Initial research suggesting a microbially mediated reaction between manganese oxides and methane in the natural environment can be traced back to Hein et al.’s (1987) investigation into the formation of ¹³C-depleted rhodochrosite (MnCO₃) deposits in the California Coast Ranges. Beal et al.’s (2009) landmark investigation, involving the introduction of birnessite and ferrihydrite to marine seep sediment from the Eel River Basin, significantly illuminated the potential for metal-dependent AOM (manganese and iron) in marine settings. While their study did not conclusively ascertain the direct reaction of manganese with methane, it prompted numerous investigations into the role of metals (Fe and Mn) in AOM processes.

Among metal-AOM reactions, Fe-AOM has attracted more extensive attention than Mn-AOM (Crowe et al., 2011; Sivan et al., 2011; Egger et al., 2015b; Rooze et al., 2016; Cai et al., 2018; Yang et al., 2023). However, studies have revealed Mn involvement in AOM alongside Fe, with observable geochemical signatures. Dissolved inorganic carbon (DIC) produced by AOM and the authigenic carbonates precipitated from it typically inherit the isotopically light carbon signature of methane (Paull et al., 1992; Kastner et al., 2008; Tong et al., 2013). In porewater profiles, high concentrations of Mn²⁺ below the SMI, together with increasing δ¹³C-CH₄, rising DIC levels, and lower δ¹³C-DIC values at corresponding depths, can be attributed to manganese reduction coupled to AOM ( Figure 1 ) (Riedinger et al., 2014; Xiao et al., 2019, 2023). Decoupling of sulfate reduction and AOM, characterized by high methane-to-sulfate ratios or higher AOM rates relative to sulfate reduction rates, suggests the use of alternative electron acceptors (Segarra et al., 2013; Su et al., 2020). The authigenic precipitation of rhodochrosite, or elevated concentrations of Mn(II) in carbonate phases exhibiting depleted δ¹³C signatures, has been interpreted as diagnostic evidence for manganese-coupled AOM (Xiao et al., 2019). Furthermore, Mn(III/IV) oxides or manganese micronodules in the solid phase have also been documented in hydrate-bearing or cold seep environments, providing suggestive evidence for Mn-AOM (Segarra et al., 2013; Xiao et al., 2019; Yang et al., 2023).

AOM driven by manganese is thermodynamically favorable and presumed to occur preferentially (Riedinger et al., 2014; Xu et al., 2021). Manganese oxide reduction as an electron acceptor provides substantially more energy (∼20 times) than sulfate reduction, and also more than iron oxide reduction, in AOM process ( Table 1 ) (Beal et al., 2009; Segarra et al., 2013; Ettwig et al., 2016). The high redox potential of manganese oxides enables them to act as terminal electron acceptors in organic matter oxidation, potentially oxidizing methane through HS⁻ and Fe(II) acting as electron shuttles (Wang and Van Cappellen, 1996; Thomsen et al., 2004; De Schamphelaire et al., 2007). Evidence from post-Marinoan Doushantuo cap dolostone formation, where δ³⁴S values of authigenic pyrite correlate positively with carbonate manganese content, suggests manganese reduction influences the HS⁻ pool. This implies manganese oxides can serve as terminal electron acceptors for methane oxidation by oxidizing HS⁻ to SO₄ ²⁻ (Cai et al., 2023).

In order to reveal the specific role of manganese in AOM, researchers have conducted controlled incubation experiments to manipulate key variables and subsequently elucidate the underlying pathways of Mn-AOM ( Table 2 ). Laboratory incubations of freshwater and brackish tidal wetland sediments amended with manganese oxides (birnessite) showed a significant increase in the Mn(II) content of the carbonate phase. A greater rate of AOM than SR was also observed, suggesting that AOM may be linked to the reduction of birnessite (Segarra et al., 2013).

