Potential for Manganese Oxide Driven Anaerobic Methane Oxidation in Sediments of a Seasonally Euxinic Coastal Basin

TL;DR

This study explores how manganese oxides can help remove methane in sediments of a coastal basin with low oxygen levels.

Contribution

The study identifies manganese oxides as potential electron acceptors for anaerobic methane oxidation in seasonally euxinic coastal sediments.

Findings

Manganese oxides like birnessite, pyrolusite, and manganite enhance methane oxidation in sediments.

Anaerobic methanotrophic archaea (ANME-2ab and ANME-3) are likely involved in manganese-driven methane oxidation.

Methanosarcina methanogens increase in abundance under manganese oxide reduction conditions.

Abstract

Methane (CH4) is a strong greenhouse gas that, in marine sediments, is produced via methanogenesis and removed via oxidation with electron acceptors such as oxygen, sulfate and metal oxides. This study assesses the potential for manganese driven anaerobic oxidation of methane (Mn-AOM) in rapidly accumulating sediments in a seasonally euxinic coastal marine basin (Scharendijke basin, Lake Grevelingen, the Netherlands). Geochemical sediment and porewater profiles demonstrate that, at the study site, Mn oxides are buried in the methanic zone. Sediment incubations amended with 13C-CH4 and various Mn forms indicate that the Mn oxide minerals birnessite, pyrolusite and manganite can enhance CH4 oxidation, whereas ligand bound dissolved Mn(III) does not. This is attributed to either direct Mn-AOM, where Mn oxides act as the electron acceptor, and/or indirect Mn-driven AOM via cryptic sulfur cycling. Results of 16S rRNA gene amplicon sequencing of the incubated sediment point towards a likely role for anaerobic methanotrophic archaea of the clades ANME-2ab and ANME-3 in direct and indirect Mn-driven AOM, respectively. An increase in relative abundance of the methanogen Methanosarcina in several of the incubations suggests that these versatile methanogens benefit from Mn oxide reduction. This study demonstrates potential for Mn-driven AOM in sediments from a eutrophic, seasonally euxinic coastal basin, and expands the range of electron acceptors involved in CH4 removal in such systems.

The online version contains supplementary material available at 10.1007/s12237-026-01685-7.

Figures

Sediment profiles of Mo, C_org_, Mn oxides, Fe oxides, total S, FeS and FeS_2_ and pore water profiles of SO_4_^2−^, CH_4_, dMn, dFe and H_2_S in October 2021. The depth intervals surrounded by a red box indicate the depths that were used for the incubations. The gray shading represents the sediment that was deposited during bottom water euxinia and the white areas represent the sediment that was deposited during oxic bottom water conditions

Headspace ^13^C-CO_2_/^12^C-CO_2_ ratios and dissolved Mn concentrations for the incubations with sediment from (a—c) 0–5 cm depth and (d—f) 15–20 cm depth amended with birnessite (a, d) pyrolusite (b, e) and manganite (c, f). The ^13^C-CO_2_/^12^C-CO_2_ and dissolved Mn concentrations of the controls are also indicated in each plot. The dashed lines indicate timepoints when new Mn oxides and new CO_2_ were added to the incubations

SO_4_^2−^ and H_2_S concentrations for the incubations with sediment from (a—c) 0–5 cm depth and (d—f) 15–20 cm depth spiked with (a, d) birnessite, (b, e) pyrolusite and (c, f) manganite over time. The SO_4_^2−^ concentrations of the controls are also indicated in each figure. The dashed line represents the time point when new Mn oxide minerals were added to all incubation bottles

Headspace ^13^CO_2_/^12^CO_2_ ratios and dissolved Mn concentrations for the incubations with sediment from (a, b) 0–5 cm depth and (c, d) 15–20 cm depth spiked with (a, c) dMn(III)-L bound to a strong ligand and (b, d) dMn(III)-L bound to a weak ligand over time. The dashed lines indicate timepoints when new dMn(III)-L was added to the incubations

Relative abundance of (a) selected archaea and (b) bacteria based on 16S rRNA gene amplicon sequencing. The entire archaeal and bacterial communities can be found in supplementary figure S5

Full text

Introduction

Methane (CH_4_) is a potent greenhouse gas with a concentration in the atmosphere that has more than doubled since the start of the Industrial Revolution (Saunois et al., 2020). Aquatic ecosystems account for approximately half of total global CH_4_ emissions (Rosentreter et al., 2021), with inland and oceanic waters estimated to contribute ~ 261 and ~ 8 Tg CH_4_ per year, respectively (median values from Rosentreter et al. (2021)). Given that global rates of CH_4_ production in coastal marine sediments range up to at least 49 Tg CH_4_ per year and are expected to increase due to eutrophication (Egger et al., 2018), there is a crucial need to understand the processes controlling CH_4_ removal in coastal systems.

In coastal sediments, methanogens produce CH_4_ via methanogenesis as the final step in anaerobic organic matter degradation (Froelich et al., 1979). A major proportion of the CH_4_ is thought to be removed via anaerobic oxidation of CH_4_ (AOM) coupled to SO_4_^2−^ reduction (S-AOM) in the sulfate methane transition zone by a consortium of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB; Knittel & Boetius, 2009). Other electron acceptors, such as nitrate and nitrite (N-AOM; Raghoebarsing et al., 2006), iron (Fe) and manganese (Mn) oxides (Fe-AOM, Mn-AOM; Beal et al., 2009; Egger et al., 2015; Cai et al., 2018; Leu et al., 2020), have also been linked to AOM, but their role is less well understood. Although Fe oxides are typically more abundant than Mn oxides in marine sediments, the Mn oxide birnessite is thermodynamically a more favorable electron acceptor for AOM, as shown with gene-based and bioenergetic modelling (Lenstra et al., 2023; Wallheimer et al., 2025).

Typically, Mn oxides are enriched in oxic surface sediments. Reductive dissolution upon burial generally leads to loss of the Mn oxides through biotic and abiotic processes, producing dissolved Mn (Burdige, 1993), either as dissolved Mn(II) (dMn(II)) or, when stabilized by organic ligands, dissolved Mn(III) bound to ligands (dMn(III)-L; Madison et al., 2011). Upward diffusion into oxygenated sediment layers can result in re-oxidation of the dissolved Mn and precipitation as Mn oxides (Adelson et al., 2001; Slomp et al., 2013). Burial of Mn oxide minerals into the methanic zone (i.e. the zone where methane is present; Canfield & Thamdrup, 2009) is promoted by non-steady state conditions such as variations in Mn oxide input, high sedimentation rates or bioturbation (Lenstra et al., 2023; März et al., 2008; Riedinger et al., 2014). Both dMn(II) and dMn(III)-L can be present in the methanic zone, as products of Mn oxide reduction, or via downward diffusion (Klomp et al., 2025). In the methanic zone, the dMn(III)-L could act as an electron acceptor for AOM.

