaerobic methanogenic bacteria in the dark, but they are both\ntogether all the time in the light and the dark, so I think this is not a good way to illustrate the concepts.\nRather, I suggest that you show them both together in the dark and the light, and use the figure to\nillustrate how the proposed mechanisms of methane production and interactions of these two cell types\nchange between light and dark.\nResponse: We appreciate the reviewer's valuable suggestion. As suggested, the Figure 1 and its\nlegend have been revised, and the relevant information has been replenished in the revised manuscript.\nRevised in text: “The results showed that CH production by the interaction of oxygenic\n4\nphotosynthetic bacteria and anaerobic methanogenic archaea was significantly enhanced through the\nredox cycling of Fe-ethylenediaminetetraacetic acid (Fe-EDTA), involving both syntrophic\nmethanogenesis and abiotic methanogenesis during the periodic dark-light cycles (Fig. 1). Specifically,\nin darkness, the organics and H produced by PCC6803 during dark fermentation were utilized as\n2\ncarbon sources and reducing equivalents by M. b for syntrophic methanogenesis under anoxic\nconditions. The significantly lowered hydrogen pressure by M. b, in turn, created more\n6\nthermodynamically favourable conditions for PCC6803. In contrast, in illumination, the\nphotosynthesized organic compounds and their intermediates by PCC6803 served as potential methyl\ndonors (-CH ). Meanwhile, the simultaneously produced O stimulated reactive oxygen species (ROS)\n3 2\nproduction by M. b. Along with the Fenton reaction with Fe-EDTA, various methyl donors were\noxidized by ROS to form methyl radicals (•CH ) as intermediates that eventually resulted in abiotic\n3\nmethanogenesisunder oxic conditions.”.\nFig. 1 | Schematic illustration of CH production by the interaction of oxygenic photosynthetic\n4\nbacteria and anaerobic methanogenic archaea through both syntrophic methanogenesis (under\nanoxic conditions in darkness) and abiotic methanogenesis (under oxic conditions in illumination)\npathways during the periodic light-dark cycles.\n7\n4) The manuscript gave the impression that across aquatic environments these processes are occurring.\nThe proposed syntrophy only occurs in micro environments that periodically become anoxic. At some\npositions in the manuscript this is clear, but in the text explaining the methane paradox this point needs\nclarification.\nResponse: We agree that the statement in the original manuscript is not clear. As depicted in the\nrevised Figure 1 of the manuscript, CH production by oxygenic photosynthetic bacteria and\n4\nmethanogenic archaea involved both syntrophic methanogenesis (under anoxic conditions in darkness)\nand abiotic methanogenesis (under oxic conditions in illumination) pathways during the periodic light-\ndark cycles, which potentially explained the prevalent methane paradox phenomenon. The relevant\ninformation has been replenished in the revised manuscript.\nRevised in text: “Thus, this light-driven methanogenesis process, involved both syntrophic\nmethanogenesis (under anoxic conditions in darkness) and abiotic methanogenesis (under oxic\nconditions in illumination) during the periodic dark-light cycles, surpasses the conventional methane\nproduction pathways (i.e., acetoclastic methanogenesis and hydrogenotrophic\nmethanogenesis), and potentially making a more significant contribution to the global CH cycle. The\n4\ninference was supported by the correlation between CH supersaturation and photosynthesis.8,9,54”.\n4\n5) My overall impression is that this manuscript is not yet ready to publish. Very complex ideas are\nadvanced to explain the observations. This makes the manuscript a challenge to write, because a\nvariety of different processes that are proposed must be proven. It's also a challenge to read, because\nof the multiple interpretations for methane production.\nResponse: We agree with the reviewer that the relevant mechanisms explaining the observations\nare somewhat complex. Like many other studies for understanding (different) complex processes, this\nstudy would serve as an initial step to reveal this complex process in the nature. To better illustrate the\nmethanogenesis processby oxygenic photosynthetic bacteria and anaerobic methanogenic archaea, we\nhave restructured the explanation into two main pathways during the periodic light-dark cycles, based\non the obtained results and reviewer’s suggestions, involving both syntrophic methanogenesis (under\nanoxic conditions in darkness) and abiotic methanogenesis (under oxic conditions in illumination).\nAccordingly, we have modified the article’s title, structure and figures, including the revision of the\nrelevant figures (e.g., Fig. 1 and Fig. 5a) and expansion of the related discussion (e.g., the role of M. b\nduring abiotic methanogenesis). We sincerely hope that our revisions have satisfactorily addressed the\nreferee’s concerns.\n8"}]

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