Metabolic, Phylogenetic, and Ecological Diversity of the Methanogenic Archaea

Metabolic, Phylogenetic, and EcologicalDiversity of the Methanogenic Archaea

YUCHEN LIU AND WILLIAM B. WHITMAN

Department of Microbiology, University of Georgia, Athens, Georgia, USA

Although of limited metabolic diversity, methanogenic archaea or methanogens possess greatphylogenetic and ecological diversity. Only three types of methanogenic pathways are known:CO2-reduction, methyl-group reduction, and the aceticlastic reaction. Cultured methanogens aregrouped into five orders based upon their phylogeny and phenotypic properties. In addition, uncul-tured methanogens that may represent new orders are present in many environments. The ecologyof methanogens highlights their complex interactions with other anaerobes and the physical andchemical factors controlling their function.

Key words: methane; methanogen; methanogenesis; hydrogenotrophic methanogenesis; methy-lotrophic methanogenesis; aceticlastic methanogenesis

Introduction

Biological methane production or methanogenesisis an important process in the global carbon cycle,processing about 1.6% of the carbon fixed every yearby plants and algae.1 Biologically generated methanecan either serve as a substrate for aerobic or anaer-obic methane oxidation or be emitted to the atmo-sphere. Methane emitted to the atmosphere is a ma-jor greenhouse gas, whose atmospheric concentrationhas increased by threefold in the last 200 years.1 Thecurrent global methane emission is 500–600 Tg CH4

y−1.2 About 74% of the emitted methane is derivedfrom biological methanogenesis.3 The major sourcesof methane emissions are listed in TABLE 1.

Methanogens are the microorganisms that producemethane as the end-product of their anaerobic res-piration. All methanogens are strictly anaerobic ar-chaea belonging to the Euryarchaeota. They are a largeand diverse group, all of which are obligate methane-producers that obtain all or most of their energy frommethanogenesis. The methanogenesis pathway is com-plex, requiring a number of unique coenzymes andmembrane-bound enzyme complexes, the details ofwhich have been recently reviewed.1

Methanogens have been cultivated from a wide va-riety of anaerobic environments. In addition to tem-perate habitats, they are also common in environments

Address for correspondence: Dr. William B. Whitman, Department ofMicrobiology, University of Georgia, 541 Biological Sciences Building,Athens, GA 30605.

[email protected]

of extreme temperatures, salinity, and pH. The com-mon methanogenic habitats include marine sediments,freshwater sediments, flooded soils, human and animalgastrointestinal tracts, termites, anaerobic digestors,landfill, geothermal systems, and heartwood of trees.

Methanogenic Substrates

Although the methanogens are very diverse, theycan only utilize a restricted number of substrates.The substrates are limited to three major types: CO2,methyl-group containing compounds, and acetate(TABLE 2). Most organic substances, for instance, car-bohydrates and long-chain fatty acids and alcohols, arenot substrates for methanogenesis. Instead, these com-pounds must first be processed by anaerobic bacteria oreukaryotes to produce the substrates actually used bythe methanogens. Thus, in most methanogenic envi-ronments, most of the energy available for growth is uti-lized by these nonmethanogenic organisms. It is an in-teresting physiological mystery as to why methanogensdo not directly utilize complex organic matter, bypass-ing their dependency on other organisms.

The first type of substrate is CO2. Mostmethanogens are hydrogenotrophs that can reduceCO2 to methane with H2 as the primary elec-tron donor. Many hydrogenotrophic methanogens canalso use formate as the major electron donor. Inthis case, four molecules of formate are oxidized toCO2 by formate dehydrogenase (Fdh) before onemolecule of CO2 is reduced to methane. In hy-drogenotrophic methanogenesis, CO2 is reduced suc-cessively to methane through the formyl, methylene,

Ann. N.Y. Acad. Sci. 1125: 171–189 (2008). C© 2008 New York Academy of Sciences.doi: 10.1196/annals.1419.019 171

172 Annals of the New York Academy of Sciences

TABLE 1. Sources of global methane emissionsfrom identified sources

Methane emission PercentageSources (Tg of CH4 per year) (%)a

Natural sourcesWetlands 92–237 15–40Termites 20 3Ocean 10–15 2–3Methane hydrates 5–10 1–2

Subtotal 127–282 21–47Anthropogenic sources

Ruminants 80–115 13–19Energy generationb 75–110 13–18Rice agriculture 25–100 7–17Landfills 35–73 6–12Biomass burning 23–55 4–9Waste treatment 14–25 2–4

Subtotal 267–478 45–80Total sources 500–600

Source: Modified from Lowe2 and Prather and Ehhalt.140

aEstimates of the relative contribution of methane emissionfrom a source to the total global emissions of 600 Tg of CH4 peryear.

bMethane deposits released by coal mining, petroleumdrilling, and petrochemical production.

and methyl levels (FIG. 1A). The C-1 moiety is car-ried by special coenzymes, methanofuran (MFR),tetrahydromethanopterin (H4MPT), and coenzyme M(CoM). Initially, CO2 binds to MFR and is reducedto the formyl level. In this first reduction step, ferre-doxin (Fd), which is reduced with H2, is the directelectron donor. The formyl group is then transferred toH4MPT, forming formyl-H4MPT. The formyl groupis then dehydrated to methenyl group, which is sub-sequently reduced to methylene-H4MPT and thento methyl-H4MPT. Reduced F420 (F420H2) is the di-rect electron donor in these two reduction steps. Themethyl group is then transferred to CoM, formingmethyl-CoM. The last reduction step reduces methyl-CoM to methane by methyl coenzyme M reductase(Mcr), which is the key enzyme in methanogenesis.Coenzyme B (CoB) is the direct electron donor inthis reduction, and the oxidized CoB forms a het-erodisulfide with CoM (CoM-S-S-CoB). Finally, theheterodisulfide is reduced to regenerate the thiols.Thermodynamically, two reactions—the methyl trans-fer from H4MPT to CoM and the reduction of theheterodisulfide—are exergonic and involved in energyconservation. The methyl transfer reaction is catalyzedby methyl-H4MPT:HS-CoM methyltransferase (Mtr),which is a membrane-bound complex. The reductionof the heterodisulfide is catalyzed by heterodisulfide re-

ductase (Hdr), which is a membrane-bound complex inMethanosarcina and coupled to F420H2 dehydrogenase(Fpo) when H2 is present. The reduction of CO2 toformyl-MFR is endergonic and driven by ion gradientvia the membrane-bound energy conserving hydroge-nase (Ech).