In freshwater systems, Mn-AOM is commonly associated with Candidatus Methanoperedens (ANME-2d; Ettwig et al., 2016; Leu et al., 2020; Su et al., 2020). In a bioreactor inoculated with freshwater sediment and enriched in Ca. Methanoperedens, for example, CH_4_ oxidation was directly coupled to Mn oxide reduction, presumably via extracellular electron transport involving multiheme c-type cytochromes (Leu et al., 2020). An indirect role for Mn in AOM was observed when Mn oxides chemically oxidized reduced sulfur (S) species, such as iron sulfide (FeS_x_) and organically bound S, forming SO_4_^2−^ in a cryptic S cycle that fueled S-AOM (Su et al., 2020). Besides solid phase Mn oxides, dMn(III)-L was also tested for AOM in freshwater sediments, but AOM was not detected (Szeinbaum et al., 2020). Mn-AOM has also been proposed to occur in marine sediments (Beal et al., 2009), but the main microbial players and pathways remain enigmatic (Wallenius et al., 2021; Xue et al., 2025). Fe-AOM in brackish and marine sediments has been linked to the ANME clades ANME-2ab (family Methanocomedenaceae), and potentially involves a bacterial metal-reducing partner (Aromokeye et al., 2020; Rasigraf et al., 2020). ANME-2ab are also hypothesized to be involved in Mn-AOM in marine sediments (Xu et al., 2021).

In the laboratory experiments to date assessing the use of solid phase Mn as an electron acceptor for AOM, only the Mn(IV) oxides birnessite (MnO_2_ * n H_2_O) and vernadite (MnO_2_ * n H_2_O) were tested (e.g. Beal et al., 2009; Ettwig et al., 2014; Su et al., 2020; Xu et al., 2021). In the natural environment, Mn oxides may also be present in more crystalline form, e.g. as manganite (Mn(III)OOH) or pyrolysite (Mn(IV)O_2_); e.g. Burdige et al., 1992; Sulu-Gambari et al., 2016; Luo et al., 2018). Since the crystallinity and redox state of Mn oxide minerals will affect their bioavailability and their reactivity, for example towards FeS (Aller & Rude, 1987; Burdige et al., 1992), variations in rates of CH_4_ oxidation and cryptic sulfur cycling involving different Mn oxide minerals are expected but have not yet been explored.

In this study, we examine the role of different forms of Mn that are typically found in marine sediments, namely the Mn oxides birnessite, pyrolusite, manganite and of dMn(III)-L in AOM to assess whether these Mn oxides can support AOM in coastal sediments and we examine the influence of their mineralogy on Mn-AOM. We focus on rapidly accumulated sediments in a seasonally euxinic marine coastal basin (Scharendijke basin, Lake Grevelingen, the Netherlands), where Mn oxides are buried in the methanic zone of the sediment and dMn(III)-L is present in the porewater (Klomp et al., 2025; Żygadłowska et al., 2023). We combine geochemical sediment and porewater profiles with results of sediment incubations using isotopically labelled CH_4_ and the various Mn oxides and dMn(III)-L to assess potential for Mn-AOM. The microbial community in the incubations was analyzed using 16S rRNA gene amplicon sequencing.

Materials and Methods

Study Area

Lake Grevelingen is a coastal marine lake in the southwest of the Netherlands. The lake is a former estuary of the river Rhine that was dammed at the landward side in 1964 and at the seaward side in 1971. The lake has an average water depth of around 5 m, but is intersected by former tidal channels with deep basins that can reach depths of up to 45 m (Egger et al., 2016; Hagens et al., 2015). Exchange with water from the North Sea is enabled by sluices in the dam at the seaward side, giving the lake a salinity of 29–33. The lake is highly eutrophic (Hagens et al., 2015).

This study focuses on sediments in the deepest part of the lake, the Scharendijke basin (51.742^◦^N, 3.849^◦^E). Here, temperature-driven stratification leads to seasonally euxinic conditions between May and September, recorded in the sediment by distinct enrichments of the redox-sensitive trace metal molybdenum (Mo; Egger et al., 2016; Żygadłowska et al., 2024a). The sedimentary Mo profiles can also be used to determine the rate of sediment accumulation, which is exceptionally high and varied between 13—20 cm yr^−1^ over the last decade (Egger et al., 2016; van Helmond et al., 2025). The geometry of Scharendijke basin, i.e. relatively narrow and deep, leads to lateral transport of material from shallower areas of the lake (in a process termed “sediment focusing”), which promotes vertical settling of suspended matter, explaining the high sediment accumulation rates (Klomp et al., 2025). High input of organic matter leads to high rates of SO_4_^2−^ reduction. Furthermore, high rates of methanogenesis lead to strong benthic release of CH_4_ via diffusion and ebullition (Klomp et al., 2025; Żygadłowska et al., 2024b). In winter, when the bottom water is oxygenated, the deposition of Fe and Mn oxides prevents the accumulation of sulfide (H_2_S) in the upper 10 cm of the sediment (Klomp et al., 2025). Through summer, due to the high sedimentation rates, the manganese oxides are buried into the methanic zone of the sediment. Besides solid phase Mn oxides, dMn(III)-L is also abundantly present in the methanic zone (Klomp et al., 2025). While the sediments record strong seasonal changes, there is little spatial variability at this location (van Helmond et al., 2025).

Sample Collection

Sediment sampling in the Scharendijke basin was performed on board the RV Navicula in October 2021. Three sediment cores were collected using a UWITEC corer and PVC core liners (120 cm length, 6 cm inner diameter). Bottom water was collected from the overlying water from one of the cores (in duplicate) using a syringe that was closed with a three-way tap and processed further as described for the porewater. One core was sectioned at a 1 cm resolution in a glove bag under an N_2_ atmosphere. The sediment from each slice was transferred into a 50 ml centrifuge tube and centrifuged at 4500 rpm for 20 min to separate the porewater from the solid phase. The supernatant from each tube was filtered over 0.45 μm pore size filters under a N_2_ atmosphere in a glove bag and subsampled for the analysis of SO_4_^2−^, total dissolved Mn and Fe and H_2_S. The samples for SO_4_^2−^ were stored at 4 °C. Samples for the analysis of total dissolved Mn and Fe were acidified with 10 μL 30% suprapur HCl per ml of sample and stored at 4 °C whereas samples for H_2_S analysis were diluted five times in a 2% Zn-acetate solution in a glass vial and stored at 4 °C. The residual sediment was stored in N_2_ purged aluminum bags at −20 °C until further processing for C_org_, total sulfur (S) and Mo, Mn and Fe oxides and FeS analysis.

