Anaerobic microbial methanol conversion in marine sediments.

Methanol is an ubiquitous compound that plays a role in microbial processes as a carbon and energy source, intermediate in metabolic processes or as end product in fermentation. In anoxic environments, methanol can act as the sole carbon and energy source for several guilds of microorganisms: sulfate-reducing microorganisms, nitrate-reducing microorganisms, acetogens and methanogens. In marine sediments, these guilds compete for methanol as their common substrate, employing different biochemical pathways. In this review, we will give an overview of current knowledge of the various ways in which methanol reaches marine sediments, the ecology of microorganisms capable of utilizing methanol and their metabolism. Furthermore, through a metagenomic analysis, we shed light on the unknown diversity of methanol utilizers in marine sediments which is yet to be explored.

Introduction

Marine sediments are rich in biomass and a source of unknown microbial diversity, with microbial cell densities as high as 109 cells per cubic centimetre (up to five orders of magnitude higher than the water column) (Jørgensen and Boetius, 2007). Marine sediments consist of deposits of clay, decaying organic matter, calciferous remains and other solids. While oxygen can diffuse in these sediments, it is rapidly consumed by aerobic organisms, in an oxic layer ranging from a few millimetres to several meters in depth, depending on a variety of factors such as organic matter input to sediment surface, sediment permeability, turbation by water currents or macrofauna, water column height, microbial activity or proximity to continental shelves (Glud, 2008; D'hondt et al., 2015). The underlying sediment remains anoxic, where microbial fermentation and anaerobic respiration are the main metabolic processes. Molecules containing no carbon–carbon bonds such as trimethylamine, dimethylsulfide, methane and methanol are suggested to be important energy sources for microorganisms in these environments (Yanagawa et al., 2016; Chistoserdova and Kalyuzhnaya, 2018; Sun et al., 2019). As the fermentation products of common osmolytes or carbohydrates, these compounds are widely present in marine systems. Besides converted by microbial activity, they can influence the climate as atmospheric aerosols and as such, their role in marine environments is in general well‐reviewed (Reisch et al., 2011; Lidbury et al., 2017; Timmers et al., 2017; Sun et al., 2019). However, there is a lack of information on the microbial utilization of methanol in anoxic marine sediments. In this review, we aim to present what is currently known about the presence and fate of methanol in anoxic marine sediments. To provide insight into anaerobic microbial methanol utilization in diverse marine sediments, we performed a metagenome mining of 246 published metagenomes of anoxic marine sediments for key methanol utilization genes. This effort reveals the ubiquitousness of several genes involved in anaerobic methanol conversion in marine sediments, further supporting the importance of methanol‐utilizing microorganisms in these environments.

Sources of methanol in marine sediments

Several studies have quantified methanol concentrations in marine systems, both in water columns and sediments. Concentrations in the water column range from less than 27 nM up to 429 nM in marine systems, although estimates vary widely even between studies on the same site (Williams et al., 2004; Kameyama et al., 2010; Dixon et al., 2011a,b; Dixon and Nightingale, 2012; Read et al., 2012; Yang et al., 2013). It should be noted, however, that most of these studies focused solely on the Atlantic Ocean and mainly on shallow, aerobic sub‐surface waters, with only one study investigating a gradient of up to 500 m depth (Dixon and Nightingale, 2012). Estimates of methanol levels in coastal area sediments range between 0.3 μM and over 100 μM in environments as diverse as the Orca Basin in the Gulf Mexico, East Japan Sea and the South China Sea (Yanagawa et al., 2016; Zhuang et al., 2014; 2019a,b). In these studies, methanol concentrations were found to increase with depth, with the lowest concentrations close to the seafloor and the highest concentrations at depths of 10–20 m below the seafloor. This increase in methanol with depth is attributed to higher methanol turnover to CO2 near the sediment‐water column interphase (Zhuang et al., 2019a,b). Table 1 gives an overview of marine anaerobic sediments where methanol concentrations have been measured.

Table 1

Measured levels of marine sediment methanol concentrations.