Some studies added both manganese oxides and molybdate (a sulfate reduction inhibitor) to determine whether sulfate reduction to HS⁻ occurred under experimental conditions. Xu et al. (2021) observed a significant decrease in the SR rate after the addition of birnessite, but an obvious increase in the AOM rate, and found that the molybdate did not affect the AOM rate. These results suggested that birnessite and sulfate might compete for the electron acceptor of AOM, and that birnessite here can act directly with methane (Xu et al., 2021). However, a similar experiment performed by Su et al. (2020) observed an evident decrease in the AOM rate after the addition of vernadite (δ-MnO₂) and molybdate. This observation suggests that SO₄ ²⁻ remains a significant factor in AOM, and further implies that manganese oxides may indirectly facilitate AOM by oxidizing reduced sulfur species, potentially linking manganese reduction to SR-AOM (Yao and Millero, 1996; Schippers and Jørgensen, 2001). The discrepancy in experimental results may be attributed to the specific mineral types of the added manganese oxides (birnessite vs. vernadite), which exhibit distinct kinetic and thermodynamic properties that can significantly influence reaction occurrence and rates (Neumann Wallheimer et al., 2025). Additionally, differences in the sediment source environments (marine for Xu et al. (2021); sulfate-rich lake for Su et al. (2020)) likely host different dominant microbial communities, potentially leading to variations in reaction pathways. In marine sediments, a cryptic sulfur cycle beneath the SMTZ, wherein manganese oxides are capable of oxidizing sulfides to sulfate [Equations 2–4], has been previously proposed as a pathway for this indirect involvement in SR-AOM (Jones et al., 2011; Treude et al., 2014):

Beyond sediment enrichments, bioreactor studies have examined Mn-AOM geochemistry ( Table 2 ). A freshwater enrichment culture obtained under anaerobic, nitrate-reducing conditions with a continuous supply of methane showed that the reduction of environmentally relevant forms of Fe(III) and Mn(IV) was coupled with the oxidation of methane (Ettwig et al., 2016). Another freshwater sediment experiment used methane and birnessite to feed the bioreactor, and biochemical profiling of the system revealed Mn(IV)-dependent AOM (Leu et al., 2020). Both studies support the direct coupling of manganese reduction to AOM.

Extant research delineates two dominant pathways of Mn-AOM: (1) direct AOM coupled to Mn oxide reduction (MnR-AOM) ( Figure 2A ) and (2) indirect AOM via Mn oxide-mediated sulfate reduction (MnR-S-AOM) ( Figure 2B ).

MnR-AOM, while thermodynamically advantageous, is kinetically less favorable than SR-AOM. The solid-phase nature of manganese minerals limits the reactive surface area compared to dissolved sulfate, resulting in slower reaction rates with methane than those observed with sulfate ( Table 1 ). This kinetic factor may promote Mn-AOM pathways that utilize sulfur as an intermediate. In addition, 1 mol of methane requires 4 mol of MnO₂ [Equation 1], four times the amount of SO₄ ^2- needed for SR-AOM, which means that MnO₂ needs a higher rate of supply.

While both pathways are recognized, the specific environmental and microbial determinants that govern their relative importance and spatial distribution in natural sediments are not yet fully understood. Electron acceptor availability is likely a significant influencing factor, encompassing the supply and bioavailability (mineral form) of manganese oxides (Neumann Wallheimer et al., 2025), as well as the concentrations of sulfate and sulfide (see section 4 for environmental conditions favoring Mn-AOM in marine sediments). The composition and metabolic preferences of microbial communities may also be crucial determinants; their preference for specific electron acceptors or metabolic strategies likely dictates the pathway utilized (see Section 3.2). For instance, microbial consortia adapted to long-term SR-AOM might preferentially utilize sulfur-mediated pathways (MnR-S-AOM) when sulfate is becoming limiting, rather than immediately switching to direct MnR-AOM (e.g., Cai et al., 2023). Additionally, ambient geochemical conditions (e.g., pH, Eh, temperature, salinity), which can significantly impact reaction kinetics and microbial activity, are likely candidates influencing pathway selection (He et al., 2018; Cai et al., 2021b; Zhao et al., 2024). Detailed and systematic studies investigating how these potential determinants influence Mn-AOM pathway selection are needed in future research.

The SR-AOM, which is the major pathway of AOM, is typically mediated by syntrophic microbial consortia of ANME and sulfate-reducing bacteria (SRB) in the natural environment (Boetius et al., 2000; Niemann et al., 2006; Wegener et al., 2015). ANME encompass three distinct phylogenetic clades: ANME-1 (subgroups a and b), ANME-2 (subgroups a, b, c, and d), and ANME-3 (Ruff et al., 2015; Cai et al., 2021b). Mn-AOM in natural systems appears to necessitate the involvement of ANME alongside some metal-reducing bacteria (MRB) partners such as Desulfuromonas and Pelobacter members, although thermochemical oxidation of methane by manganese oxides without microbes has also been observed in environments like hydrothermal sediments (Chang et al., 2012; Huang et al., 2023).