A second core was used to sample for porewater CH_4_ determination directly upon arrival of the core on deck. Through tape-covered pre-drilled holes at a 2.5 cm interval, 10 ml of sediment was transferred with plastic cutoff syringes into a 65 ml glass bottle filled with a saturated NaCl solution. The bottles were then stoppered, capped and stored upside down until analysis. Note that degassing of CH_4_ occurred during the sampling, which results in a strong underestimation of the CH_4_ concentrations (Egger et al., 2016; Jørgensen et al., 2019).

The third core was sectioned at a 5 cm resolution in a N_2_ filled glove bag to collect material for sediment incubations. The sediment was transferred into plastic bags and stored in N_2_ purged sealed aluminum bags at 4 °C until the start of the experiments.

Chemical Analyses of Sediment and Porewater

Concentrations of SO_4_^2−^ were determined via ion chromatography (Metrohm 930 Compact IC Flex; detection limit for SO_4_^2−^ of 50 µmol L^−1^). Total dissolved Mn and Fe were determined by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES, Perkin Elmer Avio; detection limit 0.1 µmol L^−1^ and 0.03 µmol L^−1^ for Fe and Mn, respectively). Concentrations of H_2_S were measured spectrophotometrically using an acidified solution of phenylenediamine and ferric chloride, where H_2_S is the sum of S^2−^, HS^−^ and H_2_S (detection limit of 1 µmol L^−1^; Cline, 1969).

The sediment was freeze-dried and ground with an agate mortar and pestle under a N_2_ atmosphere and subsequently split in an aliquot that was stored under oxic conditions and an aliquot that was stored and processed under anoxic conditions. The C_org_ content was determined on approximately 0.3 g of the oxic split that was decalcified with 1 M HCl (Van Santvoort et al., 2002), dried, weighed and powdered. Consecutively, the decalcified sample was analyzed on an elemental analyzer (Fisons Instruments model NA 1500 NCS) and the C content was corrected for the weight loss during decalcification. Accuracy and precision of this method was determined based on measurements of the internationally certified soil standard IVA2. The certified value for IVA2 is 0.732 wt.% C. The mean value that was obtained in this study for IVA2 (n = 15) was 0.739 wt.% C, with a standard deviation of 0.005 wt.% C. The analytical uncertainty based on duplicate analysis of single sample (n = 8) for C_org_ was 0.05 wt.%. The total elemental composition of the sediment was determined after digestion of approximately 0.1 g of the oxic split in 2.5 ml mixed acid (HNO_3_; HClO_4_^−^; 2:3) and 2.5 ml 40% HF at 90˚C. After evaporation of the acid mixture, the residue was redissolved in 1 M HNO_3_ and the total elemental composition was determined via analysis on the ICP-OES for the total S content and via Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Perkin Elmer NexION 2000) for the total Mo content of the sediment. The average analytical uncertainty based on duplicates (n = 8) was 106 ppm for S and 114 ppb for Mo and the average recovery rate was 103% for S and 97% for Mo. All elemental concentrations were corrected for the salt content of the freeze-dried sediment.

Two aliquots of the anoxic split (ca. 0.1 g) were subjected to a sequential extraction to determine the speciation of Mn and Fe. One of the aliquots was digested in a 1 M HCl solution for 4 h to extract easily reducible Fe(III) and Fe(II) minerals as described in Kraal et al. (2017). The concentrations of Fe(III) and Fe(II) were determined spectrophotometrically via the phenantroline method (APHA, 2005). The average analytical uncertainty based on duplicates (n = 5) was 0.14 μmol g^−1^. Speciation of Mn and further speciation of Fe was determined following the 5 step sequential extraction as described by Lenstra et al. (2021b). The steps were as follows: (1) 0.057 M ascorbic acid, 0.17 M sodium citrate and 0.6 M sodium bicarbonate (pH 7.5) which extracts poorly ordered Mn oxides and Mn associated with vivianite, (2) 1 M HCl which extracts Mn carbonates (3) 0.35 M acetic acid, 0.2 M Na citrate and 50 g L^−1^ Na dithionite, pH 4.8, which extracts crystalline Fe and Mn oxides and Mn bound to clays (4) 0.2 M ammonium oxalate and 0.17 M oxalic acid which extracts recalcitrant Fe oxides, crystalline Mn oxides and Mn bound to clays (5) 65% HNO_3_ which extracts pyrite, including any associated Mn. The analytical uncertainty based on duplicates (n = 9) for all fractions was 5.69 and 0.25 μmol g^−1^ for Fe and Mn, respectively.

Approximately 0.3 g of the anoxic splits was used to determine the FeS content, via the passive diffusion method as described by Burton et al. (2008). The FeS was extracted from the sediment via a 24 h digestion in 6 M HCl in a 50 ml polypropylene centrifuge tube. Released H_2_S was trapped in an alkaline zinc-acetate solution as zinc sulfide precipitates. Iodometric titration of the zinc-acetate solution was used to quantify the released sulfide (Burton et al., 2008). The analytical uncertainty based on duplicates (n = 4) was 2.56 μmol g^−1^.

Sediment Incubations

The potential for Mn-AOM was tested via incubations with sediment from the top layer of the sediment (0—5 cm) and from a sediment interval expected to contain Mn oxides below the sulfate methane transition zone (15—20 cm; Klomp et al., 2025). Incubations were started within 3 months after sampling. The sediment was amended with various types of Mn oxides, namely birnessite, pyrolusite and manganite, and with dMn(III)-L, where the ligand was either strong or weak (Table 1). Furthermore, isotopically labelled ^13^C-CH_4_ was added to the incubations. Oxidation of ^13^C-CH_4_ produces ^13^C-CO_2_ which is tracked via the ratio of ^13^C-CO_2_ to ^12^C-CO_2_ (Beal et al., 2009; Egger et al., 2015). We note that, in our incubations, we aimed to optimize conditions for Mn-AOM. This implies that the conditions during incubation do not reflect in-situ conditions and, for example, much higher amounts of Mn oxide were added than observed in-situ. Various chemical and microbial analysis described below were used to monitor CH_4_ oxidation and key microbes in the incubations.Table 1. Overview of the different treatments performed in the incubation experimentTreatment^13^CH_4_ addedAmount of electron acceptor addedControl, no substrate--CH_4_ oxidation control20% headspace-Birnessite; Mn(IV)O_2_ * n H_2_O20% headspace10 mmol L^−1^Pyrolusite; Mn(IV)O_2_20% headspace100 mmol L^−1^Manganite; Mn(III)OOH20% headspace100 mmol L^−1^dMn(III)-L strong ligand20% headspace1 mmol L^−1^dMn(III)-L weak ligand20% headspace1 mmol L^−1^

