Aerotolerant methanogens use seaweed and seagrass metabolites to drive marine methane emissions

Methanogenesis is classically thought to be limited to strictly anoxic environments. While ox ygenated oceans are a known methane source, it is argued that methanogenesis is driven by methylphosphonate-degrading bacteria or potentially is associated to zooplankton gut microbiomes rather than by methanogenic archaea. Here we show through in situ monitoring and ex situ manipulations that methane is rapidly produced by archaea in frequently oxygenated sandy sediments. By combining biogeochemical, metagenomic, and culture-based experiments, we show this activity is driven by aerotolerant methylotrophic methanogens (Methanococcoides spp.) broadly distributed in surface layers of sandy sediments, providing evidence of a hidden process contributing to marine methane emissions. Moreover, we show that methane emissions are driven by methylated seaweed and seagrass metabolites, revealing an unexpected feedback loop between eutrophication-driven algal blooms and greenhouse gas emissions. Main Text: Methanogens are typically considered to be strict anaerobes with high sensitivity to oxygen, and therefore restricted to stable anoxic environments (1, 2). While some methanogens have been shown to be able to survive periods of oxygen exposure (3), active methanogenesis in the environment typically recovers only over timescales of weeks to months (4–6). Of total marine emissions, the contribution of near -shore shallow coastal areas is both the largest and the most uncertain and is estimated to constitute around 75% of marine methane emissions globally, offsetting much of the CO 2 drawdown of these highly productive ecosystems (7). While the methane emissions of mangroves, salt marshes and other vegetated coastal environments have been actively studied (8–12), permeable (sandy) coasts have been largely overlooked, despite covering 50% of the world’s continental margins (13). Methane supersaturation is frequentl y observed in near -shore waters overlying permeable sediments, and has generally been explained by input of methane-rich groundwater or riverine water, or seepage of methane from below the sulfate -methane transition zone (14– .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 16, 2024. ; https://doi.org/10.1101/2024.10.14.618369doi: bioRxiv preprint 16). It has also been proposed that excess methane in these zones could be produced by aerobic bacteria during methylphosphonate degradation (17, 18) or through phytoplanktonic photosynthesis processes (19–22). Archaeal methanogenesis has been disregarded as a significant contributor given coastal permeable sediments are charact erized by sudden and unpredictable changes in redox conditions and high sulfate concentrations (23, 24). These characteristics promote the dominance of metabolically flexible facultative anaerobic bacteria (25) and are thought to exclude methanogenic archaea. Here we demonstrate methylotrophic methanogenesis is active in surface sediments under short-term anoxia and is stimulated by seaweed and seagrass (collectively macrophyte) metabolites. By pairing isolation of two strains of Methanococcoides sp. with metagenomic profiling, we show that archaeal methylotrophic methanogens are widespread and active in sandy sediments at sites of both Australia and Europe. High methane levels occur in near-shore surface waters due to sedimentary production Methane concentrations measured in near -shore surface waters over beaches around Port Phillip Bay and Westernport Bay (Australia) and Avernakø (Denmark) are consistently oversaturated with respect to the atmosphere. The extent of saturation was shown to vary over four orders of magnitude, with values ranging from 300% to 160,000% saturated with respect to the atmosphere (Fig. 1A). No relationship was found between methane and radon concentrations (Fig. 1B), indicating methane was produced in either permeable sediments or surface waters rather than through groundwater seepage, contrasting with similar previously described cases such as in the Gulf of Mexico and South Sea of Korea (26, 27). While methane was always supersaturated with respect to the atmosphere, h igher methane concentrations were observed over local (meters to tens of meters) scales adjacent to, or within, seaweed and seagrass mats at both Werribee (Fig. 1B) and Avernakø (Fig. 1A), indicating that this biomass enhances methane production in the environment. To