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Chapter 22. Geomicrobiology of Fossil Fuels

Chapter 22. Geomicrobiology of Fossil Fuels

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538



Geomicrobiology



Atmospheric CO2

6.4 × 1017 g C

a



Dissolved

CO2

3.8 × 1019 g C



r



r

a



8.3 × 1017 g C



Dissolved

organics



a



Living biomass



e,d



1.5 × 1018 g C



a

m



a,m

s



d



Organic carbon

in sediments and

soils



Limestone and other

fixed carbonates



3.5 × 1018 g C



1.8 × 1022 g C



b

Trapped organic

carbon:

natural gas, coal,

petroleum,

kerogen, and bitumen

2.5 × 1022 g C



FIGURE 22.1 Microbial and physical processes contributing to carbon transfer among different compartments in the biosphere. a, Microbial assimilation; b, burial; d, decomposition; e, excretion; m, microbial mineralization; r, respiration; s, sedimentation. (Quantitative estimates from Fenchel T, Blackburn TH, Bacteria

and Mineral Cycling, Academic Press, London, U.K., 1979; Bowen HJM, Environmental Chemistry of the

Elements, Academic Press, London, U.K., 1979.)



acceptors such as nitrate, Fe(III), Mn(IV), or sulfate. If gas pressure due to methane builds up sufficiently in anaerobic lake or coastal sediment, it may escape in the form of large gas bubbles that

break at the water surface to release their methane into the atmosphere (Martens, 1976; Zeikus,

1977). In marshes, escaping methane may be ignited (by biogenic phosphene?) to burn as the socalled will-o’-the-wisps.

Many of the methane accumulations on Earth are of biogenic origin. Methane may occur in association with peat, coal, and oil deposits, or independent of them. That which occurs in association

with coal and oil was probably microbially generated in the early stages of their formation, although

some may have been formed abiotically in later diagenetic phases. Methane associated with coal

deposits can be the cause of serious mine explosions when accidentally ignited. Such methane is

called coal damp by coal miners.

Biogenic methane formation is a unique biochemical process that appears to have arisen very

early in the evolution of life. Indeed, the methanogenesis that results from the microbial reduction



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of CO2 by H2 may represent the first process or one of the first processes on Earth that autotrophs

have harnessed for energy conservation (see Chapter 3).



22.3.1



METHANOGENS



All methane-forming bacteria, that is, methanogens, are members of the domain Archaea. As a

group, they are very diverse phylogenetically (Jones et al., 1987; Boone et al., 1993). They also

show great diversity morphologically, existing as rods, spirilla, cocci, and sarcinae (Figure 22.2).

The feature that they share in common is that of being strict anaerobes that form methane as a

product of their energy-generating metabolism (respiration and fermentation). The large majority of

them are obligate or facultative autotrophs. The majority can get their energy from the reduction of

carbon dioxide with hydrogen or its equivalent (formate or CO), whereas they obtain their carbon

exclusively by assimilating carbon dioxide. A few, like Methanosarcina (formerly Methanothrix)

x

Soehngenii (Zehnder et al., 1980; Huser et al., 1982) and Methanosaeta (formerly Methanothrix)

x

concilii (Patel, 1984), are heterotrophs that use acetate as energy and carbon source, and for this



FIGURE 22.2

Morphologies of different methanogens. (a) Methanocullens bourgensis (formerly,

Methanogenium bourgense), (b) Methanosaeta (formerly Methanothrix)

x concilii, (c) Methanospirillum hungatei OGC 16, (d) Methanosarcina barkeri OGC 35, (e) Methanobacterium formicicum OGC 55. Scale mark

represents 10 µm and applies to all panels. (Courtesy of Boone DR, Wills R)



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reason are known as acetotrophic or aceticlastic methanogens. At least one methanogen can use S0

in addition to CO2 as the terminal electron acceptor (Stetter and Gaag, 1983). As a group, most methanogens are nutritionally restricted to the following energy sources: H2, CO, HCOOH, methanol,

methylamines, and acetate (Atlas, 1997; Brock and Madigan, 1988). But exceptions exist. Widdel

reported that a freshwater strain of Methanospirillum and a strain of Methanogenium were each

able to grow on 2-propanol and 2-butanol as well as on H2 and formate. Zellner et al. (1989) found

that Methanobacterium palustre was able to grow on 2-propanol as an energy source as well as on

H2 and formate. It was able to oxidize but not grow on 2-butanol. In 1990, Zellner et al. reported that

Methanogenium liminatans can use 2-propanol, 2-butanol, and cyclopentanol as energy sources in

addition to H2 and formate (Zellner et al., 1990). Finster et al. (1992) found that strain MTP4 can use

methanediol and dimethylsulfide as well as methylamines, methanol, and acetate as energy sources.