Preparation of the Substrates

Birnessite was synthesized by reducing KMnO_4_ with C_3_H_5_NaO_3_ at ambient temperature and pressure according to the protocol by Händel et al. (2013). Pyrolusite and manganite were obtained from the inhouse collection of Utrecht University; the original sources for pyrolusite and manganite were Alfa Aesar and Ward, respectively (Lenstra et al., 2021b). The mineralogy was verified using X-ray diffraction (Fig. S1). The crystallinity of Mn oxide minerals can affect their reactivity (Burdige et al., 1992). To compensate for the potential lower reactivity of crystalline pyrolusite and manganite compared to birnessite, in case the availability of Mn oxides would be limiting for Mn-AOM, higher concentrations of these minerals were added to the incubations (Table 1). Dissolved Mn(III)-L was prepared with a weak binding ligand, pyrophosphate, and a strong binding ligand, desferrioxamine B (DFOB), as described in Oldham et al. (2015). Isotopically labelled ^13^C-CH4 was obtained from Cambridge Isotope Laboratories, Inc. (Andover, USA).

Start of the Incubations

The bottles were pre-incubated for one week with the electron acceptor as indicated in Table 1, but without labelled CH_4_, to remove as much FeS from the sediment as possible and prevent the formation of SO_4_^2−^ via oxidation of FeS by Mn oxides in the subsequent incubations with labelled CH_4_. The sediment was diluted in a 1:4 ratio with artificial HEPES buffered, SO_4_^2−^-free seawater (ASW; for composition, see Table S1) under a 98% N_2_ + 2% H_2_ atmosphere, and carefully homogenized. This mixture was equally distributed over sterile 120 ml serum bottles, so that each bottle contained 60 g of the mixture. The electron acceptors were added to the bottles (Table 1) and the bottles were stoppered and capped. The headspace was replaced with a 100% N_2_ environment and the bottles were placed on a shaking Table (90 rpm) at room temperature for one week.

After the pre-incubation, the ASW was replaced with freshly prepared ASW and new electron acceptors were added to each bottle under a 98% N_2_ + 2% H_2_ atmosphere. The headspace of the bottles was replaced with a headspace of 76% N_2_, 4% CO_2_ and 20% ^13^C-labelled CH_4_, which is based on earlier studies (Egger et al., 2015; Wallenius et al., 2025). The incubations were placed on a shaking Table (90 rpm) at room temperature for 92 days. The length of the experiment reflects the expected rate of Mn-AOM and is based on previous incubations studying metal AOM (Beal et al., 2009; Egger et al., 2015).

Sampling and Analyses of the Incubations

From the start of the incubations, the ratio of ^13^C-CO_2_/^12^C-CO_2_ was determined weekly with a GC–MS (5975C inert MSD, Agilent, Santa Clara, CA, USA). We note that direct measurements of ^13^C-CH_4_ cannot be used to quantify AOM because the changes in concentration of ^13^C-CH_4_ are too small to be detected on the GC–MS. When CO_2_ concentrations are very low, the ^13^C-CO_2_/^12^C-CO_2_ ratio measured by the GC–MS decreases, because the ^13^C-CO_2_ reaches the detection limits faster than ^12^C-CO_2_ (Fig. S2). Upon the addition of Mn oxides, CO_2_ was drawn from the headspace (Fig. S2), possibly linked to an increase in pH in the liquid due to high Mn oxide reduction rates (Silburn et al., 2017). However, the increase in pH upon Mn reduction is expected to be limited due to the use of HEPES buffer in the medium. The drawdown of CO_2_ from the headspace was highest in the incubations amended with pyrolusite and manganite, since extra Mn oxides were added to compensate for the expected lower reactivity of these minerals (Burdige et al., 1992). To compensate for the loss of CO_2_ from the headspace, new CO_2_ was injected into the headspace of all bottles amended with Mn oxide minerals after 52 days. Rates of AOM could not be calculated from the ^13^C-CO_2_/^12^C-CO_2_ ratios in the incubations because of the drawdown of CO_2_.

Every two weeks, the supernatant was sampled to analyze concentrations of SO_4_^2−^, H_2_S and total dissolved Mn. Concentrations of SO_4_^2−^, H_2_S and total dissolved Mn were analyzed as described above. During the setup of the pre-incubations and at the end of the incubations, sediment was sampled for DNA isolation. The samples for DNA isolation were stored frozen (−20°C) until analysis via 16S rRNA amplicon sequencing.

DNA was isolated from approximately 0.2 g of defrosted sediment with DNeasy PowerSoil DNA isolation kit (Qiagen, Venlo, Netherlands) after bead-beating for 10 min at 50 Hz on a TissueLyser LT (Qiagen, Venlo, Netherlands). DNA quantity and quality in the eluted sterile MilliQ were measured by NanoDrop 1000 (Thermo Fischer Scientific, Bremen, Germany) and by Qubit® 2.0 (Invitrogen, Waltham, MA, USA). The DNA was stored frozen at −20˚C until further analysis.

Amplification of 16S rRNA genes was performed on samples from the incubations from the 15–20 cm depth, using primers Arch349F (5′-GYGCASCAGKCGMGAAW-3′) and Arch806R (5′-GGACTACVSGGGTATCTAAT-3′; (Takai & Horikoshi, 2000) and Bac341F (5′CCTACGGGNGGCWGCAG-3′; Herlemann et al., 2011) and Bac806R (5′GGACTACHVGGGTWTCTAAT-3′; Caporaso et al., 2012). The amplicon sequencing was performed on the Illumina MiSeq Next Generation Sequencing platform by Macrogen (Seoul, South Korea) using Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2, yielding 2 × 300 bp paired-end reads. FastQC (v0.11.5; Andrews, 2010) was used to check the quality of the raw reads. Cutadapt (v1.158; Martin, 2011) was used to trim the paired-end reads to remove adapters. The DADA2 pipeline (v1.8; Callahan et al., 2016) was used in RStudio to cluster the reads into amplicon sequence variants (ASVs), remove chimera and determine the taxonomic classification using SILVA 16S rRNA database (v138.1; Quast et al., 2013). The package phyloseq (v1.36.0; McMurdie & Holmes, 2013) was used for microbial community data analysis. A differential analysis was performed with package DESeq2 (v.1.44.0; Love et al., 2014) to analyze the changes in microbial communities between the different incubations. Specific microbial taxa were manually selected for detailed analysis and discussion based on their established metabolic roles in the environment. The archaeal clades ANME-2ab and ANME-3 were targeted based on their known involvement in AOM. Bacterial groups were selected based on either their known association with ANME in sulfate-dependent AOM (S-AOM; e.g., Desulfosarcinaceae, Desulfobulbaceae) or their reported metal oxide reducing capacities (e.g., Geopsychrobacteraceae, Sva1033).The raw sequencing data can be accessed in the NCBI database under Bioproject number PRJNA1268434.