determine whether methane production specifically occurs in the sediment, water column, or on the surface of the seaweed or seagrass biomass , rates of methane production were compared between slurry incubations with combinations of sediment, seawater, and seaweed. The combination of sediment and seaweed stimulated the highest rates of methane production , followed by seaweed only (Fig. 1 D), whereas no methane production was detectable in seawater-only controls or sediment only. This indicates that microbes responsible for methane production are primarily located in sediment, but benefit from substrates derived from seaweed or seagrass. Methylotrophic methanogenesis driven by plant metabolites dominates emissions We initially hypothesized that bacteria or microalgae, rather than methanogenic archaea, may be responsible for methane production in permeable sediments because frequent oxygenation of the surface sand would inhibit archaeal methanogenesis (28). 2-bromoethane sulfonate (BES) was used as a targeted inhibitor of archaeal methanogenesis through its inhibition of methyl-CoM reductase, the terminal enzyme in all known pathways of archaeal methanogenesis (29, 30). BES addition completely inhibited methane production in our .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 16, 2024. ; https://doi.org/10.1101/2024.10.14.618369doi: bioRxiv preprint slurries, indicating that the only methane-producing microbes were methanogenic archaea (Fig. 1F). Methylphosphonates did not stimulate methane production in either slurries (Fig. 1G) or oxic or anoxic flow through reactors (FTRs) (Fig. S3), further ruling out a bacterial source and showing that the pathway of methane production in this environment is different to that which has been used previously to explain the oceanic methane paradox in marine surface waters (i.e. methylphosphonate degradation in the water column) (31–33). Figure 1. (A) Map of in situ surface water methane supersaturation at Australian and Danish sites. Green seaweed symbols indicate sites with high macrophyte accumulation. For detailed site information see table S1. ( B) Methane (nM) versus radon (Bq/m3) concentrations in groundwater (GW), surface water clear of drift algae (SW) and surface water from within a drift algal mat (DA) at Werribee. Error bars indicate standard deviation of triplicate samples (methane) or error given by 222Radon analyzer. (C) Flow -through reactor (FTR) ex periments show methane production after onset of anoxia and macrophyte extract addition (dashed line) in surface sediments (0 -5 cm) from a site with macrophyte accumulation (Shoreham) and sites without accumulation (Werribee and St Kilda). Error bars represent standard deviation from mean of four independent FTRs. Note broken y axis. (D) Methane production in slurry incubations of seawater, sediment and seaweed (brown drift algae) from Werribee. (E) Methane production in Werribee sediment slurry incubations with spirulina additions with and without .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 16, 2024. ; https://doi.org/10.1101/2024.10.14.618369doi: bioRxiv preprint specific archaeal methanogenesis inhibitor BES (20 mM). (F) Methane production in sediment slurry incubations with addition of 100 µM concentrations of trimethylamine (TMA), dimethyl sulfide (DMS), acetate, hydrogen (340 ± 20 ppm), methylamine and methylphosphonate (MPn) in sediments from Shoreham and Werribee. (G) Methane production in sediment slurry incubations with addition of 100 µM concentrations of trimethylamine (TMA) and choline in sediments from Avernakø, Denmark. All slurries were prepared with surface sediment (0-5 cm) and argon-purged at the start (time = 0 h). Error bars of all slurry experiments represent standard deviation from mean of three independent slurries. We investigated acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis pathways using targeted substrate addition (Fig 1 F) and found that methylotrophic methanogenesis predominated. This is likely due to the fact that many organisms such as sulfate and nitrate reducers outcompete methanogens for acetate and hydrogen, but not for methylated substrates, allowing methylotrophic methanogenesis to occur in unreduced environments with abundant alternative electron acceptors (34, 35). Additionally, surface sedime nts are often exposed to high fluxes of labile methylated compounds and may therefore be adapted to quickly utilize these substrates as described in seagrass meadows (36). Methylotrophic methanogenesis was similarly stimulated by trimethylamine (TMA) and dimethyl sulfide (DMS) (Fig. 1F); these are abundant compounds in coastal regions and are formed through the breakdown of widely occurring macrophyte osmolytes such as glycine betaine, choline, and dimethyl sulfoniopropionate (DMSP) (37, 38). The rate of potential methane production from sediment at sites with different levels of macrophyte accumulation was examined with FTR experiments to investigate the role of community composition and priming effects. FTRs more realistically emulate the adv ective flow which is caused by wave and tidal pumping in surface permeable sediments (39, 40). Remarkably, methanogenesis started within ~20 hours of the transition to anoxia and addition of macrophyte extract in FTRs from all sites (Fig. 1C), which is much faster than previously reported recovery times of weeks to months (4–6), regardless of site. However, the rate of methane production at the site with the most macrophyte accumulation (Shoreham) was both faster (within 1.5 hours) and four orders of magnitude higher (at 20 hours) than the two sites with less accumulation. This indicates that permeable sediments may generally harbor the latent potential for methanogenesis, but supports our hypothesis that macrophyte accumulation causes changes in abundance and/or activity of methanogens , resulting in high er methane production rates. While methane production rates are difficult to compare between different sediment types due to the differences in advective and diffusive flux, some comparisons can be made between our experimental rates to place them in the context of more well -studied environmental fluxes. If integrated conservatively over a 0.5 cm permeable sediment depth as the depth of advective penetration of high -substrate surface water in the intertidal zone (41), the maximum methane production rate at Shoreham is approximately 3.8 g/m2/h, nearly three orders of magnitude higher than flux rates recently reported for tropical wetlands (42, 43). The CH4:CO2 carbon remineralization ratio reached 1: 9 after 44 hours (Fig. S 4), exceeding the typical ratio for most types of wetland sediment and indicating that methanogenesis may be a quantitatively important carbon remineralization path in permeable sediments (44). These findings highlight not only the ability of methanogenic archaea to adapt highly dynamic environments and recover extremely quickly after oxygen exposure, but also that their methane .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 16, 2024. ; https://doi.org/10.1101/2024.10.14.618369doi: bioRxiv preprint production rates can be comparable to, or even exceed, those of the most active methanogenic environments studied (44–46). Together, these field data and inhibitor slurry experiments show that archaeal methanogenesis is not only feasible in surface sandy sediments, but in some cases, appears to be the major source of methane to the overlying water and subsequent emissions. Furthermore, these results reveal a mechanism linking macrophytes to elevated methane concentrations, which could be significant in the context of increasing ocean eutrophication and enhanced macrophyte growth. Novel archaeal isolates and sediment metagenomes confirm aerotolerant methanogenic potential Methanogens were isolated from both Australian and Danish sites t o investigate the aerotolerance of methanogens from permeable sediments, independent of potential anaerobic sand grain microniches caused by bacterial oxygen consumption. Two methanogens from the genus Methanococcoides (family Methanosarcinaceae) were isolated from surface sands (0-5 cm), one from Avernakø, Denmark (strain DA) the second from Shoreham, Australia (strain SH). Based on average nucleotide identity (ANI) comparisons with all representative genomes of Methanococcoides, DA was most closely related to Methanococcoides burtonii (ANI value 89.3) while SH was most closely related to Methanococcoides orientis (ANI value 90.0) . Between the two isolates , the ANI value was 80.3 . In support of the biogeochemical observations, both methanogens rapidly produced methane in the presence of TMA (Fig. 2A). Moreover, activity of these methanogens resumed immediately following transient (approximately 30 min) exposure to 6-9 mg/L oxygen (Fig 2A). Figure 2. (A) Methane production after oxygen exposure in sand isolates Methanococcoides sp. DA and SH. (B) Read mapping of metagenomic reads to isolate sequences from post-FTR samples (Shoreham) .