Some hydrogenotrophic methanogens can also usesecondary alcohols, such as 2-propanol, 2-butanol, andcyclopentanol, as electron donors. A small numbercan use ethanol.4–7 The secondary alcohols are ox-idized to ketones via coenzyme F420-dependent sec-ondary alcohol dehydrogenases (Adf).8 Ethanol is ox-idized to acetate via a nicotinamide adenine dinu-cleotide phosphate (NADP)–dependent alcohol dehy-drogenase.9 Although the growth on alcohols is poorcompared to that on H2, it is an important exceptionto the generalization that methanogens cannot directlymetabolize most organic compounds. Even in this case,where the substrate is obviously assimilated, the oxida-tion is incomplete, and methane is derived from CO2

reduction.Two species have been shown to utilize CO as a re-

ductant for methanogenesis from CO2.10,11 In Methan-

othermobacter thermoautotrophicus and Methanosarcina bark-

eri, four molecules of CO are oxidized to CO2 usingCO dehydrogenase (CODH) before one molecule ofCO2 is reduced to methane.11 H2 is an intermedi-ate in this reaction and serves as the direct electrondonor for the reduction of CO2. Growth with CO ispoor, and the doubling time is more than 200 h for M .thermoautotrophicus and 65 h for M . barkeri. In contrast,Methanosarcina acetivorans grows on CO by an entirelydifferent pathway to be discussed later in this chapter.

The second type of substrate is methyl-group con-taining compounds, including methanol, methylatedamines (monomethylamine, dimethylamine, trimethy-lamine, and tetramethylammonium), and methy-lated sulfides (methanethiol and dimethylsulfide).Methanogens that are able to use methylated com-pounds, or methylotrophic methanogens, are limited tothe order Methanosarcinales, except for Methanosphaera

species, which belong to the order Methanobacteri-ales. During methanogenesis, the methyl-groups frommethylated compounds are transferred to a cognatecorrinoid protein and then to CoM (FIG. 1B).12–14

Methyl-CoM subsequently enters the methanogen-esis pathway and is reduced to methane. Theactivation and transfer of the methyl group re-quires substrate-specific methyltransferases. Interest-ingly, all known methylamine methyltransfereasescontain a UAG (amber codon)-encoded L-pyrrolysinedesignated the twenty-second genetically encodedamino acid. This implies a connection between the

Liu & Whitman: Diversity and Habitats of Methanogens 173

TABLE 2. Free energies and typical organisms of methanogenesis reactions

Reaction �G◦′ a (kJ/mol CH4) Organisms

I. CO2-type4 H2 + CO2 → CH4 + 2 H2O −135 Most methanogens4 HCOOH → CH4 + 3 CO2 + 2 H2O −130 Many hydrogenotrophic methanogensCO2 + 4 isopropanol → CH4 + 4 acetone + 2 H2O −37 Some hydrogenotrophic methanogens4 CO + 2H2O → CH4 + 3 CO2 −196 Methanothermobacter and Methanosarcina

II. Methylated C1 compounds4 CH3OH → 3 CH4 + CO2 + 2 H2O −105 Methanosarcina and other methylotrophic

methanogensCH3OH + H2 → CH4 + H2O −113 Methanomicrococcus blatticola and Methanosphaera

2 (CH3)2-S + 2 H2O → 3 CH4 + CO2 + 2 H2S −49 Some methylotrophic methanogens4 CH3-NH2 + 2 H2O → 3 CH4 + CO2 + 4 NH3 −75 Some methylotrophic methanogens2 (CH3)2-NH + 2 H2O → 3 CH4 + CO2 + 2 NH3 −73 Some methylotrophic methanogens4 (CH3)3-N + 6 H2O → 9 CH4 + 3 CO2 + 4 NH3 −74 Some methylotrophic methanogens4 CH3NH3Cl + 2 H2O → 3 CH4 + CO2 + 4 NH4Cl −74 Some methylotrophic methanogens

III. AcetateCH3COOH → CH4 + CO2 −33 Methanosarcina and Methanosaeta

SOURCE: Modified from Hedderich and Whitman1 and Zinder.43

aThe standard changes in free energies were calculated from the free energy of formation of the most abundant ionic species atpH 7. For instance, CO2 is HCO3

− + H+ and HCOOH is HCOO− + H+.

abilities of amber codon translation and methylamineutilization.15–17 In most methylotrophic methanogens,the electrons required for the reduction of the methylgroups to methane are obtained from the oxida-tion of additional methyl-groups to CO2, which pro-ceeds stepwise as the reverse of hydrogenotrophicmethanogenesis. In methylotrophic methanogenesis,three methyl groups are reduced to methane for ev-ery molecule of CO2 formed. This process is termeda disproportionation, since the oxidation of a por-tion of the substrate is used to reduce the remain-der. Different from this mode of growth, the methy-lotrophic growth of Methanomicrococcus blatticola andMethanosphaera species is H2-dependent.18–23 Theyare obligate methylotrophic and hydrogenotrophicmethanogens that are specialized to reduce methylgroups with H2. The metabolism of Methanosphaera isrestricted to methanol, while M . blatticola can use bothmethanol and methylamine.

The third type of substrate is acetate. Acetate is amajor intermediate in the anaerobic food chain, andas much as two-thirds of the biologically generatedmethane is derived from acetate. Surprisingly, onlytwo genera are known to use acetate for methano-genesis: Methanosarcina and Methanosaeta. They carryout an aceticlastic reaction that splits acetate, oxi-dizing the carboxyl-group to CO2 and reducing themethyl group to CH4 (FIG. 1C). Methanosarcina is arelative generalist that prefers methanol and methy-lamine to acetate, and many species also utilize H2.