Investigation into microbial communities involved in Mn-AOM in marine sediments employs both direct molecular analysis (DNA/RNA extraction and sequencing) of sediment samples and cultivation-based methods aimed at enriching key microorganisms (Beal et al., 2009; Liu et al., 2023; Xiao et al., 2023). Despite the persistent lack of representative pure cultures for Mn-AOM and the resulting unclear identification of the specific microbial populations responsible, shifts in microbial community structure following manganese addition offer valuable insights into potential functional groups ( Table 2 ). For example, a 10-month culture experiment presented by Beal et al. (2009) indicated a substantial increase in manganese reducers within the bacterial community upon birnessite incubation, suggesting a vital role of bacteria in Mn-AOM. These results imply that Mn-AOM may be mediated by ANME-1 and/or Methanococcoides/ANME-3 in association with bacterial partners. House et al. (2011), using FISH and FISH-SIMS, identified that archaea-bacteria mixed aggregates were the most metabolically active entities during Mn-AOM, resembling those involved in SR-AOM. Direct DNA/RNA sequencing and analysis of sediment samples by Xiao et al. (2023) indicated that in methanic sediments, abundant active ANME groups (ANME-1 and ANME-2c) and potential dissimilatory metal reducers (e.g., ETH-SRB1) are potentially involved in metal-AOM in situ. Some studies have illuminated the capacity of ANME to perform the entire process without bacterial partners. Metagenomic sequencing has revealed that ANMEs capable of utilizing manganese solely are ANME-2a/b (Xu et al., 2021), “Ca. M. nitroreducens”-like archaea (Ettwig et al., 2016), “Ca. M. manganireducens” and “Ca. M. manganicus”; the latter three are assigned to ANME-2d (Leu et al., 2020; Cai et al., 2021b). In contrast to Leu et al.’s (2020) research, a recent bioreactor study by Liu et al. (2023) found Mn-AOM to be mediated by an anaerobic consortium comprising Candidatus Methanoperedens sp. BLZ1 and a distinct bacterial partner (Luteitalea pratensis).

Beyond direct Mn-AOM, the microbial functional roles in the MnR-S-AOM pathway are also complex. In the sediments of Cadagno Lake, where manganese is coupled to AOM through the sulfur cycle, the interdependence of ANME and SRB is likely facultative (Su et al., 2020). Analysis of microbial abundance revealed that SRB, specifically members of the Desulfobulbus group, exhibited peak abundance coinciding with only one of the two observed ANME maxima. This observation hints that ANME may engage in collaborative interactions with SRB or, alternatively, possess the capacity to execute AOM independently in MnR-S-AOM (Su et al., 2020).

It is important to note that the utilization of different parental substrates in cultivation experiments contributes to their variable effectiveness in representing in situ marine sediment conditions. Some employ a mixture of sampled marine sediment and artificial seawater, which can relatively better simulate in situ marine conditions (Beal et al., 2009; House et al., 2011; Segarra et al., 2013; Treude et al., 2014). In contrast, studies involving bioreactors primarily focused on the Mn-AOM reaction itself may diverge considerably from natural settings (Ettwig et al., 2016; Leu et al., 2020; Liu et al., 2023). Compared to cultivation experiments, results obtained from direct DNA/RNA extraction and sequencing of sediment samples provide more robust evidence for demonstrating the natural occurrence or ecological significance of microorganisms in marine sediments (Chang et al., 2012; Xu et al., 2021; Xiao et al., 2023). Therefore, future research incorporating in situ analysis of microbial communities and functions in sampled areas is essential for achieving a deeper understanding and stronger evidence of Mn-AOM.

Manganese oxides occur predominantly in solid phase in natural environments, so Mn-AOM microbes have to possess several strategies for transferring electrons between the cells and the minerals. Existing evidence suggests several potential strategies for this electron transfer process: direct contact between microbial cells and manganese oxide minerals; indirect electron transfer by electron shuttles; and direct electron transfer by nanowires (He et al., 2018; Liang et al., 2019; Leu et al., 2020; Dang et al., 2021) ( Figure 3 ).