Location	Depth	Methanol concentration	Reference
Black Sea sediment	0–700 cm below seafloor	6 μM at sea floor 1 μM 100–400 cm bsf 6 μM 500 cm bsf	Zhuang et al. (2014)
Northern Gulf of Mexico	0–30 cm below seafloor	2 μM at sea floor 65 μM 30 cm bsf	Zhuang et al. (2014)
South China Sea	0–700 cm below seafloor	4.3 μM at sea floor 111.7 μM at 700 cm bsf	Zhuang et al. (2019a,b)
Umitaka Spur, eastern Japan Sea	0–35 m below seafloor	0.3–3.5 μM at 0–3 m bsf 20 μM at 30 m bsf	Yanagawa et al. (2016)
Intertidal sediment, Lowes Cove, Maine	Seafloor	0.5–3.5 μM	King et al. (1983)
Guaymas Basin, Gulf of California	0–40 cm below seafloor	0.2–2 μM at seafloor 36.7 μM at 35 cm bsf	Zhuang et al. (2019a,b)
Western Mediterranean Sea	0–500 cm below seafloor	0.5–1.5 μM across all depths	Zhuang et al. (2018a,b)

Methanol sources in marine systems are attributed to both in situ production and external depositions from terrestrial origins. Terrestrial methanol mainly originates as a by‐product of plant growth and to a lesser degree through fermentation of pectin (Jacob et al., 2005; Millet et al., 2008). Because of its volatility, terrestrially produced methanol evaporates into the atmosphere, with an estimated annual emission of 70–350 Tg (Galbally and Kirstine, 2002). A large amount of this methanol, estimated between 8 and 101 Tg year−1 is deposited in the oceans through air–sea exchange, diffusion and rainfall (Beale et al., 2013). It should be noted that higher estimations of methanol deposition in the oceans also take sea to air emissions into account, which is estimated to be 30–85 Tg year−1, thereby diminishing total methanol deposition in the oceans (Heikes et al., 2002; Dixon et al., 2011a,b; Dixon and Nightingale, 2012; Beale et al., 2013; Yang et al., 2013).

Methanol is also produced in situ in the oceans. A study of methanol production in Atlantic waters estimated a net production of around 49 nmol L−1 day−1 (Dixon et al., 2013). Sources of this methanol are primary production by phytoplankton where methanol is an exudate by‐product and through microbial fermentation of algal carbohydrates such as galactins and pectin (Sieburth and Keller, 1989; Riemer, 1998; Dixon et al., 2013). Phytoplankton accounts for almost half of the global primary production and as such is suspected to be a major contributor to marine methanol production, the same equivalent to terrestrial primary production (Cloern et al., 2014; Mincer and Aicher, 2016). Interestingly, there have been very few studies quantifying net methanol production by phytoplankton. Mincer and Aicher (2016) assessed methanol production through 13C‐labelled bicarbonate addition to axenic phytoplankton cultures. As much as 0.3% of all assimilated carbon was sequestered in methanol, and it was implied that the genus Prochlorococcus alone could produce 846–1693 Tg of methanol per year, worldwide (Neufeld et al., 2008; Mincer and Aicher, 2016). Furthermore, phytoplankton mobilizes between 10% and 35% of their assimilated carbon into pectin, lignin and galactans, which are methoxylated polysaccharides (Sista Kameshwar and Qin, 2018). Thus, through demethoxylation of these carbohydrates by both aerobic microorganisms in the water column and oxic sediment and anaerobic microorganisms in the anoxic sediment, methanol is released (Schink and Zeikus, 1980, 1982; Sieburth and Keller, 1989; Sista Kameshwar and Qin, 2018).

Chemolithotrophic microorganisms present in anaerobic sediments are capable of producing methanol. Anaerobic oxidation of methane occurs in most anoxic systems where methane is present, by methane‐oxidizing microorganisms or syntrophic microbial communities capable of using sulfate, nitrate, manganese or iron as terminal an electron acceptor (Wegener et al., 2016; Welte et al., 2016; McGlynn, 2017; Timmers et al., 2017; Leu et al., 2020). Canonically, anaerobic methane oxidizers can metabolize methane through a reversal of the methanogenesis process (Haroon et al., 2013). This process, contrary to oxic methanotrophy (which relies on the activity of oxygen‐dependent methane monooxygenase), does not involve methanol as intermediate (Cicerone and Oremland, 1988; Oremland and Culbertson, 1992). However, in freshwater sediments, methane oxidation by nitrate‐ and nitrite‐dependent facultative anaerobic organisms has been shown to occur via particulate methane monooxygenase, with methanol as a key intermediate (Grinsven et al., 2020). Ca. Methylomirabilis oxyfera utilizes a strategy of intracellular production of oxygen from nitrite. This oxygen can then act as electron acceptor for methane oxidation, also utilizing particulate methane monooxygenase (Wu et al., 2011; Timmers et al., 2017; Vaksmaa et al., 2017). These processes with methanol as intermediary are leaky, and diffused methanol can be used by surrounding methylotrophic microorganisms (Wu et al., 2011; Chistoserdova and Kalyuzhnaya, 2018). Thus far, these processes have only been described in freshwater environments, which are richer in nitrite and nitrate than marine sediments (Ettwig et al., 2009; Grinsven et al., 2020). However, in an enrichment culture of marine origin with methane as substrate, the NC10 phylum to which Ca. M. oxyfera belongs was abundant (He et al., 2015). Furthermore, phylogenetic studies on marine oxygen minimum zones report detection of 16S rRNA genes closely related to NC10 bacteria as well as transcripts of particulate methane monooxygenase and nitric oxide reductase genes in oxygen minimum zones off the coast of northern Mexico and Costa Rica (Padilla et al., 2016). These findings suggest an environmental role of the NC10 phylum in marine environments as well.