Results

Sediment and Porewater Chemistry

At the time of sampling in October 2021, two sediment intervals were enriched in Mo and C_org_. These were the top 7 cm of the sediment and the layer between 19 and 25 cm depth, which was characterized by maximum Mo and C_org_ contents of 38 ppm and 3.4 wt%, respectively (Fig. 1a, b). Mn oxide contents varied between 1 and 5 μmol g^−1^, with a maximum content at 8 cm depth (Fig. 1c). Fe oxide content varied between 0 and 4 μmol g^−1^, with the highest content at 10 cm depth (Fig. 1d). Total S concentrations varied from 200 to 380 μmol g^−1^, with the lowest concentrations between 7 and 10 cm depth and the highest content near the sediment–water interface (Fig. 1e). FeS followed a similar trend with depth as total S and varied between 35 and 90 μmol g^−1^. Pyrite (FeS_2_) contents were below 4 μmol g^−1^. The sediment was depleted in SO_4_^2−^ below 10 cm depth and CH_4_ was present throughout the sediment (Fig. 1f, g). Concentrations of dMn varied around 80 μmol L^−1^, with maxima near the sediment water interface and around 10 cm depth (Fig. 1h). Dissolved Fe was always below 11 µmol L^−1^ in the porewater while H_2_S was present in the top 17 cm and reached a maximum concentration of 3.5 mmol L^−1^ (Fig. 1i, j).Fig. 1. Sediment profiles of Mo, C_org_, Mn oxides, Fe oxides, total S, FeS and FeS_2_ and pore water profiles of SO_4_^2−^, CH_4_, dMn, dFe and H_2_S in October 2021. The depth intervals surrounded by a red box indicate the depths that were used for the incubations. The gray shading represents the sediment that was deposited during bottom water euxinia and the white areas represent the sediment that was deposited during oxic bottom water conditions

Sediment Incubations

Based on sediment geochemistry, two sediment depths (i.e. 0–5 cm and 15–20 cm; Fig. 1) were chosen to study Mn-AOM. In the incubations with sediment from 0–5 cm depth, all bottles amended with Mn oxides showed a signal for CH_4_ oxidation (Fig. 2a-c), observed as an increase in the ^13^C-CO_2_/^12^C-CO_2_ ratio relative to the control after 22—29 days (with maximum values of 0.019, 0.028 and 0.027 for birnessite, pyrolusite and manganite, respectively, compared to 0.013 in the control). Soon after the increase, the CO_2_ isotope ratio decreased to the same values as the background ratio in all incubations, due to the drawdown of CO_2_ as detailed above (Sect. 2.4.3 and illustrated in Fig. S2). In the incubations with sediment from 15–20 cm depth, addition of birnessite led to an immediate increase in the ratio of ^13^C-CO_2_/^12^C-CO_2_, reaching a value of 0.056, compared to 0.015 in the control, before leveling off after 36 days of incubation (Fig. 2d). The ratio increased again in this incubation only after the addition of both new Mn oxide and new CO_2_. In the incubations where pyrolusite and manganite were added, the ^13^C-CO_2_/^12^C-CO_2_ ratio also increased compared to the control (Fig. 2e, f). However, the increase was much smaller compared to the increase with birnessite (0.019 and 0.018 for pyrolusite and manganite, respectively, relative to 0.015 in the control incubation) and ratios decreased to below that in the control after a few weeks.Fig. 2. Headspace ^13^C-CO_2_/^12^C-CO_2_ ratios and dissolved Mn concentrations for the incubations with sediment from (a—c) 0–5 cm depth and (d—f) 15–20 cm depth amended with birnessite (a, d) pyrolusite (b, e) and manganite (c, f). The ^13^C-CO_2_/^12^C-CO_2_ and dissolved Mn concentrations of the controls are also indicated in each plot. The dashed lines indicate timepoints when new Mn oxides and new CO_2_ were added to the incubations

Concentrations of dMn increased to 3–4 mmol L^−1^ in all bottles where Mn oxides were added and remained below 0.08 mmol L^−1^ in the controls (Fig. 2a-f). In the incubations with sediment from 0–5 cm, dMn increased after 22–29 days. In the incubations with sediment from 15–20 cm depth, dMn increased from the start of the experiment. Accumulation of SO_4_^2−^ occurred in the incubations with sediment from 0–5 cm depth where birnessite and pyrolusite were added, reaching concentrations around 1 and 4 mmol L^−1^ with birnessite and pyrolusite, respectively (Fig. 3a-c). In the incubation with manganite (0—5 cm), a SO_4_^2−^ concentration of only 0.2 mmol L^−1^ was observed. In the incubations with sediment from 15–20 cm depth, the SO_4_^2−^ concentration reached 2 mmol L^−1^ and 6 mmol L^−1^ in the incubations with birnessite and pyrolusite, respectively, but no SO_4_^2−^ was detected in the incubation with manganite (Fig. 3d-f). In the controls, SO_4_^2−^ concentrations were below 0.1 mmol L^−1^ (Fig. 3). The only incubation where H_2_S accumulated (up to 500 µmol L^−1^) was the incubation with birnessite for sediments from the 0–5 cm depth layer, (Fig. 3a).Fig. 3SO_4_^2−^ and H_2_S concentrations for the incubations with sediment from (a—c) 0–5 cm depth and (d—f) 15–20 cm depth spiked with (a, d) birnessite, (b, e) pyrolusite and (c, f) manganite over time. The SO_4_^2−^ concentrations of the controls are also indicated in each figure. The dashed line represents the time point when new Mn oxide minerals were added to all incubation bottles