Yang et al. (1992) found that Methanococcus voltae, M. maripaludis, and M. vannielii can each use

pyruvate as an energy source in the absence of H2.

For methanogens to be able to draw on the wide range of oxidizable carbon compounds that may

be available in their environment but that cannot be metabolized by them directly, they associate

with heterotrophic fermenters or anaerobic respirers that do not completely mineralize their organic

energy sources (see, e.g., Jain and Zeikus, 1989; Sharak Genthner et al., 1989; Grbic-Galic, 1990).

To optimize access to the microbially generated energy sources and the electron acceptor CO2 that

these methanogens need, some of them form intimate consortia (syntrophic associations)

s with other

anaerobic bacteria that can furnish them with these energy sources (H2, acetate) and CO2 through

their metabolic end products (see, e.g., Bochem et al., 1982; MacLeod et al., 1990; McInerney et al.,

1979; Winter and Wolfe, 1979, 1980; Zinder and Koch, 1984; Wolin and Miller, 1987). Frequently,

the metabolites that are the basis for these syntrophic associations are not readily detectable when

all the members of the consortium are growing together in mixed culture. This is because the

metabolites are consumed as quickly as they are formed. When hydrogen is the metabolite, the

process is called interspecies hydrogen transfer (Wolin and Miller, 1987).

Among the most widely recognized genera of methanogens are Methanobacterium, Methanothermobacter, Methanobrevibacterium, Methanococcus, Methanomicrobium, Methanogenium,

Methanospirillum, Methanosarcina, Methanoculleus, and Methanosaeta (see Brock and Madigan,

1988; Bhatnagar et al., 1991; Boone et al., 1993; Atlas, 1997). Methanogens may be mesophilic or

thermophilic. They are found in diverse anaerobic habitats (Zinder, 1993), including some marine

environments such as salt marsh sediments (Oremland et al., 1982; Jones et al., 1983b), coastal sediments (Gorlatov et al., 1986; Sansone and Martens, 1981), anoxic basins (Romesser et al., 1979),

geothermally heated seafloor (Huber et al., 1982), hydrothermal vent effluent on the East Pacific

Rise (Jones et al., 1983a), sediment effluent channel of the Crystal River Nuclear Power Plant

(Florida; Rivard and Smith, 1982), lakes (Deuser et al., 1973; Jones et al., 1982; Giani et al., 1984),

soils (Jakobsen et al., 1981), desert environments (Worakit et al., 1986), solfataric fields (Wildgruber

et al., 1982; Zabel et al., 1984), oil deposits (Nazina and Rozanova, 1980; Rubinshtein and Oborin,

1986; Stetter et al., 1993), the digestive tract of insects and higher animals, especially ruminants and

herbivores (Breznak, 1982; Brock and Madigan, 1988; Wolin, 1981; Zimmerman et al., 1982; Atlas,

1997), and as endosymbionts (van Bruggen et al., 1984; Fenchel and Finlay, 1992). Thus, despite

their obligately anaerobic nature, methanogens are fairly ubiquitous.

Methanogens play an important but not exclusive role in anaerobic mineralization of organic

carbon compounds in soil and aquatic environments, especially freshwater sediments (Wolin and

Miller, 1987). In marine sediments, where methanogens have to share hydrogen and acetate as

sources of energy with sulfate-reducing bacteria, they tend to be outcompeted by the sulfate reducers because of the latter’s higher affinity for hydrogen and acetate (Abrams and Nedwell, 1978;

Kristjansson et al., 1982; Schönheit et al., 1982; Robinson and Tiedje, 1984). Thus, in many estuarine

or coastal anaerobic muds, sulfate-reducing activity and methanogenesis occur usually in spatially

separated zones in the sediment profile, with the zone exhibiting sulfate-reducing activity overlying

the zone exhibiting methanogenesis (e.g., Martens and Berner, 1974; Sansone and Martens, 1981).



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Recent evidence indicates that some sulfate-reducing bacteria can also use methane as electron

donor (Section 22.3.5.2).