It is unclear if and how methanol produced in surface water or exchanged with the air reaches the sediment of marine systems. As discussed above, methanol levels in the sediment are substantially higher than in the water column. Gradual deposition of methanol in the water column into the sediment is possible, but relatively high turnover rates of methanol in the water column (<1 day) suggests metabolization before it reaches anoxic sediments (Dixon et al., 2011a,b). This high methanol turnover can also explain its lower concentrations in the water column (Dixon et al., 2011a,b). In marine sediments, methanol turnover is estimated to be between 22 and more than 100 days (Zhuang et al., 2019a,b).

Biochemistry of microbial methanol utilization

In anaerobic environments, methanol can be converted by three distinct processes: oxidation to CO2 (e.g. by sulfate‐reducing microorganisms (SRM), Equation 1), conversion with CO2 to acetate (by acetogens, Equation 2), or conversion to methane and CO2 (by methanogens, Equation 3) (Goorissen et al., 2004; Chistoserdova, 2015; Yanagawa et al., 2016, Gibbs free energy changes calculated with data from Thauer et al., 1977). The distinct pathways employed for methanol conversion by these organisms are described below and are summarized in Fig. 1.

Fig. 1

Methanol degradation pathways as outlined in this review.

A. Respiratory methanol oxidation. Abbreviations: xoxF, lanthanide‐dependent methanol dehyrdogenase; MxA, calcium‐dependent methanol dehydrogenase; RuMP pathway, ribulose monophosphate pathway for carbon fixation; WLP, Wood–Ljungdahl pathway for carbon fixation.

B. Acetogenesis pathway. Abbreviations: THF, tetrahydrofolic acid; CODH/ACs, carbon monoxide dehydrogenase/Acetyl‐coA synthetase

C. methanogenesis; MT, methyl‐transferase 1; CoM, co‐enzyme M; H MPT, tetrahydromethanopterin; Mcr, methyl‐coenzyme M reductase; MFR, methanofuran. [Color figure can be viewed at wileyonlinelibrary.com]

The methanol methyltransferase system is a major pathway for methanol metabolism in anoxic environments. This pathway is catalysed by the methanol:coenzyme M methyltransferase MtaABC, found in methanogens, acetogens and SRM (Sauer et al., 1997; Visser et al., 2016; Sousa et al., 2018). Subunit MtaB cleaves the C‐O bond of the methanol and transfers the methyl group to a second subunit, MtaC, using cobalamin as a cofactor (Sauer et al., 1997). MtaC requires the cobalamin to contain the highly reduced cobalt(I), which is only possible in strict anoxic conditions (van der Meijden et al., 1984; Daas et al., 1996). The methylated group is subsequently transferred to either coenzyme‐M (HS‐CoM) in methanogens or tetrahydrofolic acid (THF) in acetogens and SRM by MtaA, forming CH3‐CoM or CH3‐THF respectively (van der Meijden et al., 1983; Stupperich, 1994; Daas et al., 1996; Sousa et al., 2018; Evans et al., 2019). It has been proposed that MtaA can be replaced with THF‐methyltransferase in Sporomusa strain An4 (Visser et al., 2016).

In methanogenic methanol conversion, one‐quarter of the substrate is oxidized to CO2 through a reversal of the hydrogen/CO2 methanogenesis pathway. This process yields six electrons per molecule of methanol, generating sufficient reducing power to reduce the remaining three‐quarters of the substrate, which is shuttled to CoM‐CH3. This CoM‐CH3 is further reduced by methyl coenzyme M reductase (Mcr) to methane and HS‐CoM (Gottschalk and Thauer, 2001; Welander and Metcalf, 2005). Furthermore, some methanogens of the order Methanomassiliicoccales couple H2 oxidation to methanol reduction, yielding solely methane as product (Costa and Leigh, 2014; Lang et al., 2015; Nobu et al., 2016).