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 16, 2024. ; https://doi.org/10.1101/2024.10.14.618369doi: bioRxiv preprint and in situ samples from three sites collected at the same time as the FTR sediment samples. (C) mcrA gene abundance in all metagenome samples. (D) Maximum -likelihood phylogenetic genome tree showing both isolates (bold) and reference genomes. Amino acid sequences from the isolated genomes were aligned with reference genomes. Coloured boxes indicate family (dark shade) and phylum (light shade). Legends for tree scale and bootstrap of 100 are reported in the bottom left corner. (E) Maximum- likelihood phylogenetic gene tree of mcrA sequences, displaying isolates (bold), contigs from metagenomic analyses of in situ and post-FTR sand samples (bold), and reference sequences. Amino acid sequences of isolates and contigs were aligned with reference sequences. Coloured boxes indicate family (dark shade) and class (light shade). Legends for tree scale and bootstrap o f 90 and 100 are reported in the bottom left corner. Sequence alignments used to generate these phylogenetic trees are provided and source trees are available in tree file format (see data and materials availability below). Read mapping of metagenomic reads to the isolate genomes revealed that both strains were present in in situ samples from all sites and their abundance increased 9 to 10 -fold with the macrophyte extract treatment in FTRs (Fig. 2B). Furthermore, a nalyses of the genome sequences of both isolates revealed remarkable similarities in their methanogenesis pathways and antioxidant systems, despite being isolated from geographically and climatically distinct locations, suggesting that these traits are important for adaptation in this environmental niche. Both isolates encoded a complete methylotrophic methanogenesis pathway with methyltransferases for tri -, di - and monomethylamines and methanol ( mttABC, mtbABC, mtmABC and mtaABC respectively). No methylthiol methyltransferases ( mts) were found, despite DMS equally stimulating methane production to TMA in slurries (47). It has been proposed that some methanogens may use mtt and mta methyltransferases to metabolize DMS (47). Both genomes encode various genes involved in oxidative stress protection, including the F420H2-dependent oxidase that detoxifies O 2 to water and is unique to methanogens (48), as well as those typically associated with aerobic organisms such as superoxide reductase and catalase-peroxidase (49). Additionally, both genomes encoded a variety of other proteins with potential antioxidant roles such as rubredoxins (50), thioredoxins (51), and peroxiredoxins (3, 52, 53). This abundance and diversity of genes involved in oxidative stress protection supports the finding of remarkable robustness to, and quick recovery from, oxygen exposure. Shotgun metagenomics was undertaken to determine if methanogens and their functional genes were present in the permeable sediments. Samples for analysis were collected at the beginning of the FTR experiment for all sites, as well as at the end of the experiment for Shoreham samples. The marker gene for archaeal methanogenesis, McrA (encoding methyl- CoM reductase), were found in samples from all sites (Fig. 2C). This gene was encoded by a relatively small proportion of the community (0. 094 – 1.33%) in line with previous observations for surface permeable sediments (25, 54, 55); however, it’s well-established that methanogens are highly transcriptionally and biogeochemica lly active even in niches where their abundance is low (11). Furthermore, McrA gene abundance increased from 0.22% to 1.33% (a six-fold increase) in the Shoreham FTR experiment macrophyte extract treatment but not in the control, indicating growth of methanogens. Most sequences were affiliated with Methanosarcinaceae (Fig. 2E), all of which are known to utilize methylated substrates (35), giving a genetic basis for the experimental finding that all sites harbor the latent potential for methylotrophic methanogenesis. This suggests a dormant pool of these methanogens exists in sands, with growth stimulated by macrophyte metabolites and anoxic conditions. .