Under two special circumstances, methanogenesis and sulfate reduction can be compatible

in an anaerobic marine environment. One circumstance is the existence of an excess supply of

a shared energy source (H2 or acetate; Oremland and Taylor, 1978). The other circumstance is

one where sulfate reducers and methanogens use different energy sources, namely, products of

decaying plant material and methanol or trimethylamine, respectively (Oremland et al., 1982). In

anaerobic freshwater sediments and soils where sulfate, nitrate, ferric oxide, and manganese(IV)

oxide concentrations are very low, methanogenesis is usually the dominant mechanism of organic

carbon mineralization. Yet, even here, certain sulfate-reducing bacteria may grow in the same

niche as methanogens (e.g., Koizumi et al., 2003). Indeed, they may form a consortium with them.

In the absence of sulfate, these sulfate reducers ferment suitable organic carbon with the production of H2, which the methanogens then use in their energy metabolism to form methane (e.g.,

Bryant et al., 1977).

A few methanogens, in particular, Methanosarcina barkeri grown with H2/CO2 or methanol,

have the ability to reduce Fe(III) in place of CO2 (van Bodegom et al., 2004). This ability may

explain in part the inhibition of methanogenesis in soil and sediment by Fe(III).



22.3.2 METHANOGENESIS AND CARBON ASSIMILATION BY METHANOGENS

22.3.2.1 Methanogenesis

One kind of autotrophic methane formation represents a form of anaerobic respiration in which

hydrogen (H2 ) is the electron donor and CO2 is the terminal electron acceptor, with the CO2 being

transformed to CH4 according to the overall reaction

4H2 + CO2 → CH4 + 2H2O



(22.1)



This reaction is exothermic and yields energy (∆G 0 = –33 kcal or –137.9 kJ) that can be used by the

organism to do metabolic work.

In a few instances, secondary alcohols were found to serve as electron donors, with CO2 as the

terminal electron acceptor. The CO2 was therefore the source of the methane formed (Widdel, 1986;

Zellner et al., 1989). In these reactions, the alcohols were replacing H2 as the reductant of CO2. At

least one instance is known in which ethanol served as the electron donor for methane formation

from CO2, the ethanol being oxidized to acetate (Frimmer and Widdel, 1989):

2CH 3CH 2OH ϩ HCOϪ3 → 2CH 3COOϪ ϩ CH 4 ϩ H 2O ϩ Hϩ

(∆G 0 ϭ Ϫ27.8 kcal/mol CH 4 or Ϫ116.3 kJ/mol CH 4 )



(22.2)



The organism in this instance was a nonautotrophic methanogen, Methanogenium organophilum

growing in a medium containing 0.05% tryticase peptone and 0.05% yeast extract as nitrogen

sources among other ingredients (Frimmer and Widdel, 1989).

Some methanogens can form methane by a disproportionation reaction, that is, by fermentation,

in which a portion of the substrate molecule acts as the electron donor (energy source) and the rest as

the electron acceptor. For example, they can produce methane from carbon monoxide, formic acid,

methanol, acetate, and methylamines without H2 as the electron donor (Brock and Madigan, 1988;

Atlas, 1997; Mah et al., 1978; Smith and Mah, 1978; Zeikus, 1977),



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4HCOOH → CH4 + 3CO2 + 2H2O (∆G 0 = –35 kcal or –146.3 kJ)



(22.3)



4CH3OH → 3CH4 + CO2 + 2H2O (∆G 0 = –76 kcal or 317.7 kcal)



(22.4)



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CH3COOH → CH4 + CO2



(∆G 0 = –9 kcal or –37.6 kJ)



4CH3NH2 + 2H2O → 3CH4 + CO2 + 4NH3 (∆G 0 = –75 kcal or –313.5 kJ)

4CO + 2H2O → CH4 + 3CO2



(∆G 0 = –44.5 kcal or –186 kJ)



(22.5)

(22.6)

(22.7)



Although methanogenesis from acetate by the disproportionation reaction of aceticlastic methanogens (Reaction 22.5) is fairly common during anaerobic degradation of organic matter in the

absence of a plentiful external supply of external electron acceptors such as Fe(III), Mn(IV),

or sulfate, a consortium of anaerobic acetate oxidizers like Clostridium spp., which generate

H2 and CO2 from the acetate, and hydrogenotrophic methanogens like Methanomicrobium or

Methanobacterium may form methane in the absence of aceticlastic methanogens (Karakashev

et al., 2006).

Some methanogens can form methane from pyruvate by disproportionation (Yang et al., 1992).