In acetogens and SRM generated CH3‐THF can be integrated into the Wood–Ljungdahl pathway (WLP, Fig. 1). One in four molecules of CH3‐THF is used in a reversal of the methyl branch of the WLP, oxidizing the methylated group to CO2 and generating 2 mol of NAD(P)H, 1 mol of ATP and 1 mol of H2. The generated H2 is then utilized in a bifurcating mechanism to generate 0.5 mol of reduced ferredoxin (Fd2−). The microorganisms invest ATP to produce a proton/sodium gradient to generate an additional 2.5 mol of reduced ferredoxin through an RNF complex. This ferredoxin is subsequently used for the reduction of three molecules of CO2 to 3 mol of CO in the carbonyl branch of the WLP. The WLP carbonyl branch then converts this CO and the three remaining moles of CH3‐THF through acetyl‐CoA to acetate, generating 3 mol of ATP (Kremp et al., 2018).

Methanol dehydrogenase pathways are organized in three distinct clusters: MxaFI, Mdh2 and XoxF. Both MxaFI and XoxF methanol dehydrogenase clusters occur in a wide range of microorganisms and environments, including oceans, soils, or the human microbiome (Rusch et al., 2007; Lidbury et al., 2014; Dinasquet et al., 2018; Pietzke et al., 2020). Mdh2‐type is less widespread and has been detected in soil environments only (Kalyuzhnaya et al., 2008; Kolb, 2009). Although functionally similar, MxaFI and Mdh2 only share about 35% of amino acid identity, while Mdh2 shares up to 80% identity to other alcohol dehydrogenases with a low affinity for methanol (Anthony and Williams, 2003; Kalyuzhnaya et al., 2008; Lu et al., 2012; Keltjens et al., 2014; Chistoserdova and Kalyuzhnaya, 2018). MxaFI and Mdh2 methanol dehydrogenases catalyse the conversion of methanol to formaldehyde, releasing two electrons which are then shuttled to cytochrome c, whereas XoxF catalyses the conversion of methanol to formate. Mdh2 utilizes NAD(P) as cofactor to shuttle electrons to cytochrome c (Zhang et al., 2017). Both MxaFI and XoxF enzyme systems utilize pyrroloquinoline quinone (PQQ) as cofactor for electron transport to cytochrome c, but they differ in the active site metal. MxaFI utilizes calcium, whereas XoxF utilizes a range of rare earth elements called lanthanides (Picone and Op den Camp, 2019). Incorporation of strontium in vitro instead of calcium by MxaFI methanol dehydrogenase has been reported, resulting in increased reaction rates (threefold over calcium) and lower activation energy (by 13.4 kJ mol−1) (Harris and Davidson, 1994). While calcium is abundantly present in seawater, with concentrations measured in the millimolar range, strontium is available with concentrations around 150 μM. Whether all microorganisms containing these methanol dehydrogenases are able to incorporate strontium in vivo and whether this has ecological meaning in regards to methanol competitiveness for microorganisms utilizing this strategy requires further research. XoxF enzymes can work with any element of the lanthanide group, although the specific activity is higher with the two lightest lanthanides, lanthanum and cerium, compared with heavier lanthanides like neodymium or promethium (Kiene et al., 1986; Keltjens et al., 2014; Chistoserdova and Kalyuzhnaya, 2018; Picone and Op den Camp, 2019). While XoxF is mostly described in aerobic marine microorganisms, Ca. Methylomirabilis oxyfera utilizes XoxF in its metabolism, which is intracellular aerobic (Ramachandran and Walsh, 2015; Taubert et al., 2017; Chistoserdova and Kalyuzhnaya, 2018; Howat et al., 2018). Under high calcium and low lanthanide concentrations, the methanotroph Methylomicrobium buryatense, which contains both methanol dehydrogenase pathways in its genome, has a higher expression of XoxF than of MxaFI (Chu and Lidstrom, 2016). Interestingly, while metagenomes of anaerobic terrestrial environments revealed multiple XoxF‐type alcohol dehydrogenases, PQQ biosynthesis requires molecular oxygen (Velterop et al., 1995; Magnusson et al., 2004; Martins et al., 2019). Diffusion of PQQ from aerobic marine sediments, where XoxF is the most abundant methanol dehydrogenase could provide anoxic sediments with this cofactor (Ramachandran and Walsh, 2015; Taubert et al., 2019). Furthermore, it cannot be excluded that alternative, unknown pathways for PQQ biosynthesis that may not involve oxygen exist.