Methanosaeta is a specialist that uses only acetate.Methanosaeta is a superior acetate utilizer in that it canuse acetate at concentrations as low as 5–20 μM, whileMethanosarcina requires a minimum concentration ofabout 1 mM.24 The difference of acetate affinity isprobably due to differences in the first step of acetatemetabolism. Methanosarcina uses the low-affinity acetatekinase (AK)-phosphotransacetylase (PTA) system to ac-tivate acetate to acetyl-CoA, while Methanosaeta usesthe high-affinity adenosine monophosphate (AMP)–forming acetyl-CoA synthetase.24–27 Moreover, basedon their genome sequences, these two genera proba-bly have different modes of electron transfer and en-ergy conservation, even though the main steps in themethanogenesis pathway are likely to be similar.27

The metabolism of M. acetivorans grown on CO isunconventional. It is distinct from the CO metabolismin M. barkeri and M. thermoautotrophicus. First, althoughthe oxidation of CO to CO2 via CODH is commonlyrequired for CH4 production, H2 is not generated asan intermediate in M . acetivorans.28 M . acetivorans is alsounable to grow with H2/CO2 and lacks hydrogenaseactivities. Second, proteomic analyses suggest that CO-dependent methanogenesis in M . acetivorans requires anunusual mechanism for energy conservation. Althoughthe steps of CO2 reduction to methyl groups are simi-lar as those in hydrogenotrophic methanogens, a coen-zyme F420H2:heterodisulfide oxidoreductase system isresponsible for energy conservation.29,30 This systemis used in Methanosarcina during methanogenesis with


FIGURE 1.


methylated compounds in the absence of H2, but it hasnot been identified in CO2-dependent methanogene-sis. Third, novel methyltransferases, which are usedin place of methyl-H4MPT:CoM methyltransferase(Mtr), are involved in the transfer of methyl groupfrom H4MPT to CoM.29 Fourth, substantial amountsof acetate and formate are produced in addition tomethane during the growth on CO in M . acetivorans.Mutational analysis suggests that acetate is formed viathe AK-PTA system, which is the reverse of the initialsteps of methanogenesis from acetate in Methanosarcina.Acetate production may generate adenosine triphos-phate (ATP) via substrate level phophorylation. Thus,the growth on CO in M . acetivorans may conserve moreenergy than that in M . barkeri and M . thermoautotrophi-

cus, which can be an explanation of the faster growthof M . acetivorans on CO (doubling time about 24 h).The mechanism of formate production is unclear yet.Since the formate dehydrogenase activity is absent inM . acetivorans, novel reactions are presumably involved.

Systematics of Methanogenic ArchaeaDespite their restricted substrate range,

methanogens are phylogenetically diverse. Theyare classified into five well-established orders:Methanobacteriales, Methanococcales, Methanomi-crobiales, Methanosarcinales, and Methanopyrales.Organisms from different orders have less than82% 16S rRNA sequence similarity. Methanogensbelonging to different orders also possess different cell-envelope structure, lipid composition, substrate range,and other biological properties. The methanogen or-ders are further divided into 10 families and 31 genera.Organisms with less than 88–93% and less than 93–95% 16S rRNA sequence similarity are separated intodifferent families and genera, respectively. Detaileddescriptions of the systematics and characteristics ofthe methanogen taxa are reviewed in Bergey’s Manual of

Systematic Bacteriology31 and The Prokaryotes.3,32–35 Somecharacteristics of the methanogenic taxa are listed inTABLE 3.

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−FIGURE 1. Pathways of methanogenesis. (A) Methanogenesis from H2/CO2 or formate.

(B) Methanogenesis from methanol. (C) Methanogenesis from acetate. ABBREVIATIONS: Fdred

= reduced form of ferredoxin; Fdox = oxidized form of ferredoxin; F420H2 = reducedform coenzyme F420; MFR - methanofuran; H4MPT = tetrahydromethanopterin; CoM-SH =coenzyme M; CoB-SH = coenzyme B; CoM-S-S-CoB = heterodisulfide of CoM and CoB; CoA-SH = coenzyme A. Enzymes: 1. formyl-MFR dehydrogenase (Fmd); 2. formyl-MFR:H4MPT formyl-transferase (Ftr); 3. methenyl-H4MPT cyclohydrolase (Mch); 4. methylene-H4MPT dehydrogenase (Hmd);5. methylene-H4MPT reductase (Mer); 6. methyl-H4MPT:HS-CoM methyltransferase (Mtr); 7. methyl-CoMreductase (Mcr); 8. heterodisulfide reductase (Hdr); 9. formate dehydrogenase (Fdh); 10. energy-conserving hydrogenase (Ech); 11. F420-reducing hydrogenases; 12. methyltransferase; 13. acetate ki-nase (AK)-phosphotransacetylase (PTA) system in Methanosarcina; AMP-forming acetyl-CoA synthetasein Methanosaeta; 14. CO dehydrogenase/acetyl-CoA synthase (CODH/ACS).

Recent culture-independent studies have revealedthe presence of novel phylogenetic groups ofmethanogens. They are not closely related to knownorganisms and probably represent new orders. For in-stance, the rice cluster I (RC-I), which is abundant inrice-field soil, forms a separate lineage within the phy-logenetic radiation of Methanosarcinales and Metha-nomicrobiales. The 16S rRNA sequences of RC-Ihave similarities of less than 82% with known organ-isms.36–39 Investigations of rumen methanogens havefound a novel monophyletic group of at least two fam-ilies.40 The 16S rRNA sequences of this group havesimilarities closest to, but less than 80%, with those ofMethanosarcinales. These studies illustrate our incom-plete understanding of methanogen diversity.

MethanobacterialesMembers of the order Methanobacteriales generally

produce methane using CO2 as the elector acceptorand H2 as the electron donor. Some species can use for-mate, CO, or secondary alcohols as electron donors.The genus Methanosphaera can only reduce methanolwith H2. In most genera, the cells are short to longrods with a length of 0.6–25 μm. They often formfilaments up to 40 μm in length. The cell wall con-tains pseudomurein. The cellular lipids contain caldar-chaeol, archaeol, and, in some species, hydoxyarchaeolas core lipids. The polar lipids can contain glucose,N -acetylglucosamine, myo-inositol, ethanolamine, andserine, depending on the species. Most species arenonmotile. However, members of the genus Methan-

othermus are motile via peritrichous flagella. Theyare widely distributed in anaerobic habitats, such asmarine and freshwater sediments, soil, animal gas-trointestinal tracts, anaerobic sewage digestors, andgeothermal habitats.

The order of Methanobacteriales is divided into twofamilies, Methanobacteriaceae and Methanotherma-ceae. The family Methanobacteriaceae contains threemesophilic genera, Methanobacterium, Methanobrevibacter,and Methanosphaera, and one extremely thermophilic


TABLE

3.