Resting cells of Methanococcus spp. grown in a pyruvate-containing medium under an N2 atmosphere were shown to transform pyruvate to acetate, methane, and CO2 according to the following

stoichiometry (Yang et al., 1992):

4CH 3COCOOH ϩ 2H 2O → 4CH 3COOH ϩ 3CO2 ϩ CH 4 (∆G 0 ϭ Ϫ74.9 kcal or 313.1 kJ)



(22.8)



This stoichiometry is attained if the organism oxidatively decarboxylates pyruvate:

4CH3COCOOH + 4H2O → 4CH3COOH + 4CO2 + 8(H)



(22.8a)



and uses the reducing power [8(H)] to reduce one-fourth of the CO2 to CH4:

CO2 + 8(H) → CH4 + 2H2O



(22.8b)



Bock et al. (1994) found that a spontaneous mutant of Methanosarcina barkeri could grow by fermenting pyruvate to methane and CO2 with the following stoichiometry:

CH3COCOOH + 0.5H2O → 1.25CH4 + 1.75CO2



(22.9)



To achieve this stoichiometry, the authors proposed the following mechanism based on known

enzyme reactions in methanogens. Pyruvate is oxidatively decarboxylated to acetyl∼SCoA

and CO2:

CH3COCOOH + CoASH → CH3CO∼SCoA + CO2 + 2(H)



(22.9a)



The available reducing power [2(H)] from this reaction is then used to reduce one-fourth of the CO2

formed to methane:

0.25CO2 + 2(H) → 0.25CH4 + 0.5H2O



(22.9b)



and the acetyl∼SCoA is decarboxylated to methane and CO2:

CH3CO∼SCoA + H2O → CH4 + CO2 + CoASH



(22.9c)



The standard free energy yield at pH 7 (∆G 0) was calculated to be –22.9 kcal mol−1 or –96 kJ mol–1

of methane produced (Bock et al., 1994).



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Although Reactions 22.1 through 22.7 look very disparate, they share a common metabolic pathway (Figure 22.3). The reason methanogens differ with respect to the methane-forming reactions

they can perform is that not all of them possess the same key enzymes that permit entry of particular methanogenic substrates into the common pathway (Vogels and Visser, 1983; Zeikus et al.,

1985; Stanier et al., 1986; Brock and Madigan, 1988; Atlas, 1997). The pathway involves stepwise

reduction of carbon from the +4 to the –4 oxidation state via bound formyl, methylene, and methyl

carbon. The operation of the methane-forming pathway requires some unique coenzyme and carrier molecules (Table 22.1). Coenzyme M (2-mercaptoethylsulfonate) is unique to methanogens and

CO



CO2

(+H2O)



+ MF, +2(H)



Methanofuran



CHO

+ THMP, F420red., −H2O

CH2



Tetrahydromethanopterin

+2(H)



CH3



Tetrahydromethanopterin

+ CoM



Acetate

+CoASH



+B12

Acetyl ~ SCoA −CoASH, −CO

2

Methanol

Methylamines



Coenzyme M



CH3



+F420red.

CH4 + CoM



Secondary alcohols



FIGURE 22.3 Pathways of methanogenesis from CO, CO2, acetate, methanol, secondary alcohols, and

methylamines. (THMP, Tetrahydromethanoptein.)



TABLE 22.1

Unusual Coenzymes in Methanogens

Coenzymea

Methanofuran

Methanopterin (coenzyme F342)

Coenzyme M (2-mercaptoethane sulfonate)

Coenzyme F430

Coenzyme F420 (nickel-containing tetrapyrrole)



a



Function

CO2 reduction factor in first step of

methanogenesis

Formyl and methene carrier in methanogenesis

Methyl carrier in methanogenesis

Hydrogen carrier for reduction of methyl

coenzyme M

Mediates electron transfer between hydrogenase

or formate and NADP, reductive carboxylation

of acety1∼CoA, and succinyl∼CoA



For structures of these coenzymes, see Brock and Madigan (1988) and Blaut et al. (1992).



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may be used to identify them as methane formers. The large majority of methanogens synthesize

this molecule de novo.