Addition of dMn(III)-L did not lead to an increase in the ^13^C-CO_2_/^12^C-CO_2_ ratio relative to the ^13^C-CO_2_/^12^C-CO_2_ ratio in the background control (Fig. 4). Concentrations of dMn in the experiment with sediment from 0–5 cm depth first increased and then decreased during the experiment, to a minimum of 0.1 mmol L^−1^ (Fig. 4a, b). In the incubations with sediment from 15–20 cm depth, dMn concentrations were stable, around 0.6 mmol L^−1^ in the incubation with strong ligands (around 1.8 mmol L^−1^ after the addition of new dMn(III)-L solution) and around 0.1 mmol L^−1^ in the incubation with weak ligands (Fig. 4c, d). No SO_4_^2−^ accumulated in the bottles (Fig. S3). Concentrations of H_2_S were mostly around 500 μmol L^−1^ in the incubations with sediment from 0–5 cm depth and generally < 50 μmol L^−1^ in the bottles with sediment from 15–20 cm (Fig. S3). After the addition of substrate, H_2_S concentrations increased in all but the dMn(III) strong ligand incubation (0–5 cm; Fig. S3).Fig. 4. Headspace ^13^CO_2_/^12^CO_2_ ratios and dissolved Mn concentrations for the incubations with sediment from (a, b) 0–5 cm depth and (c, d) 15–20 cm depth spiked with (a, c) dMn(III)-L bound to a strong ligand and (b, d) dMn(III)-L bound to a weak ligand over time. The dashed lines indicate timepoints when new dMn(III)-L was added to the incubations

Diversity of ANME and SRB in the Incubations

To determine which microorganisms were involved in Mn-AOM, the samples from incubations with sediment from 15–20 cm depth were analyzed with 16S rRNA gene amplicon sequencing (Fig. 5; Fig. S4) as this depth showed the most pronounced signal of CH_4_ oxidation in the ^13^C-CO_2_/^12^C-CO_2_ ratio in the incubations with birnessite (Fig. 2d). At the start of the incubations, ANME-2ab reads covered only 0.2% of the total archaeal reads, but their reads increased in all incubations. The largest increase in ANME-2ab reads was observed in the incubations with manganite (8.6% ± 2.4) and pyrolusite (9.0% ± 1.9). The relative abundance of ANME-3 was also low in the beginning (0.8%), but ANME-3 were most enriched in the incubations with birnessite (13.6% ± 0.2%) and pyrolusite (13.2% ± 1.2%). The relative abundance of methanogenic Methanosarcina increased from 4% at the start to 14% in the control and pyrolusite-amended incubations and comprised 21.6% of the total archaeal reads in the incubations with manganite (Fig. 5a). For the bacterial community, the changes were more substrate specific. Below, we discuss the changes of the most important bacterial families, which we selected based on differential abundances (Fig. S5, S6). The relative abundance of potential metal-reducing bacterial families, Desulfobulbaceae, Sva 1033 and Geopsychrobacteraceae increased relative to the start and control in all incubations where Mn oxides were added, especially with pyrolusite and manganite (Fig. 5b; Fig S4). Desulfosarcinaceae, a potential partner for ANME in SRB, increased in abundance in the incubations amended with birnessite and pyrolusite, but decreased in the presence of manganite. Members of S-oxidizing Sulfurimonadaceae increased from 3 to 6% in the control incubations, but decreased in all incubations where Mn oxides were added (Fig. 5b).Fig. 5. Relative abundance of (a) selected archaea and (b) bacteria based on 16S rRNA gene amplicon sequencing. The entire archaeal and bacterial communities can be found in supplementary figure S5

Discussion

Burial of Mn Oxides Creates Potential for Mn-AOM

Burial of Mn oxides below the sulfate methane transition zone at the study site is the result of the seasonal variation in bottom water redox conditions in combination with very high rates of sedimentation (20 cm yr^−1^) and organic matter input (~ 86 mol m^−2^ yr^−1^; Klomp et al., 2025). When bottom waters are oxic in winter and spring, the surface sediment is enriched in Mn and Fe oxides and devoid of H_2_S (Klomp et al., 2025; van Helmond et al., 2025; Żygadłowska et al., 2024a). When the bottom waters become euxinic, the supply of metal oxides from the water column decreases strongly, and the ongoing production of H_2_S in the sediment leads to a removal of around 95% of the easily reducible Fe oxides from the upper part of the sediment via the precipitation of FeS_x_ (Fig. 1; van Helmond et al., 2025). Due to the high sedimentation rates and efficient H_2_S removal by Fe oxides, a part of the Mn oxides deposited in winter ends up buried below the sulfate methane transition zone in the absence of H_2_S in October (Fig. 1). This sediment layer (15–20 cm) is also part of the zone where substantial numbers of ANME have been observed (Wallenius et al., 2025), enabling the potential for Mn-AOM.

Potential for Mn-AOM, based on the ^13^C-CO_2_/^12^C-CO_2_ ratios in the incubations, was detected in the controls for both studied sediment intervals (0–5 cm and 15–20 cm) and enhanced upon addition of several different Mn oxide minerals.

The CH_4_ oxidation signal observed in the controls indicates that some residual electron acceptors were still present at the start of the incubations (Fig. 2). Since SO_4_^2−^ was not detected in the control incubations (Fig. 1, 2) and nitrate and nitrite are unlikely to have been present (Rigutto et al., 2025), Mn or Fe oxides could have acted as electron acceptors in AOM in the control incubations. However, an increase in dMn or dFe in the control incubations, which would provide a strong indication for active metal oxide reduction, was not observed (Table S2). As an alternative, organic compounds such as humic acids in the sediment could act as an electron acceptor for AOM (Pelsma et al., 2023; Valenzuela et al., 2019, 2022).

Differences in AOM potential occurred between the two depths and between the different Mn oxide minerals (Fig. 3). The strongest signal for CH_4_ oxidation was observed with birnessite as an electron acceptor in the sediment depth interval of 15–20 cm, which is also the sediment layer where ANME were most abundant in the previous year (Wallenius et al., 2025). This suggests that the initial biomass at the start of the incubation affected the rates. We also have indications that pyrolusite and manganite can be used for AOM, although in these incubations the signal for methane oxidation in the ^13^C-CO_2_/^12^C-CO_2_ ratio was masked by the drawdown of CO_2_ from the headspace (see methods). In both cases, the drawdown was higher in the incubations with pyrolusite and manganite due to the higher concentrations of Mn supplied. In the incubation with the top 5 cm of the sediment, dissolved Mn formed after a lag phase and simultaneously with the onset of the CH_4_ oxidation signal, which subsequently faded away (Fig. 2). The decline in dissolved Mn might be related to Mn adsorption to Mn oxides or Mn mineral precipitation. The delay in the onset of the CH_4_ oxidation signal was possibly linked to a very low initial ANME biomass (Wallenius et al., 2025). Importantly, CH_4_ oxidation was enhanced by all of the supplied Mn oxide minerals and the timing of its onset coincided with the onset of dissolved Mn production, pointing towards a potential link via AOM.