Numerous multiheme c-type cytochrome-encoding genes are found in abundance in ANME-2d members. The genome of “Ca. M. nitroreducens” encodes a great number of multiheme c-type cytochromes that may contribute to electron transfer in Mn-AOM (Haroon et al., 2013; Arshad et al., 2015). However, “Ca. M. nitroreducens” has yet to be directly confirmed to have an intermediate interspecies electron transfer. Besides, 43 and 25 putative multiheme c-type cytochromes were found to be encoded in “Ca. M. manganicus” and “Ca. M. manganireducens,” respectively. In the transcriptomes of “Ca. M. manganicus” and “Ca. M. manganireducens,” 23 and 9 multiheme c-type cytochromes were substantially expressed, respectively (Leu et al., 2020). These c-type cytochromes were previously found to transfer electrons from intracellular to extracellular and therefore facilitate the direct electron transfer between microorganisms and minerals (Cai et al., 2021b). They also have been recognized as shuttles for extracellular electron transfer (Shi et al., 2007; Wang et al., 2014), and it is hypothesized that they perform a similar function in ANME-2d members (Ettwig et al., 2016).

Conductive nanowires constitute another Mn-AOM electron transfer mechanism ( Figure 3 ) (Leu et al., 2020). These conductive structures of “Ca. M. manganicus”, similar to the “Ca. M. nitroreducens” Mnv1 strain (Guerrero-Cruz and Cremers, 2018), show upregulation of archaellum-related genes under oxidative stress. Nanowires are suggested to allow electrons to transfer over greater distances relative to multiheme c-type cytochromes independently (Krukenberg et al., 2018). Mn-AOM-related microbes “Ca. M. manganicus” and “Ca. M. manganireducens” genomes encode for genes of the principal subunit flagellin (flaB), a component of the archaellum. During Mn-AOM experiments in “Ca. M. manganicus,” 2 of the 4 flaB genes were significantly expressed, suggesting the involvement of these conductive appendages in electron transfer (Leu et al., 2020).

In terms of electron acceptor utilization, ANME populations are recognized as versatile methanotrophs, exhibiting metabolic flexibility depending on environmental availability (Wankel et al., 2012; Ettwig et al., 2016; Scheller et al., 2016). While ANME-1, -2, and -3 clades are primarily associated with SR-AOM (Milucka et al., 2012), evidence indicates their involvement in Mn-AOM as well (Beal et al., 2009; Chang et al., 2012; Xu et al., 2021). However, studies suggest a preferential utilization of sulfate over metal oxides by these ANME clades (Riedinger et al., 2014; Egger et al., 2015b). Modeling of the Haima cold seep by Xiao et al. (2023) further supports this preference, indicating a minor contribution of Mn-AOM and Fe-AOM (1.5% each) relative to the dominant SR-AOM (97%). In contrast to these findings, laboratory experiments have demonstrated that some anaerobic methanotrophs prefer metals over sulfate as electron acceptors. Enriched from freshwater sediment, “Ca. M. ferrireducens,” “Ca. M. manganicus,” and “Ca. M. manganireducens” performed Fe(III)- or Mn(IV)-dependent AOM over a prolonged period, even in the presence of sulfate (Cai et al., 2018; Leu et al., 2020). Likewise, “Ca. M. nitroreducens” and “Ca. M. oxyfera” exhibit a preference for nitrate/nitrite reduction, irrespective of sulfate availability in culture media (Haroon et al., 2013; Ettwig et al., 2009).

The versatility of ANME to utilize electron acceptors is likely to be greatly affected by the availability of electron acceptors in the environment. Alterations in external environmental conditions not only induce functional adaptations within existing microbial populations but also contribute to shifts in community composition. For instance, the nitrate-dependent AOM archaeon “Ca. M. nitroreducens” is able to execute Fe- and Mn-AOM when nitrate concentrations are limiting (Haroon et al., 2013; Ettwig et al., 2016). Furthermore, laboratory bioreactor experiments, with conditions modified from initial incubations, further revealed significant shifts in dominant microbial types and the emergence of novel Mn-AOM microbes (e.g., Ca. M. manganicus and Ca. M. manganireducens) (Leu et al., 2020). It is hypothesized that microorganisms in these systems may mutate and/or evolve in response to variations in electron acceptor supply (Guerrero-Cruz et al., 2021). These findings underscore the complexity and under-exploration of microbial diversity and function in Mn-AOM.