Methanogenic reactions with hydrogen as the electron donor that utilize formic or acetic acid,

methanol, or methylamines as electron acceptors instead of CO2 such as

3H2 + HCOOH → CH4 + 2H2O (∆G 0 = –42 kcal or –175.6 kJ)

4H2 + CH3COOH → 2CH4 + 2H2O



(∆G 0 = –49 kcal or 204.8 kJ)



H2 + CH3OH → CH4 + H2O (∆G 0 = –26.9 kcal or –112.4 kJ)

H2 + CH3NH2 → CH4 + NH3



(∆G 0 = –9 kcal or –37.6 kJ)



(22.10)

(22.11)

(22.12)

(22.13)



are not known to occur.

New evidence suggests that Reaction 22.1 can occur abiotically in the presence of a nickel–iron

alloy under hydrothermal conditions (e.g., 200–400°C, 50 MPa), conditions met in parts of the oceanic crust, as reported, for instance, by Horita and Berndt (1999).



22.3.3 BIOENERGETICS OF METHANOGENESIS

As an anaerobic respiratory process, methane formation is performed to yield useful energy to

the cell. Evidence to date indicates that adenosine 5-triphosphate (ATP) is generated by chemiosmotic energy-coupling metabolism (e.g., Mountford, 1978; Doddema et al., 1978, 1979; Blaut and

Gottschalk, 1984; Sprott et al., 1985; Gottschalk and Blaut, 1990; Blaut et al., 1990, 1992; Müller

et al., 1993; Atlas, 1997; Li et al., 2006). The chemiosmotic coupling mechanism seems to involve

pumping of protons or sodium ions across the plasma membrane, depending on the methanogen.

Membrane-associated electron transport constituents required in chemiosmotic energy conservation involving proton coupling in Methanosarcina strain Gö1 include reduced factor F420 dehydrogenase, an unknown electron carrier, cytochrome b, and heterodisulfide reductase (see Blaut

et al., 1992). The heterodisulfide consists of coenzyme M covalently linked to 7-mercaptoheptanoylthreonine by a disulfide bond (Blaut et al., 1992). A proton-translocating ATPase associated with

the membrane catalyzes ATP synthesis in this organism.

An example of a methanogen that employs sodium ion coupling is Methanococcus voltae (Dybas

and Konisky, 1992; Chen and Konisky, 1993). It appears to employ Na+-translocating ATPase that

is insensitive to proton translocation inhibitors. A scheme for pumping sodium ions from the cytoplasm to the periplasm that depends on membrane-bound methyl transferase was proposed by

Blaut et al. (1992). Methanosarcina acetivorans is another example of a methanogen that employs

sodium ion coupling in its ATP synthesis (Li et al., 2006).



22.3.4



CARBON FIXATION BY METHANOGENS



When methanogens grow autotrophically, their carbon source is CO2. The mechanism by which

they assimilate CO2 is different from that of most autotrophs (Simpson and Whitman, 1993). Most

autotrophs in the domain Bacteria use the pentose diphosphate pathway (Calvin-Benson-Bassham

cycle). Among the exceptions are green sulfur bacteria, which use a reverse tricarboxylic acid cycle;

Chloroflexus aurantiacus, which uses bicyclic CO2-fixation involving 3-hydroxypropionate as a key

intermediate; and the methane-oxidizing bacteria, which use either the hexulose monophosphate

or the serine pathway (see Section 22.3.7). In methanogens, as in homoacetogens (see Chapter 6)

and some sulfate-reducing bacteria (see Chapter 19), the chief mechanism of carbon assimilation

is by reduction of one of the two molecules of CO2 to methyl carbon and the second to a formyl

carbon followed by the coupling of the formyl carbon to the methyl carbon to form acetyl∼SCoA



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(see Figure 6.8 in Chapter 6). To form the important metabolic intermediate pyruvate, they next

carboxylate the acetyl∼SCoA reductively. All other cellular constituents are then synthesized from

pyruvate and may utilize incomplete reductive or oxidative tricarboxylic acid cycles (Simpson and

Whitman, 1993).

Examination of the genome sequence of Methanocaldococcus (Methanococcus

(

s) jannaschii and

Methanosarcina acetivorans has revealed the presence of genes for a ribulose 1,5-bisphosphate

carboxylase/oxidase (Finn and Tabita, 2003), but these organisms do not use the Calvin-BensonBassham cycle for primary CO2 fixation (see introduction of the article by Finn and Tabita, 2004,

and Sprott et al., 1993).