A cryptic S-cycle as a driver of CH_4_ oxidation (Holmkvist et al., 2011; Su et al., 2020) likely occurred in the incubations where birnessite and pyrolusite were added, since SO_4_^2−^ accumulated (Fig. 4). A possible source for this SO_4_^2−^ is the oxidation of FeS present in the sediment by the added Mn oxides (Fig. 2; Luo et al., 2018). Another source for the SO_4_^2−^ could be the release of S from the degradation of organic matter and consecutive oxidation of this S to SO_4_^2−^ when Mn oxides are added to the sediment (Canfield et al., 2005). The observed lower accumulation of SO_4_^2−^ in the incubation with sediment from 0—5 cm depth, when compared to the incubations with sediment from 15—20 cm depth (Fig. 3), might be explained by the expected higher abundance of SO_4_^2−^-reducing bacteria in the shallower sediment, based on microbial abundances in the previous year (Wallenius et al., 2025). The presence of SO_4^2−^, even after a pre-incubation with Mn oxides, shows how difficult it is to exclude other electron acceptors than Mn oxides when studying Mn-AOM. The emergence of H_2_S in the incubations with birnessite (0–5 cm; Fig. 4) was likely related to depletion of Mn oxide. This is supported by the removal of H_2_S upon resupply of birnessite. Our results emphasize the importance of measuring SO_4^2−^ and H_2_S when incubating marine sediment with alternative electron acceptors. Accumulation of SO_4_^2−^ did not occur when manganite was added as an electron acceptor, which confirms the observations of Aller and Rude (1987) that, in marine sediments, Mn(III) minerals are less efficient in oxidizing reduced S species than Mn(IV) minerals. In laboratory experiments, manganite was also less efficient than birnessite in oxidizing FeS, likely because the former mineral solely consists of Mn(III) (Luo et al., 2018). The contribution of Mn(III) minerals to AOM is therefore expected to be through direct Mn-AOM, as a cryptic sulfur cycle is likely not possible due to the lower efficiency of Mn(III) in oxidizing reduced S species.

Incubations that were amended with dMn(III)-L did not show enhanced CH_4_ oxidation, which is in accordance with previous findings regarding dMn(III)-L and CH_4_ oxidation in a freshwater environment (Szeinbaum et al., 2020). During the incubations, concentrations of dMn were a factor 1.7 to 10 lower than the 1 mmol L^−1^ added to the bottles (Fig. 4). However, dMn was still present at concentrations comparable to porewater dMn(III)-L (Klomp et al., 2025). Metal–ligand complexes can vary over time, due to, for example, ligand exchange (Luther et al., 2015). Such exchange could contribute to degradation of Mn(III)-L complexes and/or Mn precipitation over time. H_2_S was observed in all incubations amended with dMn(III)-L (Fig. S3) and has been shown to inhibit AOM (Dalcin Martins et al., 2024). However, in the depth interval from 15–20 cm H_2_S concentrations were only 50 µmol L^−1^, and thus were unlikely to have been high enough for full inhibition of AOM (Dalcin Martins et al., 2024). We speculate that the lack of enhanced AOM could also result from shielding of the Mn(III) from the methanotrophic archaea by the organic ligand surrounding it, making the Mn(III) less available for the microbes. Furthermore, using dissolved Mn as an electron acceptor might require a different metabolic pathway than the extracellular electron transfer proposed for metal oxide reduction coupled to AOM in freshwater sediments (Leu et al., 2020). In summary, while we did not observe enhanced AOM with dissolved Mn(III)-L, our results do show evidence for a coupling of Mn oxide reduction and CH_4_ oxidation, which may be either direct or indirect (i.e. coupled to the sulfur cycle).

Microbial Players in Mn Driven AOM

Based on 16S rRNA genes recovered from the incubations from 15–20 cm depth after 92 days, we show that two methanotrophic archaea enriched in the incubations. These were ANME-2ab and ANME-3, with ANME-3 being present in a higher abundance than ANME-2ab (Fig. 5). The higher increase in relative abundance of ANME-2ab in the incubations where Mn oxide minerals were added compared to the control incubations indicates that these ANMEs may benefit from the addition of Mn oxides (Fig. 5). ANME-2ab have previously been linked to other electron acceptors than SO_4_^2−^, such as organic compounds and Fe oxides (Aromokeye et al., 2020; Pelsma et al., 2023; Tu et al., 2017). It is proposed that their large multi-heme cytochromes facilitate extracellular electron transfer, potentially allowing the ANME to directly reduce metal oxides (Chadwick et al., 2022; McGlynn et al., 2015; Scheller et al., 2016). ANME-2ab may also use a bacterial partner to couple CH_4_ oxidation to the reduction of metal oxides (Slobodkin et al., 2023). The main bacterial species that increased when Mn oxides were added, in parallel to the increase in ANME-2ab, were Desulfobulbaceae, Sva 1033 and Geopsychrobacteraceae (Fig. 4). Desulfobulbaceae are known bacterial partners for ANME (Green-Saxena et al., 2014; Lösekann et al., 2007) and some members have the potential to reduce metal oxides (Lovley et al., 1993; Müller et al., 2020). It is therefore possible that Desulfobulbaceae and ANME-2ab form a consortium in which CH_4_ oxidation by ANME-2ab is coupled to Mn oxide reduction by Desulfobulbaceae.

The relative abundance of ANME-3 also increased when compared to the start of the incubations. Although the overall increase of ANME-3 was higher compared to that of ANME-2ab, the increase of ANME-3 in Mn oxide amended incubations relative to the control samples was not as pronounced (Fig. 5). In the incubation where manganite was added, the increase in relative abundance of ANME-3 was even smaller than in the control incubations. The highest ANME-3 relative abundances were observed in the incubations where SO_4_^2−^ was present, especially the incubations where birnessite and pyrolusite were added (Figs. 3, 4). We acknowledge that the 16S rRNA gene amplicon data provided here yields relative abundances, allowing only for qualitative comparisons rather than quantitative correlations with process rates. However, the consistent co-occurrence of ANME-3 enrichment with SO_4_^2−^ accumulation strongly suggests that these methanotrophs perform S-AOM that is fueled by Mn oxides via a cryptic S cycle. ANME-3 can be independent from SRBs or in a consortium with members of the Desulfobulbaceae and Desulfosarcinaceae families (Lösekann et al., 2007). In our incubations, both Desulfosarcinaceae and Desulfovibrionaceae families show a similar pattern in relative abundance as ANME-3, indicating that these could be potential bacterial partners for ANME-3 in S-AOM.