Pro

per

ties

of

majo

rta

xonom

icgro

ups

of

met

hanogen

s

Met

hano

gene

sis

Tem

pera

ture

Typ

ical

Ord

erFa

mily

Gen

usSu

bstr

ates

aO

ptim

um(◦ C

)H

abita

ts

Met

hano

bact

eria

les

Met

hano

bact

eria

ceae

Met

hano

bact

eriu

mH

2,(

form

ate)

37–4

5A

naer

obic

dige

stor

s,fr

eshw

ater

sedi

-m

ents

,mar

shy

soils

,rum

enM

etha

nobr

evib

acte

rH

2,fo

rmat

e37

–40

Ani

mal

gast

roin

test

inal

trac

ts,

anae

r-ob

icdi

gest

ors,

rice

padd

ies,

deca

ying

woo

dytis

sues

Met

hano

spha

erab

H2+

met

hano

l37

Ani

mal

gast

roin

test

inal

trac

tsM

etha

noth

erm

obac

ter

H2,(

form

ate)

55–6

5A

naer

obic

dige

stor

sM

etha

noth

erm

acea

eM

etha

noth

erm

usH

280

–88

Hot

spri

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Met

hano

cocc

ales

Met

hano

cocc

acea

eM

etha

noco

ccus

H2,f

orm

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35–4

0M

arin

ese

dim

ents

Met

hano

ther

moc

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sH

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60–6

5M

arin

ege

othe

rmal

sedi

men

ts

Met

hano

cald

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aeM

etha

noca

ldoc

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sH

280

–85

Mar

ine

geot

herm

alse

dim

ents

Met

hano

torr

isH

288

Mar

ine

geot

herm

alse

dim

ents

Met

hano

mic

robi

ales

Met

hano

mic

robi

acea

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etha

nom

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H2,f

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s,gr

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wat

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20–5

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men

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H2,f

orm

ate

37–4

0A

naer

obic

dige

stor

sM

etha

noge

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H2,f

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15–5

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arin

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sedi

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s,an

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gast

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acts

Met

hano

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nia

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arin

ese

dim

ents

Met

hano

plan

usH

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32–4

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Met

hano

spir

illac

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Met

hano

spir

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mH

2,f

orm

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30–3

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naer

obic

dige

stor

s,m

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di-

men

ts

cont

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TABLE

3.

Continue

d.

Met

hano

gene

sis

Tem

pera

ture

Typ

ical

Ord

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mily

Gen

usSu

bstr

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corp

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2),

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Ana

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mar

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sedi

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ents

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men

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Met

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cocc

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sM

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223

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Mar

ine

sedi

men

tsM

etha

noha

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umM

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240

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Hyp

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line

sedi

men

tsM

etha

noha

loph

ilus

MeN

H2

35–4

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ese

dim

ents

Met

hano

lobu

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237

Hyp

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line

sedi

men

tsM

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nom

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lovo

rans

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20–5

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stro

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acts

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hano

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umM

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235

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line

sedi

men

tsM

etha

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hano

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gest

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men

ts

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subs

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nesi

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met

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min

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sm

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me,

butn

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cies

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taxo

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genus Methanothermobacter. The family Methanoth-ermaceae is represented by one hyperthermophilicgenus, Methanothermus, which has only been isolatedfrom thermal springs.

MethanococcalesMembers of the order Methanococcales produce

methane using CO2 as the electron acceptor and H2

or formate as the electron donor. The cells are ir-regular cocci with a diameter of 1–3 μm. The cellwall is composed of S-layer proteins. Glycoproteinsand cell-wall carbohydrates are generally absent or oflow abundance. The cellular lipids contain archaeol,caldarchaeol, hydroxyarchaeol, and macrocyclic ar-chaeal, depending upon the species. The polar lipidscan contain glucose, N -acetylglucosamine, serine, andethanolamine. They are motile by means of flag-ella. The temperature range for growth varies frommesophilic to hyperthermophilic. They have all beenisolated from marine habitats and require sea salts foroptimal growth.

The order of Methanococcales has been dividedinto two families distinguished by their growth temper-atures. The family Methanocaldococcaceae includestwo hyperthermophilic genera, Methanocaldococcus andMethanotorris. The family Methanococcaceae includesthe mesophilic genus Methanococcus and the extremelythermophilic genus Methanothermococcus.

MethanomicrobialesMembers of the order Methanomicrobiales use

CO2 as the electron acceptor and H2 as electron donor.Most species can use formate, and many species alsouse secondary alcohols as alternative electron donors.Their morphology is diverse, including cocci, rods,and sheathed rods. Most cells have protein cell walls,and some cells are surrounded by a sheath contain-ing glycoproteins. The cellular lipids contain archaeoland caldarchaeol as core lipids. Glucose, galactose,aminopentanetetrol, and glycerol are common polarlipids. Motility varies between species. They are widelydistributed in anaerobic habitats, including marine andfreshwater sediments, anaerobic sewage digestors, andanimal gastrointestinal tracts.

The order of Methanomicrobiales is di-vided into three families, Methanomicrobiaceae,Methanospirillaceae, and Methanocorpusculaceae.The Methanospirillaceae is distinguished from theother two families by the curved rod shape of the cells.The Methanomicrobiaceae and Methanocorpuscu-laceae are difficult to distinguish by their physiologicalcharacteristics.

MethanosarcinalesMembers of the order Methanosarcinales have the

widest substrate range among methanogens. Most canproduce methane by disproportionating the methyl-group containing compounds or by splitting acetate.Some species can reduce CO2 with H2, but formateis not used as an electron donor. Their cellular mor-phologies are diverse, including cocci, pseudosarci-nae, and sheathed rods. Most cells have protein cellwalls, and some cells are surrounded by a sheath oracidic heteropolysaccharides. The cellular lipids con-tain archaeol, hydroxyarchaeol, and caldarchaeol. Po-lar lipids can contain glucose, galactose, mannose,myo-inositol, ethanolamine, serine, and glycerol, de-pending upon the species. All cells are nonmotile.They are widely distributed in marine and freshwatersediments, anaerobic sewage digestors, and animalgastrointestinal tracts.

The order of Methanosarcinales is divided intotwo families, Methanosarcinaceae and Methanosae-taceae. All members of the family Methanosarcinaceaeare able to grow with methyl group–containing com-pounds. The family Methanosaetaceae is representedby only one genus, Methanosaeta, and all cells producemethane by splitting acetate.

MethanopyralesThe order of Methanopyrales is represented by only

one species, Methanopyrus kandleri. Cells reduce CO2

with H2 for methanogenesis. They are rod-shaped.Cell walls contain pseudomurein, and lipids containarchaeol. The cells are motile via flagella arrangedas polar tufts. M. kandleri is hyperthermophilic with agrowth temperature range of 84–110◦C. It inhabitsmarine hydrothermal system.