22.3.5



MICROBIAL METHANE OXIDATION



22.3.5.1 Aerobic Methanotrophy

Methane can be used as a primary energy source by a number of aerobic bacteria. Some of these are

obligate methanotrophs; others are facultative (Higgins et al., 1981; see also Theisen and Murrell,

2005). Methane is also oxidized by some yeasts (Higgins et al., 1981). Except for the anaerobic

methanotrophic consortia described in Section 22.3.5.2, most, if not all, known methanotrophs are

aerobes. Examples of obligate methanotrophs are Methylomonas, Methylococcus, Methylobacter,

Methylosinus, and Methylocystis (Figure 22.4). All are gram-negative and feature intracytoplasmic

membranes. On the basis of the organization of these membranes, each obligate methanotroph can

be assigned to one of two types (Davies and Whittenbury, 1970). Members of Type I have stacked

membranes, whereas members of Type III have paired membranes concentric with the plasma membrane and forming vesiclelike or tubular structures (Figure 22.5). Facultative methanotrophs feature internal membranes with an appearance like those of the Type III obligate methanotrophs. All

methanotrophs can also use methanol as primary energy source, but not all methanol oxidizers can

grow on methane as primary energy source. Methanol oxidizers that cannot oxidize methane are

called methylotrophs.

Recently, the surprising discovery was made that the filamentous, sheathed bacteria Crenothrix

polyspora Cohn 1870 and Clonothrix fusca Roze 1896 have methanotrophic ability (Stoecker et al.,

2006; Vigliotta et al., 2007). C. polyspora was found to have an unusual methane monooxygenase

(Stoecker et al., 2006). C. fusca was shown to possess an elaborate internal membrane system to

oxidize and assimilate methane (Vigliotta et al., 2007).



FIGURE 22.4 Aerobic methane-oxidizing bacteria (methanotrophs) (×19,000). (a) Methylosinus trichosporium in rosette arrangement. Organisms are anchored by visible holdfast material. (b) Methylococcus

capsulatus. (From Whittenbury R, Phillips KC, Wilkinson JF, J. Gen. Microbiol., 61, 205–218, 1970. With

permission.)



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FIGURE 22.5 Fine structure of methane-oxidizing bacteria (×80,000). (a) Section of Methylococcus (subgroup minimus) showing Type I membrane system. (b) Peripheral arrangement of membranes in Methylosinus

(subgroup sporium) characteristic of Type II membrane systems. (From Davies SL, Whittenbury R,

J. Gen. Microbiol., 61, 227–232, 1970. With permission.)



Methanotrophs are important for the carbon cycle in returning the carbon of methane, which is

always generated anaerobically, to the reservoir of CO2 (e.g., Vogels, 1979). Obligate methanotrophs are found mainly at aerobic/anaerobic interfaces in soils and aquatic environments that are

crossed by methane (e.g., Alexander, 1977; Reeburgh, 1976; Ward and Brock, 1978; Sieburth et al.,

1987; Hyun et al., 1997; Horz et al., 2002; Inagaki et al., 2004; Sundh et al., 2005) and also in coal

and petroleum deposits (Ivanov et al., 1978; Kuznetsov et al., 1963). Some methanotrophs are also

important intracellular symbionts in mussels from marine hydrocarbon seeps and in other benthic

invertebrates that encounter methane in their habitat. Cavanaugh et al. (1987) found evidence of

the presence of such symbiotic methanotrophs in the epithelial cells of the gills of some mussels

from reducing sediments at hypersaline seeps at abyssal depths in the Gulf of Mexico at the Florida

Escarpment. MacDonald et al. (1990) made similar observations in mussels that occurred in a large

bed surrounding a pool of hypersaline water rich in methane at a depth of 650 m on the continental slope south of Louisiana. Transmission electron microscopic examination by Cavanaugh

et al. (1987) showed that the symbionts feature typical intracytoplasmic membranes of Type I methanotrophs. They possess the key enzymes associated with methane oxidation in the group (see

Section 22.3.6). The basis for the symbiosis between the invertebrate host and the methanotrophs

is the sharing of fixed carbon derived from methane taken up by the host and metabolized by the

methanotroph (Childress et al., 1986). This activity is similar to that of the symbiotic H2S-oxidizing

bacteria in some invertebrates of hydrothermal vent communities, which share with their host the

carbon they fix from CO2 taken up by the host (see Chapter 20). Whether the source of the methane

issuing from Gulf of Mexico seeps is biogenic or abiogenic is unclear at this time. The invertebrate

fauna in the vicinity of the hydrocarbon seeps on the Louisiana slope in the Gulf of Mexico features

intracellular methanotrophic bacterial endosymbionts only in mussels. Vestimentiferan worms and

the clam species in these locations feature autotrophic sulfur-metabolizing symbionts (Brooks et al.,

1987). Interestingly, the mussel Bathymodiolus sp. from a methane seep on the Gabon continental

margin in the southeast Atlantic hosts not only methane-oxidizing symbionts on its gills but also

sulfide-oxidizing symbionts (Duperron et al., 2005). The methane-oxidizing symbionts in this mussel are associated with the basal region of the gill epithelium, whereas the sulfide-oxidizing symbionts are associated with the apical region. Some animals at the Oregon subduction zone have also

formed associations with methanotrophs that enable them to feed on methane (Kulm et al., 1986).