Microbial ecology of methanol degradation in marine environments

The processes mentioned in the previous paragraph occur in many phyla of both Bacteria and Archaea, including SRM, nitrate‐reducing microorganisms, acetogens and methanogens which all occur in marine sediments. Furthermore, metals such as ferric iron or manganese can be present in these environments and can act as an electron acceptor for some methanol oxidizers (Leu et al., 2020). However, near neutral pH these compounds are insoluble and occur mainly as minerals, severely limiting their use as electron acceptor without specialized extracellular electron transfer (EET) systems, as found in genera like Geobacter or Shewanella (Shi et al., 2016). Although microorganisms capable of EET utilizing methanol have been described, such as Shewanella putrefaciens, there is very little known on their role in marine sediments (Daniel et al., 1999). Multiple marine species capable of growing on methanol in anaerobic conditions have been isolated and characterized, and these are described in Table 2.

Table 2

Isolated marine microorganisms capable of growing on methanol and their growing conditions.

Name organism	Original isolation source	Temperature range (°C)	pH range	Reference
Sulfate‐reducing microorganisms
Desulfallas arcticus	Marine surface sediment, Svalbard, Norway	26–46.5	7.1–7.5	Vandieken et al. (2006); Watanabe et al. (2018)
Desulfoconvexum algidum	Marine surface sediment, Svalbard, Norway	0–20	7.2–7.4	Konneke et al. (2013)
Desulfospira joergensenii a	Marine sediment, Arcachon Bay, France	8–30	7.4‐ND	Finster et al. (1997)
Desulfatiglans anilini	Marine sediment, North Sea coast, Germany	12–40	6–8	Schnell et al. (1989)
Desulfosporosinus nitroreducens	Baltic sea sediment,	10–30	6.4–8.1	Vandieken et al. (2017)
Nitrate‐reducing microorganisms
Methylophaga nitratireducenticrescens	Seawater denitrification reactor, Montreal Canada	15–37	6–11	Labbé et al. (2007)
Acetogens
Acetobacterium woodii b	Oyster pond, Massachusetts	2–45	5.9–8.5	Balch et al. (1977)
Methanogens
Methanococcoides methylutens	Scripps Canyon, California	30–35	7–7.5	Sowers and Ferry, 1983; Watkins et al. (2014)
Methanococcoides burtonii	Saltwater lake in Antarctica	5.6–29.5	6.8–8	Franzmann et al. (1992); Watkins et al. (2014)
Methanococcoides alaskense	Skan Bay, Alaska	−2.3‐28.4	6.3–7.5	Singh et al. (2005); Watkins et al. (2014)
Methanococcoides vulcani	Napoli mud volcano, Mediterranean Sea	ND‐35	6–7.8	L'Haridon et al. (2014); Watkins et al. (2014)
Methanosarcina acetivorans	Scripps Canyon, California	10–50	5.5–8.5	Ferry (1999); Sowers et al. (1984)
Methanosarcina baltica	Baltic sea	−22.3‐27	4.9–8.5	Von Klein et al. (2002)
Methanosarcina semesiae	Dar es Salaam mangrove, Tanzania	30–35	6.5–7.5	Lyimo et al. (2009)
Methanosarcina siciliae	Scripps canyon, California	15–40	5–7.5	Elberson and Sowers (1997)
Methermicoccus shengliensis	Shengli oilfield, South China Sea	50–70	5.5–8	Cheng et al. (2007)
Methanohalophilus halophilus	Shark Bay, Australia	26–36	6.3–8	Wilharm et al. (1991)
Methanolobus bombayensis	Arabian sea	15–43	6.2–8.3	Kadam et al. (1994)
Methanolobus vulcani	San Francisco Bay, California	13–45	6–7.5	Kadam and Boone (1995)
Methanolobus profundi	Deep subsurface sediments, Movara, Japan	9–37	6.1–7.8	Mochimaru et al. (2009)
Methanolobus taylorii	San Francisco Bay, California	5–45	5.7–9.2	Oremland and Boone (1994)
Methanolobus tindarius	Tindari, Sicily	7–50	5.5–8	König and Stetter (1982)

D. joergensenii showed sulfide production on methanol but no growth.

A. woodii was isolated from an ocean inlet that was closed off to the sea.

SRMs occur amongst seven lineages, five of which are within the domain Bacteria and two in the domain Archaea, although methanol‐utilizing SRM have only been described in Bacteria. Bacterial SRM belong to Deltaproteobacteria, Clostridia, Nitrospirae, Thermodesulfobacteria and Thermodesulfobiaceae (Muyzer and Stams, 2008). However, based on single‐copy marker gene analysis, the phylum Deltaproteobacteria was recently proposed to be divided into the sulfate‐reducing phylum Desulfobacterota and the non‐sulfate reducing phyla Myxococcota, Bdellovibrionota and SAR324. Additionally, this assessment places the phylum Thermodesulfobacteria within the phylum Desulfobacterota (Waite et al., 2020). Furthermore, based on metagenomic datasets, 13 bacterial and archaeal phyla were identified to have the genes for sulfate reduction (DsrABCD), thereby doubling the number of known taxa (Anantharaman et al., 2018). Only a few marine sulfate reducers have been proven to grow with methanol as the sole electron donor and carbon source (Table 2). However, methanol is not conventionally tested as substrate in the characterization of newly isolated marine SRM.