Sequenced Genomes of Methanogens

Genome sequencing facilitates an understanding ofthe physiology of methanogens. Hitherto completedmethanogen genomes include 18 cultured strains(TABLE 4). The genome sizes vary from 1.6 Mbp to5.8 Mbp. The Methanosarcina species have the largestgenomes, presumably due to their relatively versa-tile lifestyle. A large portion of genes in methanogengenomes have unknown functions, which will have tobe characterized experimentally.

Metagenomic approaches lend insight into the phys-iology of uncultured methanogens. The genome ofan uncultured RC-1 representative was reconstructed(TABLE 4) and revealed genes encoding enzymesfor oxygen detoxification, carbohydrate metabolism,and assimilatory sulfate reduction, which are rare


TABLE 4. Currently completely sequenced methanogen genomes

GC ProteinLength content coding RNA

Species Strain (bp) (%) genes genesa References

Methanothermobacter thermautotrophicus Delta H 1751377 49.54 1845 46 141Methanobrevibacter smithii ATCC 35061 1853160 31.03 1795 42 111Methanosphaera stadtmanae DSM 3091 1767403 27.63 1534 52 22Methanococcus maripaludis S2 1661137 33.10 1728 48 142Methanococcus maripaludis C5 1789046 32.98 1845 46Methanococcus maripaludis C7 1766675 33.21 1819 37Methanococcus vannielii SB 1693095 31.19 1694 38Methanococcus aeolicus Nankai-3 1568349 30.02 1510 40Methanocaldococcus jannaschii DSM 2661 1739916 31.29 1789 43 143Methanoculleus marisnigri JR1 2478101 62.06 2506 51Methanospirillum hungatei JF-1 3544738 45.15 3239 65Methanocorpusculum labreanum Z 1804962 50.01 1765 61Methanosaeta thermophila PT 1879471 53.55 1730 50Methanosarcina barkeri Fusaro 4873766 39.23 3758 71 144Methanosarcina mazei Go1 4096345 41.48 3398 67 145Methanosarcina acetivorans C2A 5751492 42.68 4721 56 146Methanococcoides burtonii DSM 6242 2575032 40.76 2431 61 147Methanopyrus kandleri AV19 1694969 61.16 1727 38 148Uncultured methanogenic archaeon RC-1MRE50 3179916 54.60 3103 63 38

aInclude genes for the rRNAs and tRNAs.

in methanogens.38 The metagenome derived fromhuman gut suggested lower divergence betweenMethanobrevibacter smithii strains than Bifidobacterium

longum strains, but differences in metabolic capacity andsurface properties between different M . smithii strainswere still substantial.41,42

Ecology of Methanogens

Methanogens are abundant in habitats where elec-tron acceptors such as O2, NO3

−, Fe3+, and SO42−

are limiting. When electron acceptors other than CO2

are present, methanogens are outcompeted by the bac-teria that utilize them, such as sulfate-reducing bac-teria, denitrifying bacteria, and iron-reducing bacte-ria. This phenomenon probably occurs because thesecompounds are better electron acceptors, and their re-ductions are thermodynamically more favorable thanCO2 reduction to methane. However, because CO2

is generated during fermentations, it is seldom limit-ing in anaerobic environments. Besides methanogens,homoacetogens are another group of anaerobes thatcan reduce CO2 for energy production. During ac-etate production or acetogenesis, CO2 is reduced withH2 or other substances, for instance, sugars, alco-hols, methylated compounds, CO, and organic acids.However, acetogenesis with H2 is thermodynamicallyless favorable than methanogenesis. Therefore, ho-moacetogens do not compete well with methanogens

in many habitats. However, homoacetogens outcom-pete methanogens in some environments, such as thehindgut of certain termites and cockroaches. Possibleexplanations may be their metabolic versatility as wellas lower sensitivity to O2.

In methanogenic habitats, complex organic matteris degraded to methane by the cooperation of differentgroups of anaerobes. A general scheme of the anaero-bic food chain is shown in FIGURE 2. The organic poly-mers are initially degraded by specialized bacteria tosimple sugars, lactate, volatile fatty acids, and alcohols.These products are further fermented by syntrophsand related bacteria to acetate, formate, H2, and CO2,which are substrates for methanogenesis. Methanogenscatalyze the terminal step in the anaerobic food chainby converting methanogenic substrates to methane.The reactions catalyzed by the syntrophic bacteria, forinstance, the conversion of volatile fatty acids and alco-hols to acetate, CO2, and H2, is only favorable at H2

partial pressures below 102 Pa.43 When methanogensare present, H2 is rapidly metabolized and main-tained at concentrations below 10 Pa.1 Therefore,these syntrophic bacteria depend on the associa-tion with methanogens or another hydrogenotrophicorganisms for energy production. The interactionbetween H2-producing organisms (e.g. propionate-oxidizing bacteria) and H2-consuming organisms (e.g.hydrogenotrophic methanogens) is named interspecieshydrogen transfer.


FIGURE 2. Anaerobic food chain for the conversion oforganic matter to methane. Major microbial groups catalyz-ing the reactions are in ellipses.

Marine SedimentsMethanogenesis in marine habitats is a significant

process that produces between 75 and 320 Tg CH4

year−1, but nearly all the methane is anaerobically oxi-dized to CO2 instead of escaping to the atmosphere.44

Sulfate, which is commonly present at 20–30 mM inseawater, is an important factor that controls the dis-tribution of marine methanogens.45 Sulfate-reducingbacteria outcompete methanogens in sulfate-rich ma-rine sediments for H2 and acetate. For instance, in thesediments of Cape Lookout Bight (North Carolina),the H2 in the sulfate-rich sediments is mainly utilizedby sulfate-reducing bacteria and kept at a level of 0.1–0.3 Pa, which is below the minimum values utilizedby hydrogenotrophic methanogens.46,47 Therefore inupper layers of sediments, or sulfate-reducing zones,methanogenesis is limited and accounts for less than0.1% of total carbon turnover.45 In sediments with highinput of organic matter, sulfate can be depleted withdepth, and methanogenesis can become the predomi-nant terminal process in the anaerobic food chain.