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22.3.5.2 Anaerobic Methanotrophy

Methane may also be anaerobically oxidized by some microbes. Most of the evidence for this activity derives from the study of marine environments, although in at least two instances, evidence was

obtained from two freshwater lakes, one being Lake Mendota, Madison, Wisconsin, United States,

and the other being Plußsee, Germany. Initially, sulfate-reducing bacteria were implicated in this

oxidation on the assumption that some of them can use methane as electron donor for the reduction

of sulfate (e.g., Oremland and Taylor, 1978; Panganiban et al., 1979; Reeburgh, 1980; Martens and

Klump, 1984; Iversen and Jørgensen, 1985; Gal’chenko et al., 1986; Henrichs and Reeburgh, 1987;

Ward et al., 1987). A presumably pure, sulfate-reducing culture with methane-oxidizing ability was

obtained from sediment in Lake Mendota (Panganiban and Hanson, 1976; Panganiban et al., 1979),

but it was incompletely characterized. A consortium of a methanogen and a sulfate reducer, which

together oxidize methane anaerobically to CO2, has been detected in the anoxic zone in Plußsee

(Eller et al., 2005). A similar relationship probably exists in the upper sediments (0–20 cm) of Lake

Biwa, Japan, as described by Koizumi et al. (2003).

As to marine environments, Thomsen et al. (2001), who studied anaerobic sediments from

Aarhus Bay, Denmark, suggested that an archaeal sulfate reducer with methane-oxidizing capacity could have been active in their samples but were unable to rule out other possibilities. Hoehler

et al. (1994, 1998), Hansen et al. (1998), Niewöhner et al. (1998), Hinrichs et al. (1999), Boetius

et al. (2000), and Pancost et al. (2000) suggested that anaerobic methane oxidation may involve a

consortium of methanogens and sulfate reducers. Biogeochemical evidence in support of anaerobic

methane oxidation was presented by Schouten et al. (2003).

It was an observation by Orphan et al. (2001a,b) that clarified what kind of organisms were

responsible for the anaerobic methane oxidation. By means of rRNA gene and lipid analysis of

anoxic methane seep sediments from the California continental margin, they implicated a consortium of Methanosarcinales and Desulfosarcinales in anaerobic methane oxidation. Orphan

et al. (2002) recognized involvement of at least two archaeal groups: ANME-1 and ANME-2. Group

ANME-1, unlike ANME-2, was often encountered in monospecific aggregates or single filaments

without a bacterial partner closely related to Desulfosarcina. Teske et al. (2002) found evidence for

the occurrence of anaerobic methanotrophic communities that showed affiliation to the ANME-1

and ANME-2 groups in the hydrothermal sediments in the Guaymas Basin. Niemann et al. (2006)

identified an additional archaeal group, ANME-3, in the Haakon Mosby Mud Volcano, Barents Sea.

In this group, Methanococcoides and Methanolobus are partnered with Desulfobulbus. Nauhaus

et al. (2002) demonstrated in vitro, anaerobic methane oxidation at the expense of sulfate reduction by a consortium of members of the Methanosarcinales and the Desulfosarcina-Desulfococcus

cluster in marine sediments associated with a gas hydrate deposit at the hydrate ridge. Treude

et al. (2005) quantified anaerobic oxidation of methane (AOM) and sulfate reduction occurring

along the upwelling region at the Chilean continental margin. They measured AOM rates between

7 and 1124 mmol m−2 a−1, individual measurements at various depths showing the highest rates at a

depth of 800 m. Their data suggested that in the sulfate–methane transition zone, sulfate reduction

was mainly coupled to anaerobic methane oxidation. They associated the high methane turnover

rates they found with elevated input of organic matter that was converted to methane in this region.