As the most energetically favourable electron acceptor after oxygen that is abundantly available, nitrate reduction is common at the oxic–anoxic interphase, carried out by anaerobes and facultative anaerobes (Kuypers et al., 2018). However, there is a lack of investigation on the use of methanol by nitrate reducers in oxygen minimum zones or marine anoxic sediments. While dissolved organic matter is often not limiting in these systems, methanol is available and yields comparable energy as DOM per reducing equivalent (Nurse, 1980). While freshwater denitrification with methanol as electron donor is widely studied due to its biotechnological relevance, whether this process occurs in marine sediments is less clear (Labbé et al., 2007; Villeneuve et al., 2013).

Acetogens are a diverse group of bacteria, occurring in 22 genera comprising over 100 species (Drake et al., 2008). Their key defining trait is the utilization of the Wood–Ljungdahl pathway to fix CO2 for both energy and biomass production. This metabolism is widely dispersed amongst many habitats, and acetogens are metabolically flexible, incorporating a broad range of electron donors besides C1 compounds, such as sugars or organic acids (Drake et al., 2008). Currently, there are few well‐characterized marine acetogens capable of growing on methanol (Table 2). Acetobacterium woodii was isolated from a former ocean inlet that was closed off but had characteristics similar to the sea (Drake et al., 2008; Kremp et al., 2018). Furthermore, Schuppert and Schink (1990) isolated an acetogen from the marine Rio Marin in Venice, Italy (Schuppert and Schink, 1990). However, this strain was never deposited in a culture collection. About 67% of all cultivated acetogens have been shown to grow on methanol (Lever et al., 2010). Acetogenesis has been described in enriched cultures from marine sediments, and genetic evidence for genes in the Wood–Ljungdahl pathway, such as the presence of formyltetrahydrofolate synthetase and methenyltetrahydrofolate cyclohydrolase coding genes, have been found in metagenomes from a diverse range of marine systems, for example the Juan de Fuca Ridge, the Guaymas Basin, the Baltic Sea and the Arctic Sea (Lovell and Leaphart, 2005; Lever et al., 2010; Kirchman et al., 2014; He et al., 2016; Beulig et al., 2018; Jochum et al., 2018; Marshall et al., 2018). These findings suggest that acetogenesis and the Wood–Ljungdahl pathway are widely spread in marine systems.

Methanogens were canonically only described within the archaeal phylum Euryarchaeota, comprising seven orders and 29 genera (Bapteste et al., 2005; Holmes and Smith, 2016). However, recent genomic discoveries have found organisms containing genes coding for methanogenic pathways in the distantly related archaeal phyla Bathyarchaeota and Verstraetearchaeota, suggesting a much wider phylogenetic range of this metabolism than previously thought (Evans et al., 2015; Vanwonterghem et al., 2016; Adam et al., 2017; Evans et al., 2019). Besides marine systems, methanogens have mainly been isolated from animal rumen, rice paddies, soils and freshwater systems (Lyu et al., 2018). In marine anaerobic systems, methanogens occur in the sulfate‐depleted zone of the sediment, usually several meters below the seafloor, as most methanogens are outcompeted for their common substrates hydrogen and acetate by SRM (Harrison et al., 2009). However, methylotrophic methanogenesis has been described to occur in the sulfate zone of the sediment and attributed to the utilization of non‐competitive substrates such as methanol (Sela‐Adler et al., 2017). Furthermore, through interspecies hydrogen transfer, syntrophic interactions between methanogens and SRM for substrates are possible, leading to methane production in the sulfate reduction zone (Dolfing et al., 2008; Ozuolmez et al., 2015). It is uncertain whether methanol can be utilized in syntrophic interactions in marine environments. A recent study of Mediterranean Sea sediments suggested methanol as being the primary source of methane in sulfate‐rich sediments as hydrogenotrophic methanogenesis appeared to be outcompeted by sulfate reduction, with up to 98% of total methane produced in the top sediment deriving from methanol, suggesting methanol to be the main source of methanogenesis (Zhuang et al., 2018a,b). Cultured methylotrophic methanogens comprise three orders, Methanosarcinales, Methanobacteriales and Methanomassiliicoccales, although only Methanosarcinales have marine representatives. An overview of methanol‐utilizing methanogens currently isolated is shown in Table 2. Based on genomic information, methylotrophic methanogenesis has been found outside of the canonical methanogenic phyla, such as Verstraetearchaeota, which suggests a much broader phylogeny of methylotrophic methanogenesis than previously thought (Vanwonterghem et al., 2016).