Based on studies of carbon- and hydrogen-stable iso-topes, CO2-reduction by H2 is a predominant sourceof methanogenesis in deep marine sediments.48–50 Inmethanogenic zones, which are usually beneath the

sulfate-reducing zones, the dissolved bicarbonate poolis replenished from oxidation of carbon compoundsin upper sediments and can obtain concentrationsgreater than 100 mM.49 CO2-reducing methanogensin marine sediments include members within theorders Methanococcales, Methanomicrobiales, andMethanobacteriales.51–53 They gain energy strictly byCO2 reduction coupled with H2 or formate oxidation.Moreover, hydrogenotrophic methanogens belongingto Methanomicrobiales can be detected from all depthsof the sediments under certain circumstances53. Pos-sibly, these methanogens gain energy from syntrophicgrowth with H2 producers.54

Methylotrophic methanogens are major contribu-tors to the limited methane production in sulfate-rich sediments. Identified organisms include mem-bers of the genera Methanococcoides, Methanosarcina, andMethanolobus.52,53,55 Methylated compounds are gen-erated in marine sediments from osmolytes of ma-rine bacteria, algae, phytoplankton, and some plants.For instance, dimethylsulfide and trimethylamine arederived from dimethylsulfoniopropionate and betaineglycine, respectively. These compounds are not uti-lized efficiently by the sulfate-reducing bacteria andare termed noncompetitive substrates.56 Because ofthe presence of a considerable amount of these sub-strates, obligately methylotrophic methanogens, suchas Methanococcoides, can be cultivated from all depths inthese sediments.53

Acetate is a minor substrate for methanogenesis inmost marine sediments.48,51 Only a few aceticlasticmethanogens belonging to Methanosarcina have beenisolated.57–59 The minimum concentration of acetateutilized by Methanosarcina is typically 1 mM.43 However,pore water acetate concentrations in marine sedimentsare usually below 20 μM.50,52 Therefore, the aceticlas-tic methanogens that have been isolated probably alsouse methylated compounds for methanogenesis underenvironmental conditions. As measured by isotopes,the rate of acetate oxidation to CO2 exceeds that ofCH4 production by the aceticlastic reaction in sulfate-reducing sediments.50 Therefore, in these sediments,acetate is mainly metabolized by acetate oxidizers, forinstance, sulfate-reducing bacteria.

Freshwater SedimentsFreshwater environments have lower sulfate con-

centrations (100–200 μM) than marine sediments.45

Therefore methanogenesis in freshwater sedimentsproceeds uninhibited in anoxic zones and replaces sul-fate reduction as the most important terminal pro-cess in the anaerobic degradation of organic matter.Due to the absence of competition by sulfate-reducing


bacteria, the acetate pool is available for methano-genesis and is the dominant substrate. In most in-vestigated freshwater environments, aceticlastic andhydrogenotrophic methanogenesis are responsible forabout 70% and 30% of the CH4 production, respec-tively.49,60 The relative contribution of acetate andH2/CO2 in methanogenesis is close to the theoret-ical expectation. The fermentation of hexose yields4 H2, 2 acetate, and 2 CO2. In addition, 4 H2 are re-quired to reduce CO2 to methane. Thus, the expectedratio of methane from H2 and acetate is 1:2. Methano-genesis from methylated compounds is minor, possiblyreflecting the absence of these substrates in freshwatersediments.45 The methanogenic communities are usu-ally dominated by the aceticlastic family Methanosae-taceae and the hydrogenotrophic families Methanomi-crobiaceae and Methanobacteriaceae. Methanosarci-naceae may also be present, and they may utilize eitherH2/CO2 or acetate.61–65

A few factors have been investigated that af-fect the relative contribution of aceticlastic and hy-drogenotrophic methanogenesis as well as the relativeabundance of different types of methanogens in fresh-water sediments. First, hydrogenotrophic methano-genesis decreases at low pH.66 For instance, in LakeKnaack sediments (pH 6.8), only 4% of the CH4 pro-duction is derived from H2/CO2.66 In Lake GrosseFuchskuhle (pH < 5), only aceticlastic methanogensbelonging to Methanosarcinaceae can be detected.67

Low pH provides a selective advantage to homoaceto-gens, which reduce CO2 to acetate instead of methane.This limits hydrogenotrophic methanogenesis. Sec-ond, the relative contribution of hydrogenotrophicmethanogenesis decreases with temperature, eventhough the absolute rate of aceticlastic methano-genesis also decreases.62,68–70 A similar effect oftemperature is also observed in rice paddies.71 Thiseffect can be explained by better adaptation of ho-moacetogens to low temperatures.68,69 Moreover, H2

production by syntrophic bacteria decreases, as H2-producing processes are less favorable at low temper-ature. Therefore, hydrogenotrophic methanogenesis isinhibited at low temperature due to an insufficient sup-ply of substrates. Third, the relative contribution of hy-drogenotrophic methanogenesis changes with depthin some cases. In Lake Dagow sediments, the rela-tive contribution of hydrogenotrophic methanogenesisincreases from 22% to 38% from 0 to 18 cm.61 Co-incidently, the relative abundance of Methanomicro-biales increases slightly with depth, while the abun-dance of Methanosaetaceae decreases. In contrast, inLake Rotsee sediments, hydrogenotrophic methano-genesis is only observed in the upper 2 cm of the sedi-

ments.72 In this case, hydrogenotrophic methanogenspossibly live as symbionts of ciliates. Fourth, the abun-dance of other H2 and acetate-consumers greatlyinfluences the methanogenic communities. In LakeConstance sediments, the presence of H2-consumingsulfate-reducing bacteria and absence of acetate-consuming sulfate-reducing bacteria, together with lowtemperature (4◦C), results in 100% CH4 productionfrom aceticlastic methanogenesis.68,73 In Lake Kin-neret sediments, CH4 is produced exclusively by hy-drogenotrophic methanogens (Methanomicrobiaceaeand Methanobacteriaceae) through syntrophic associ-ation with acetate-oxidizers.74

Rice-field SoilsRice fields are major anthropogenic sources of

methane emission. In rice paddies, upon flooding, oxy-gen, nitrate, ferric iron, and sulfate are rapidly ex-hausted due to high input of plant carbon, developingthe conditions favoring methanogenesis. Methanogen-esis is a significant process in rice fields, and about 3–6% of the photosynthetically fixed CO2 is convertedto methane.75 Methanogens inhabit both the soils andthe root surfaces. Methane is emitted mostly throughthe vascular system of the rice plant, which also sup-plies O2 to the roots and adjacent rhizosphere soil.76

Since rice-field soils are transiently oxic and exposedto drying, soil methanogens are proposed to be moreresistant to O2 and desiccation than methanogens inaquatic habitats.77