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 16, 2024. ; https://doi.org/10.1101/2024.10.14.618369doi: bioRxiv preprint Potential feedback pathways amid ongoing local and global pressures on coastal ecosystems By integrating in situ and ex situ data, spanning culture -independent and culture -based experiments, we observe a previously undescribed mechanism for methane production in coastal environments. Methanogens can be adapted to more frequently oxygenated environments than previously thought and sandy coasts broadly harbor the potential to convert macrophyte-derived substrates to methane. These methanogens may help to explain the oceanic methane paradox in marine surface waters of coastal settings , in addition to the previous explanation of methylphosphonate degradation in the water column (31–33). The shallow and turbulent nature of waters overlying coastal permeable sediments , combined with advective transport in the sediments, gives the study additional significance. In deeper waters and cohesive sediments, the balance of methanogenesis and methanotrophy is such that in the bulk of the ocean volume is undersaturated in methane with respect to the atmosphere (56). However, in rippled permeable sediments the redox seal is broken, with flow from reduced reaction zones exported directly through ripple peaks or lee sides depending on bedform and flow interactions (57, 58). This allows methane produced in shallow anoxic regions to bypass the methane oxidation filter and reach the shallow overlying water where low residence times and high turbulence causes high rates of export t o the atmosphere (14, 59). Therefore, the contribution of methane production in shallow permeable sediments to total marine methane emissions is likely disproportionately large. Furthermore, the link between macrophyte biomass and methane emissions has significance in the context of current and predicted ocean changes due to climate change and other anthropogenic impacts. Eutrophication and rising sea temper atures in particular are linked to increased algal growth and the frequency of large algal blooms in coastal zones (60– 63). Here, we have shown that depo sition of this excess algal biomass on sandy coasts may

in increasingly large and frequent pulses of methane to the atmosphere and should be accounted for in future marine methane budgets and modelling. As well as unintentional excess macrophyte growth caused by eutrophication, the results of this study further complicate CO 2 removal by macrophytes or “blue carbon” as a climate change mitigation strategy (7), as enhanced methane emissions may offset much of the CO2 removal by these ecosystems.

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12, 59–60 (2015). 78. H. R. Gruber-Vodicka, B. K. B. Seah, E. Pruesse, phyloFlash: Rapid Small-Subunit rRNA Profiling and Targeted Assembly from Metagenomes. mSystems 5, 10.1128/msystems.00920-20 (2020). 79. W. E. Wentworth, S. V. Vasnin, S. D. Stearns, C. J. Meyer, Pulsed discharge helium ionization detector. Chromatographia 34, 219–225 (1992). Acknowledgments: The authors would like to thank Rodney Hall , Vera Eate, and Michael Tolj for their technical assistance in sample analysis. Thanks to Anni Glud for experimental help and Isabell Schlangen and Annabell Moser for field assistance. Funding: Australian Research Council grant DP210101595 (PLMC, CG, WWW) National Health & Medical Research Council fellowship APP1178715 (CG) Australian Government Research Training Program Scholarship (NH) European Research Council ERC 101045149 (AER, SK) Danish Research Council DFF 1026-00159 (AER) Danish National Research Foundation, DNRF145 (RNG, NH) .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 16, 2024. ; https://doi.org/10.1101/2024.10.14.618369doi: bioRxiv preprint Author contributions: Conceptualization: NH, PLMC, CG Methodology: NH, WWW, RL, SK, FR, AER Investigation: NH, RL, FR Visualization: NH, FR Funding acquisition: CG, PLMC, WWW, AER Project administration: PLMC, CG, RNG Supervision: CG, PLMC, RNG Writing – original draft: NH Writing – review & editing: NH, PLMC, CG, RL, FR, AER, SK, WWW, RNG Competing interests: Authors declare that they have no competing interests. Data and materials availability: All scripts for bioinformatic analysis, supplementary trees and tree files available at https://github.com/GreeningLab/Sand-methanogen- manuscript Sequence data including metagenomes, isolate genomes, and contigs used for analysis available at NCBI S equence Read Archive BioProject ID: PRJNA1165813 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1165813 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted October 16, 2024. ; https://doi.org/10.1101/2024.10.14.618369doi: bioRxiv preprint