They did not determine phylogenetically which methanogens and sulfate reducers were active in

the methane turnover. Kallmeyer and Boetius (2004) reported that AOM in hydrothermal sediments

from the Guaymas Basin was observable from 35 to 90°C but accounted for less than 5% of observable sulfate reduction. Sulfate reduction in these sediments was maximal between 60 and 95°C.

Treude et al. (2007) detected methane as well as CO2 consumption in methanotrophic microbial

mats from gas seeps of the anoxic Black Sea. The anaerobic methanotrophy involved differentially

distributed members of the ANME-1 and ANME-2 groups.

Ecologically interesting is the finding by Lösekann et al. (2007) of the coexistence of aerobic

and anaerobic microbial methanotrophs in the Haakon Mosby Mud Volcano—site of a prominent



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548



Geomicrobiology



methane seep. The aerobic methanotrophs dominated the active volcano center, whereas the anaerobes (ANME-3) dominated in the sediment 2–3 cm below a Beggiatoa mat that occurred in a circle

at the rim of the center of the mud volcano.

In anaerobic methane oxidation by consortia of methanogens and sulfate reducers, the methanogens are thought to oxidize methane anaerobically by reversing the methanogenic reaction involving CO2 and H2 (Reaction 22.1):

CH4 + 2H2O → CO2 + 4H2



(22.14)



Sulfate reducers would immediately consume the H2 formed in Reaction 22.14:

HSO4– + 4H2 → HS– + 4H2O



(22.15)



It is difficult to visualize Reaction 22.14 as a simple reversal of the energy-yielding Reaction 22.1

that would enable hydrogen-oxidizing methanogens to grow. Reaction 22.14 should consume energy

if it uses the same enzymes that are used in the formation of methane from CO2 and H2. Zehnder

and Brock (1979) did observe such a reaction with a methanogenic culture under laboratory conditions, but it was very weak. Reaction 22.14 as written may merely represent an overall reaction of a

unique enzymatic pathway in those methanogens that have an ability to oxidize methane anaerobically as well as to form it. Although the combination of Reactions 22.14 and 22.15 is inconsistent

with an incompatibility of methanogenesis and sulfate reduction (Section 22.3.1), it would explain

the absence of very low concentrations of methane in sulfate-reducing zones overlying methanogenic zones. It is noteworthy that the results from a genomic study of methane-oxidizing Archaea

from deep-sea sediments were consistent with an anaerobic reverse-methanogenesis hypothesis

(Hallam et al., 2004).

Unlike the members of anaerobic methane-oxidizing marine consortia, which appear to be in

physical contact, the members of the anaerobic methane-oxidizing consortium in Plußsee are not

in direct physical contact, as determined by the staining technique involving catalyst-amplifiedreported-deposition, fluorescence in situ hybridization (CARD-FISH; Pernthaler et al., 2002).



22.3.6



BIOCHEMISTRY OF METHANE OXIDATION IN AEROBIC METHANOTROPHS



Obligate aerobic methanotrophs can use methane, methanol, and methylamines as energy sources

by oxidizing them to CO2, H2O, and NH3, respectively. When methane is the energy source, the

following steps are involved in its oxidation:

0.5O



0.5O



0.5O



0.5O



2

2

2

2

CH 4 

→ CH 3OH 

→ HCHO 

→ HCOOH 

→ CO2 ϩ H 2O



(22.16)



The first step in this reaction sequence is catalyzed by a monooxygenase that causes the direct

introduction of an atom of molecular oxygen into the methane molecule (Anthony, 1986). This

step is generally considered not to yield useful energy to the cell. A report by Sokolov (1986),

however, suggests the contrary, at least with Methylomonas alba BG8 and Methylosinus trichosporium OB3b. Because monooxygenase requires pyridine nucleotide (NADH + H+) in its catalytic

process to provide electrons for reduction of one of the two oxygen atoms in O2 to H2O (the other

oxygen atom is introduced into methane to form methanol), a proton motive force is generated in

the electron transfer from the reduced pyridine nucleotide, which the cell may be able to couple to

ATP synthesis. The enzyme that catalyzes methanol oxidation is methanol dehydrogenase, which

in Methylococcus thermophilus, as in other methanotrophs, does not use pyridine nucleotide as

the cofactor (Anthony, 1986; Sokolov et al., 1981). Instead, the enzyme contains pyrroloquinone

(+90 mV) as its prosthetic group, which feeds electrons from methanol into the electron transport

chain. The formaldehyde resulting from methanol oxidation is oxidized to formate (Reaction 22.16;



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