Methane in rice fields is derived from bothH2/CO2 and acetate. Dominant methanogens are af-filiated with Methanomicrobiaceae, Methanobacteri-aceae, Methanosarcinaceae, Methanosaetaceae, andRC-I.78–80 A few factors have been suggested thataffect methanogenesis and/or methanogenic popu-lations in rice fields. First, the relative populationsof methanogens remain constant upon flooding andseasonal drying.76,80–82 However, hydrogenotrophicmethanogenesis dominates immediately after flood-ing, while aceticlastic methanogenesis increases later,reaching a maximum at about 70–80 days after flood-ing.80,83 This shift may be due to activation of hy-drogenotrophic methanogens immediately upon flood-ing and not changes of the methanogenic popu-lations. Second, the methanogen community struc-tures on rice roots differ from those in the bulksoils. On rice roots, RC-I is usually the domi-nant organism, and CH4 is mainly derived fromH2/CO2 (60–80%).78,79 In the bulk soil, aceticlas-tic methanogens are dominant, and CH4 is mainlyderived from acetate cleavage (50–83%).60,76,79,84,85

One possible reason for this difference is that the


hydrogenotrophic methanogens on rice roots could bemore resistant to O2, which has a higher concentrationon the roots than in the bulk soils. Third, the populationof hydrogenotrophic methanogens increases with tem-perature (≥30◦C).86,87 Fourth, phosphate (≥20 mM)specifically inhibits aceticlastic methanogens.78,79,88

RC-I methanogens form a distinct phylogeneticlineage within the radiation of Methanosarcinalesand Methanomicrobiales, based upon analyses ofboth 16S rRNA and mcrA genes (review in Ref. 37).These hydrogenotrophs are ubiquitous and abundantin rice paddies, representing 20–50% of the totalmethanogens. Incubations of rice roots or rice-fieldsoils with RC-I methanogens show that they are pref-erentially active at low H2 concentrations, moderatelyhigh temperatures (45–50◦C), and with additional sup-plies of sugars or amino acids. Other speculations toexplain their prevalence in rice fields include their su-perior adaptations to temporary drought and oxic con-ditions. Indeed, the genome sequence of RC-I suggeststhe presence of enzymes that detoxify highly reactiveoxygen species.38 Recently, isolation of RC-I in pureculture has been reported.89 This novel isolate, strainSANAE, can utilize H2/CO2 or formate as methano-genesis substrates. Acetate cannot be used as an en-ergy source. The doubling time with H2/CO2 is about4.2 days.

Human and Animal Gastrointestinal TractsIn the human colon, organic substrates that have

escaped digestion in the upper intestinal tract arefermented by anaerobic microbial communities intoshort-chain fatty acids and CO2. H2 is produced fordisposing of reducing equivalents and kept at verylow partial pressures to meet the thermodynamicrequirements for anaerobic fermentation processes.H2 is mainly removed through reutilization by hy-drogenotrophic microorganisms. For instance, in someindividuals about 1 L of H2 is excreted in breath andflatus per day, while 16 L of H2 is used to make 4 L ofCH4 per day.90 The predominant H2-consuming pop-ulations in the human colon are methanogens, aceto-gens, and sulfate-reducing bacteria.

About one-third of healthy human adults are strongmethane-producers and possess breathe methane lev-els >1 ppm above the atmospheric methane level.90,91

Most of the produced CH4 gas is excreted as flatus andsome is absorbed in the blood and excreted in breath.Methanogens number 108–1010 cells/g feces in theseindividuals. Individuals with low levels of methane intheir breathe contain <102 to 5 × 106 methanogencells/g feces.90,92 Acetogens predominate in these in-dividuals, and the numbers of acetogens have a nega-

tive correlation with that of methanogens.93 Althoughacetogens offer nutritional benefits to the hosts by pro-ducing acetate, methanogens can successfully outcom-pete acetogens in the colon of some individuals, possi-bly due to their lower threshold of H2 utilization.94

Sulfate-reducing bacteria outcompete methanogensin many other habitats, but they do not excludemethanogens in human feces.95–98 Neither the cellnumbers of sulfate-reducing bacteria nor the concen-trations of sulfate and sulfide in feces are significantlydifferent between methane-producing and nonproduc-ing individuals.96,97 Moreover, methane-producing fe-ces consumes H2 more rapidly than nonproducingfeces, suggesting that methanogens can outcompeteother H2-consumers in the human colon.95,97,99 Whatdetermines methanogen abundance in feces is still amystery. Methanogens are not detected in feces ofchildren under 27 months of age; while in methane-producing adults, the levels of methanogens are stableover time.91,100 The development of methanogens isnot directly related to the introduction of particularfoods.100 No significant autosomal genetic effects haveyet been identified.101 Shared environment is one of thedeterminants of methane production.101 Methanogen-esis is negatively correlated with the frequency of bowelmovements.91 Likewise, methanogen abundance isnegatively correlated to the fecal concentrations ofbutyrate, but not to acetate or total short-chain fattyacids.102 The lack of significant correlation betweenmethanogen abundance and the concentration of ac-etate is unexpected, since low methanogen abundanceenhances acetate production by hydrogenotrophic ace-togens. Low acetate concentration in the colon isprobably due to efficient absorption by the host andconsumption by other anaerobes, such as butyrate-producing bacteria.103–105

Methane produced in the human colon is mainlyderived from H2/CO2. Aceticlastic methanogene-sis is generally limited, because the short reten-tion time of the intestine does not allow the slowgrowth of methanogens on acetate.90 Only twomethanogens species have been isolated from hu-man feces: Methanobrevibacter smithii and Methanosphaera

stadtmanae.106,107 Both of them belong to the orderMethanobacteriales. M . smithii is the predominantmethanogen species and constitutes up to 10% ofall anaerobes in the human colon.90,108 It producesmethane from H2/CO2 or formate, but grows poorlywith formate.106 M . stadtmanae is present in lower num-bers than M . smithii. It can only produce methane byreducing methanol with H2 and depends on acetateas a carbon source. Methanol in human gut is de-rived from pectin degradation by Bacteroides and other


TABLE 5. Methanogens cultured from human and animal gastrointestinal tracts

Hosts Representative methanogen species

Human Methanobrevibacter smithii; Methanosphaera stadtmanae

Bovine Methanobrevibacter ruminantium; Methanobrevibacter thaueri; Methanobrevibacter smithii; Methanosarcina barkeri; Methanobac-

terium formicicum; Methanobrevibacter millerae; Methanobacterium mobilis

Ovine Methanosarcina barkeri; Methanobacterium formicicum; Methanobrevibacter olleyae; Methanobrevibacter wolinii

Termite Methanobrevibacter cuticularis; Methanobrevibacter curvatus; Methanobrevibacter filiformis; Methanobacterium bryantii

Cockroach Methanomicrococcus blatticola

anaerobes.109,110 The restricted metabolism ofM . stadtmanae can be explained by the absence ofgenes for CO dehydrogenase/acetyl-CoA synthase(CODH/ACS) and molybdopterin biosynthesis pro-teins, which are common among other methanogens.22

The genome of both M . smithii and M . stadtmanae

encode enzymes for the synthesis of surface glycansresembling the components of the mucosal layer ofthe hosts.111 Their genomes also encode adhesion-likeproteins.111 These features are shared among bacteriain gastrointestinal tracts and may contribute to theiradaptation to the human colon.

