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

Atmospheric methane (CH₄) is a potent greenhouse gas, and its rising concentration is a significant climate change concern. Freshwater ecosystems contribute substantially to global CH₄ emissions, with the traditional understanding being that CH₄ production primarily occurs through anaerobic processes in sediments. However, the observation of CH₄ supersaturation in oxic surface waters presents a "methane paradox." This paradox suggests the existence of yet-undefined CH₄ production processes. Photosynthetic bacteria, dominant in surface waters, are known to interact with other microorganisms, influencing biogeochemical cycles. While previous studies hinted at a link between photosynthetic bacteria and CH₄ production under illumination, the underlying mechanisms remained elusive. This study investigates the role of oxygenic photosynthetic bacteria in this phenomenon, challenging the long-held belief that methanogenic archaea are highly oxygen-sensitive. The coexistence of oxygenic photosynthetic bacteria and methanogenic archaea in various oxygenated, methane-rich environments, alongside findings indicating methanogen oxygen tolerance, suggests a potential for interaction and co-metabolism. A comprehensive understanding of this interaction is crucial for accurate assessment of the global CH₄ cycle and its implications for climate change.

The literature review highlights the existing knowledge on methane production in freshwater ecosystems, focusing on the traditional view that anaerobic processes in anoxic sediments are the primary source. It also examines previous research demonstrating a correlation between photosynthetic bacteria and CH₄ production under illumination, but notes the lack of a clear understanding of the mechanisms involved. The review discusses the prevailing paradigm that methanogens are highly sensitive to oxygen and the challenges presented by the observed methane supersaturation in oxic surface waters—the methane paradox. Studies suggesting the coexistence of oxygenic photosynthetic bacteria and methanogens in oxygenated environments and evidence of methanogen oxygen tolerance are presented, laying the foundation for the study's hypothesis. The review concludes with the significance of understanding the photosynthetically regulated CH₄ production for accurate modeling of the global CH₄ cycle and its impact on climate change.

The study employed cocultures of *Synechocystis* sp. strain PCC 6803 (a model oxygenic photosynthetic bacterium) and *Methanosarcina barkeri* (a model methanogen) in a defined medium with CO₂ as the sole electron acceptor. Experiments were conducted under controlled light-dark cycles (4 hours light, 20 hours dark initially; 12-hour cycles were also performed for comparison), simulating natural conditions. Fe-ethylenediaminetetraacetic acid (Fe-EDTA) was included as a representative iron species present in many open water systems. CH₄ production was quantified using gas chromatography. Isotopic labeling experiments with ¹³C-labeled NaHCO₃ were performed to trace the carbon source of CH₄. Microscopy techniques (optical microscopy, fluorescence in situ hybridization (FISH), confocal laser scanning microscopy (CLSM)) were used to characterize the biofilm formation and cell interactions in the cocultures. Nuclear magnetic resonance (NMR) spectroscopy was employed to identify organic substances produced during syntrophic methanogenesis. Transcriptomic analysis (RNA sequencing) was conducted to investigate gene expression patterns related to photosynthesis, metabolism, and iron transport in both organisms under different light conditions. Electron paramagnetic resonance (EPR) spectroscopy and UV-Vis spectroscopy were used to detect reactive oxygen species (ROS) and measure H₂O₂ concentration. Gas chromatography-mass spectrometry (GC-MS) was employed to identify methyl donors. Scavenger trapping tests were conducted to determine the role of ROS in methanogenesis. The universality of light-driven methanogenesis was explored by testing other oxygenic photosynthetic bacteria and anaerobic methanogenic archaea, with different energy conservation modes, and various iron species. The experiments were also conducted under natural sunlight to assess the relevance of the findings to real-world conditions. Detailed protocols are provided in the supplementary material.

The coculture of PCC6803 and *M. barkeri*, in the presence of Fe-EDTA, showed significantly enhanced CH₄ production compared to monocultures under light-dark cycles. CH₄ production was observed both in light and dark periods, unlike previously reported light-dependent methanogenesis using anoxygenic bacteria. Isotopic labeling experiments confirmed that CO₂ was the primary carbon source for CH₄. Microscopy revealed the formation of dense biofilms with intimate contact between the two species. NMR analysis identified several organic compounds (lactate, pyruvate, acetate) produced by PCC6803, serving as substrates for methanogenesis. Transcriptomic analysis indicated the upregulation of genes involved in photosynthesis, CO₂ fixation, carbohydrate metabolism, and iron uptake in the light, and the upregulation of genes related to organic compound oxidation and CO₂ reduction in the dark, indicating both syntrophic and abiotic pathways. The study showed evidence of both syntrophic methanogenesis (in the dark, utilizing organic compounds and H₂) and abiotic methanogenesis (in the light, driven by ROS oxidation of methyl donors). EPR and UV-Vis spectroscopy confirmed the production of various ROS (•OH, O₂⁻, H₂O₂), with H₂O₂ levels significantly elevated in light conditions. Methyl donors, including DMS (likely derived from DMSP oxidation), were identified, providing substrates for abiotic methanogenesis. Scavenger tests showed that ROS played a crucial role in CH₄ production. Abiotic methanogenesis was estimated to contribute 65.4% to the total CH₄ produced. Light-driven methanogenesis was demonstrated with other photosynthetic bacteria from different phyla and methanogens with diverse energy conservation modes, along with various Fe species, highlighting the potential universality of this process.

The study's findings significantly advance our understanding of the global CH₄ cycle by revealing a previously unrecognized pathway for CH₄ production in oxygenated surface waters. The interplay between oxygenic photosynthesis and methanogenesis, involving both syntrophic and abiotic processes, explains the "methane paradox." The observed light-driven methanogenesis, with its dual pathways, is more efficient than conventional methanogenic pathways. The synergistic interaction between the two microorganisms, facilitated by iron redox cycling, highlights the complex microbial interactions within aquatic environments. The broad applicability of light-driven methanogenesis, demonstrated with diverse organisms and iron species, underscores its potential significance in various natural environments. The study emphasizes the necessity of reconsidering the contribution of this pathway to the global CH₄ budget.

This research unveils a novel, potentially widespread mechanism of CH₄ production driven by the interaction between oxygenic photosynthetic bacteria and methanogenic archaea. The process involves both syntrophic and abiotic pathways, occurring under alternating oxic and anoxic conditions during light-dark cycles. The study's findings necessitate reevaluation of the global CH₄ cycle and the role of photosynthetic organisms in this biogeochemical process. Future research should focus on validating the findings in diverse natural environments and quantifying the exact contribution of this pathway to global CH₄ emissions. Investigation into the involvement of other metal elements in this process is also warranted.

The study primarily used laboratory cocultures under controlled conditions. While experiments were conducted under natural sunlight, extrapolation to all natural environments requires further validation. Quantifying the precise contribution of abiotic and biotic methanogenesis in natural settings remains a challenge. The study focused on a limited set of model organisms and iron species, and further research is needed to assess the generality of the findings across a wider range of organisms and environmental conditions.