Methanol was considered to be a non‐competitive substrate for methanogens, explaining the co‐occurrence of methanogens in sulfate‐rich sediments, where methanogens would be outcompeted by SRM for common substrates such acetate or hydrogen (Oremland and Polcin, 1983). However, in situ oxidation rates of methanol to CO2 were much higher than solely methanogenesis rates would allow (Zhuang et al., 2018a; 2019a,b). Furthermore, radiotracer experiments have shown that in intertidal sediments in Maine, United States, methanol is mostly oxidized through sulfate reduction with methanogenesis only contributing 2.5% of all methanol oxidized (King et al., 1983). Coexistence of methanogenesis along with acetogenesis and sulfate reduction is thermodynamically possible, as all methanol conversion reactions have relatively similar Gibbs free energy, i.e. −105, −120 and −133 kJ mol −1 for acetogenesis, sulfate reduction and methanogenesis respectively (values calculated for hydrogen partial pressure of maximum 10 nM) (Meulepas et al., 2010; Lever, 2011; Lyu et al., 2018). Due to the similar energy gains for the different processes, the driving forces for competitive advantage on growth on methanol is the affinity of the metabolic interaction for the substrate and kinetics of the enzymes involved in methanol oxidation, as well as auxotrophy, and the availability of cofactors and catalytic metals in the environment.

It is assumed that microorganisms containing methanol methyltransferase pathways outcompete microorganisms containing methanol dehydrogenases under optimal conditions for both pathways, due to the higher affinity of methyltransferases for methanol (Florencio et al., 1993; Florencio et al., 1994). This could mean methanogens and acetogens might be able to outcompete SRM for methanol. Several studies on environmental systems have indicated that in anaerobic methanol degradation, cobalt is often the limiting factor (Florencio et al., 1993). As cobalt is the active metal in methyltransferases, this corroborates the theory of competitive advantage of methanogens and acetogens. However, Sousa et al. (2018) showed the presence of methanol methyltransferase genes in the genome of the sulfate‐reducing bacterium Desulfofundulus kuznetsovii (formerly Desulfotomaculum kuznetsovii) as well as proteomic evidence for an upregulation of the MT system in the presence of cobalt (Sousa et al., 2018; Watanabe et al., 2018). This would mean that under cobalt‐rich conditions, this SRM would be able to compete with methanogens and acetogens for the available methanol. Under cobalt‐limiting conditions, it has a competitive advantage as the methanol dehydrogenase is not dependent on cobalt. While D. kuznetsovii is not a marine organism, the genes encoding for the methyltransferase pathway in D. kuznetsovii have orthologues in many other closely related SRM, for example the marine Desulfallas arcticus, Desulfosporosinus fructosivorans or the freshwater species Desulfosporosinus merideii and Desulfosporosinus lacus (Ramamoorthy et al., 2006; Vandieken et al., 2006, 2017; Pester et al., 2012). Anoxic marine sediments have been found to be relatively rich in cobalt, while it is depleted in the oxic sediment and pore waters above (Heggie and Lewis, 1984). This suggests the possibility of rapid consumption in the oxic–anoxic interphase, indicating competitive advantages for microorganisms containing both pathways.

Metagenomic assessment of key genes involved in methanol metabolism in marine sediments

To assess the dispersion of methanol conversion amongst marine sediments worldwide, we mined core metabolic genes involved in the cycling of methanol in deposited annotated metagenomes in the Integrated Microbial Genomes & Microbiomes (IMG) v.5.0 database following the procedure described by Chen et al. (2019). Six genes involved in methanol utilization pathways were selected: three methanol methyltransferase genes: mtaA; mtaB; mtaC), one lanthanide‐dependent alcohol dehydrogenase (xoxF), one methanol‐derived formaldehyde dehydrogenase (fdhA) and one pectin methanolesterase (pesT) (Kanehisa et al., 2016). Formaldehyde dehydrogenase was chosen over canonical methanol dehydrogenase due to the high chance of methanol dehydrogenases mistakenly being annotated as other alcohol dehydrogenases, as there is a high similarity between residues between these enzymes and thus increasing the risk of false positives (Reid and Fewson, 1994). Pectin esterase was included to test the hypothesis whether methanol is produced in sediments from pectin.