Besides the taxa typically associated with AOM, several other bacterial families increased in relative abundance in the Mn-amended incubations (Fig. S5, S6), suggesting they are part of a wider microbial consortium stimulated by the presence of Mn oxides. Most notably, Thioalkalispiraceae, a family of obligate chemolithoautotrophic sulfur-oxidizers, were enriched. Their presence supports the hypothesis of a cryptic sulfur cycle, where they likely oxidize the H_2_S produced during S-AOM back to SO_4_^2−^ using the added Mn oxides as electron acceptors. Additionally, we observed increases in Syntrophotaleaceae and Peptococcaceae. These groups are generally characterized as syntrophic fermenters or heterotrophs capable of degrading complex organic matter. Their enrichment suggests that the addition of Mn oxides may have stimulated the turnover of sedimentary organic matter, creating niches for fermenters and generalist heterotrophs such as Acanthopleuribacteraceae within the community (Henkel et al., 2022; Marshall et al., 2023; Ozuolmez et al., 2020).

The relative abundance of Methanosarcina increased in the incubations where birnessite and manganite were added compared to the start and control (Fig. 5). A positive effect of Mn oxide on the relative abundance of Methanosarcina has been observed before, during anaerobic digestion (Chen et al., 2023; Tian et al., 2017). Methanosarcina is a metabolically versatile methanogen that can produce CH_4_ from various organic compounds, but has also been shown to use extracellular electron transfer to obtain electrons to perform methanogenesis in syntropy with a bacterial partner (Huang et al., 2021; Rotaru et al., 2014) and may also reduce Fe oxides (Ferry, 2020; Yang & Lu, 2022). Another possibility is that the addition of Mn oxides enhanced organic matter degradation, releasing substrates on which Methanosarcina could grow. Our results indicate that, in marine sediments, Methanosarcina may benefit from the presence of Mn oxides, regardless of the redox state, i.e. with both Mn(III) and Mn(IV) oxides.

In future work using experimental incubations, inhibitors of SO_4_^2−^ reduction could be used to exclude indirect Mn-AOM via cryptic sulfur cycling. We recommend the addition of lower amounts of Mn oxides to avoid strong drawdown of CO_2_. More details on the microbial players and metabolic pathways involved in Mn-AOM could be obtained via metagenomic analysis.

The Contribution of Mn-AOM to CH4 Removal

Our experiments highlight the potential for Mn-AOM, either via direct coupling of CH_4_ oxidation to Mn reduction, or via the production of SO_4_^2−^ when Mn oxides oxidize reduced S species in the sediment. If sufficient Mn oxides are present, Mn-AOM could contribute a few percent to total CH_4_ oxidation in coastal sediments (Lenstra et al., 2023; Xiao et al., 2023). In a seasonally euxinic coastal setting, the contribution of Mn-AOM is expected to vary depending on the redox conditions in the water column and sediments. Generally, Mn oxide input into sediments is highest in coastal regions, especially near river mouths, as a result of the continental origin of the Mn oxides (Lenstra et al., 2022). Since these are also areas where methanogenesis rates are highest, Mn-AOM is likely especially important in these river-dominated coastal regions. High input of Mn oxides can also occur in coastal basins where the basin geometry contributes to focusing of sediment (Dijkstra et al., 2016; Lenz et al., 2015). Recycling of Mn, i.e. repeated oxidation and reduction of Mn at redox interfaces in the water column or sediment, contribute to retention of Mn in marine systems (Adelson et al., 2001; Sulu-Gambari et al., 2017). This implies that Mn can be oxidized and reduced many times before the Mn is either transported away in the water column or buried in the sediment as Mn carbonate (Lenstra et al., 2021a; Sulu-Gambari et al., 2017). Eutrophic systems where Mn recycling is strong and CH_4_ is present close to the sediment–water interface, have a particularly high potential for interactions between CH_4_ and Mn oxides.

Burial of Mn oxides into the methanic zone is promoted by non-steady state events like sedimentation pulses in large river delta systems (Kasten et al., 1998) or extensive reworking of the sediment during mass movements along continental margins (Riedinger et al., 2014). For example, burial of Mn oxides below the sulfate methane transition zone upon rapid shoaling of the SO_4_^2−^ penetration depth has been reported for the Bothnian Sea (Lenstra et al., 2023; Slomp et al., 2013). Notably, ANME-2ab make up for a significant part of the archaeal community in Bothnian Sea sediments (Rasigraf et al., 2020). Our work shows that, in marine systems where Mn oxides are buried in the methanic zone, ANME-2ab and/or ANME-3 could be involved in AOM coupled to reduction of Mn oxides.

Conclusions

We studied the interactions between Mn oxides and CH_4_ in coastal marine sediments of a seasonally euxinic basin. We combined geochemical sediment and porewater profiles with results of batch incubations using various forms of oxidized Mn, namely birnessite (Mn(IV)O_2_ * n H_2_O), pyrolusite (Mn(IV)O_2_), manganite (Mn(III)OOH) and dissolved Mn(III) bound to organic ligands. Our field data show burial of Mn oxides below the sulfate methane transition zone, resulting from seasonal variation in bottom water redox conditions combined with a high sedimentation rate. Birnessite stimulated anaerobic oxidation of CH_4_ in sediment incubations. Pyrolusite and manganite appeared to do the same, while dissolved Mn(III) bound to organic ligands did not affect CH_4_ oxidation. In the incubations where birnessite and pyrolusite were added, SO_4_^2−^ formed, likely via the oxidation of FeS in the sediment. This SO_4_^2−^ could have driven the CH_4_ oxidation via a cryptic sulfur cycle. When manganite was added, no SO_4_^2−^ formed, hence CH_4_ oxidation occurred via Mn-AOM. Methanotrophs of the ANME-2ab clades increased in relative abundance in all incubations where Mn oxide minerals were added compared to the control incubation, indicating that these organisms are likely involved in Mn driven methane oxidation. In the incubations where SO_4_^2−^ formed upon addition of Mn oxides, ANME-3 increased in relative abundance, which suggests that these methanotrophs perform S-AOM and benefit from a cryptic sulfur cycle driven by Mn oxides. Furthermore, enrichment of the methanogen Methanosarcina in incubations where Mn oxides were added showed that these methanogens benefit from the presence of Mn oxides. Our results highlight the versatility of methanotrophs and show a potential role for Mn oxides in CH_4_ cycling in sediments of a seasonally euxinic basin.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (PDF 563 KB)Supplementary file2 (XLSX 44 KB)