Utilizing the search function of IMG and manual curation, all metagenomes of anoxic marine sediment samples were exported. In total, 246 metagenomes were selected for this assay. Of these metagenomes, annotation data and metadata containing information on sampling location and sample site water column depth were also collected (Supplementary Table S1). Supplementary Fig. S1 shows a world map with sample locations of all metagenomes grouped by water column height (Fig. S1). Most of the metagenomes derived from datasets close to shores, based on the metadata supplied by IMG (Table S1). This is unsurprising, as deep‐sea sampling is logistically difficult and expensive (Clark et al., 2016). Likewise, most datasets originate from either the US coasts or the North Sea in Europe, as they are relatively close to large, well‐funded marine institutes. Thus most information on methanol cycling as discussed in this review is biased to these environments. There is only a very limited set of metagenomes from deep ocean samples, for example IMG3300010241‐3 obtained from the South China Sea or IMG3300016982 obtained from the Southern Atlantic Ocean. Thus, it is difficult to differentiate between coastal ecosystems, which are generally richer in organic material and have a higher carbon turnover than deeper sea ecosystems (Rowe et al., 1990; Böer et al., 2009).

Using a hidden Markov model –based approach, all metagenomes were scanned for the listed genes. Detailed workflow and tools used can be found in Supplementary Text 1. Total data output was visualized using the R package pheatmap (Kolde, 2019; R Core Team, 2013). Figure 2 displays the results of this metagenome mining effort. Fig. S2a–f shows world maps with corresponding positive locations for each of the six mapped genes.

Fig. 2

Metagenomic mining heatmap. Red indicates presence, blue absence. Gene abbreviations: xoxF, lanthanide dependent methanol dehydrogenase F; mtaA, methanol methyltransferase A; mtaB, methanol methyltransferase B; mtaC, methanol methyltransferase C; fdhA, formaldehyde dehydrogenase A; pest, pectin methanolesterase. Each cluster denotes 50 metagenomes screened. [Color figure can be viewed at wileyonlinelibrary.com]

Based on the prevalence of genes involved in methanol conversion, the utilization is widespread in diverse anoxic marine sediments. Out of 246 published metagenomes, 78% mapped positively for at least one of the selected marker genes in this study. Over 50% of metagenomes mapped positively for at least three selected marker genes, suggesting utilization of methanol by all trophic groups discussed in this review. Interestingly, mtaB is much less present than mtaC; 117 positive hits in all metagenomes versus 172 for mtaC, even though for the utilization of methanol MtaB and mtaC form a complex (Ferry, 1999). Whether this represents evolutionary artefacts or alternative pathways involving orthologues of these genes is not known. As MtaC contains a corrinoid centre to which the methyl group of methanol is bound, it is possible it acts as a carrier for methyl groups derived from non‐methanol origins, for example trimethylamine, which is widely present in marine sediments and utilized by methanogens (Ferguson et al., 2011; Sun et al., 2019). Lanthanide‐dependent methanol dehydrogenase could be detected in 112 of the 246 datasets, whereas formaldehyde dehydrogenase was found in 87 datasets. As xoxF produces formaldehyde, it is possible other, currently unknown types of formaldehyde incorporation are present in these metagenomes. Furthermore, the presence of xoxF as artefacts in these metagenomes that are not actively utilized is also possible. Pectin esterase was present in 52 out of 246 metagenomes. While this was the least present of all studied genes, this still supports the hypothesis that complex carbohydrate degradation can occur in marine sediments, rather than only in the upper water column.

Concluding remarks

Methanol is an important compound in the global biogeochemical cycles, which has received little attention in marine sediments. There are still many gaps in our knowledge on the prevalence of methanol in these environments, how it becomes available and which microorganisms are involved in its cycling. The utilization of non‐canonical metals in methanol dehydrogenases requires a rethinking of bio‐active metals involved in this process. Cultivation approaches fail to recover the vast majority of microorganisms from marine sediments. The use of metagenomic data provides a reliable indication of the diversity and potential functions of specific microorganisms. In this regard, our approach of data mining marine sediment metagenomes for key methanol metabolism genes indicated this compound is released and utilized in anoxic marine sediments worldwide in a variety of ways. Thus, it can be concluded that methanol utilization is an active force in anoxic marine sediments, based on their genomic presence in these environments. This methodology can aid in the further understanding of the ecology of marine anoxic systems.

Author contributions

P.Q.F.: Conceptualization (supporting), data curation (lead), formal analysis (lead), writing—original draft preparation (lead). I.S.‐A.: Writing—review and editing (supporting). A.J.M.S.: Writing—review and editing (supporting), validation (equal), conceptualization (supporting). L.V.: Conceptualization (equal), validation (equal), writing—review and editing (supporting). D.Z.S. Conceptualization (equal), validation (equal), writing—review and editing (lead).

Appendix S1. Supporting Information.

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