The rumen of herbivores is the primary location formicrobial fermentation of plant material that cannotbe digested by the host enzymes. A wide variety of mi-croorganisms, such as bacteria, fungi, and protozoa,ferment plant biomass into H2, short-chain fatty acids,CO2, and CH4. Methane production from the rumencontributes to up to 20% of the global methane emis-sions to the atmosphere. In addition, 6–10% of theenergy value of the food ingested by host animal is lostas methane.112 Therefore, manipulation of methaneproduction in rumen serves as a way to limit methaneemission from anthropogenic sources as well as im-prove animal production. Daily methane productionis different among ruminant species. For instance, anadult cow produces about 200 L of CH4 per day, whilesheep under generous grazing conditions produce 35–50 L of CH4 per day.90,113

In the rumen, hydrogenotrophic methanogens pre-dominate. Methanogenesis limits hydrogenotrophicacetogenesis by lowering the H2 concentration to be-low the minimal level required for acetogenesis.114

Methanogens in the bovine rumen typically number108–1010 cells/g of rumen content, and the populationdensities of methanogens are influenced by the typeof diet, especially the fiber content.115,116 Methanobre-

vibacter species, including M . ruminantium, M . thaueri,and M . smithii, are the predominant methanogens iso-lated and cultured from the bovine and ovine rumen117

(TABLE 5). Recent 16S rRNA gene-based studies re-veal that, although Methanobrevibacter are usually dom-

inant, a wide diversity of ruminal methanogens co-exist in the rumen.117–119 Methanosphaera species sim-ilar to M. stadtmanae are likely to be common.112,117

Methanogens belonging to the Methanosarcinales andMethanococcales are present in the rumen of some in-dividuals, but they are not detectable in most individu-als.118 Substantial numbers of the 16S rRNA sequencesobtained from the rumen are not related to knownmethanogens, and probably represent a novel order ofarchaea.40,119,120 Some of these uncultured microor-ganisms can be established on methanogen-selectivemedium with H2/CO2, but do not compete well withMethanobrevibacter.40 These studies indicate that uncul-tured methanogens may have significant populationdensities in the rumen.

The insect hindgut is another important habitat ofmethanogens. About 3% of the total global methaneemissions or 11% the methane emissions from natu-ral sources are from the termite hindgut. Methano-genesis and acetogenesis occur simultaneously in thetermite hindgut, and the relative activities are relatedto host diets. In wood-feeding termites, acetogenesisoutpaces methanogenesis; by contrast, in soil-feedingand fungus-feeding termites, methanogenesis outpacesacetogenesis.121,122 It is interesting that acetogens canoutcompete methanogens in termite guts, though themechanism is still under study and may vary be-tween different termite species. A few proposed mech-anisms include: (1) Acetogens are metabolically versa-tile and able to use organic substrates in addition toH2. Their mixtrophic lifestyles give them competitiveadvantages over methanogens. However, it is neces-sary to explain why these advantages would not beapplied in other habitats. (2) The H2 levels in ter-mite guts are not limiting for acetogenesis. For in-stance, in Reticulitermes flavipes, a wood-feeding lowertermite, the H2 partial pressure is about 5 kPa, whichis two orders of magnitude higher than the mini-mum level of H2 for acetogenesis.123 (3) Acetogensand methanogens may be spatially separated in differ-ent compartments of the termite gut to avoid directcompetition for H2. Termite guts are highly structured


and characterized by steep gradients of O2, H2, andpH. Some regions may form microniches that favoracetogenesis.122,124

Methanogens identified from termite hindguts in-clude members of the Methanobacteriaceae andMethanosarcinaceae species.125,126 The relationshipbetween methanogen community compositions andtermite diet is not clear yet. Methanobacteriaceaeare predominant in most termite species, but in fourstudied termite species Methanosarcinaceae are thedominant methanogen group.125 The dominance ofMethanosarcinaceae is uncommon in gastrointestinaltracts of animals. Possibly, their ability to use a varietyof substrates, including H2/CO2, acetate, and methy-lated compounds, may provide an advantage in thetermite hindgut.

Anaerobic DigestorsAnaerobic digestors are widely used for the degra-

dation of organic wastes such as animal manure andmunicipal wastewater to methane. Compared withaerobic treatments, the anaerobic treatments have theadvantage of generating large quantities of renewablefuel in the form of biogas. Moreover, they producemuch less sludge, a costly by-product of the aerobicprocess.

The anaerobic conversion of wastes to methane re-quires cooperation of at least three groups of microor-ganisms.127 The first step, hydrolysis, involves enzyme-mediated conversion of organic polymers, such aspolysaccharides, lipids, proteins, and fats, into solu-ble organic monomers. This step is carried out byanaerobic bacteria such as Bacterioides, Clostridium, andStreptococcus. The second step, acidogenesis, involvesanaerobic fermentation of monomers into acetate, H2,CO2, as well as short chain fatty acids (propionateand butyrate), which are subsequently converted toacetate and H2. The third step, methanogenesis, in-volves conversion of acetate, H2, and CO2 into CH4 bymethanogens. Methanogenesis is considered the rate-limiting step, and high activity of the methanogens isimportant for maintaining efficient anaerobic diges-tion and avoiding the accumulation of H2 and shortchain fatty acids. Moreover, this step is most vulnerableto parameters such as temperature, pH, and inhibitorychemicals. Therefore, enhancement of methanogene-sis is a major route for improving the performance ofanaerobic digestors.

Acetate is a major product of the fermentation andaccounts for two-thirds of the methane productionin anaerobic digestors.43 Usually, only one aceticlas-tic methanogen group, Methanosaeta or Methanosarcina,dominates each digestor, depending upon the type

of waste and digestor.128 Methanosaeta have slowergrowth rates and higher affinity for acetate, whileMethanosarcina have faster growth rates and lower affin-ity for acetate. The relative abundance of these twogroups is not only regulated by acetate concentra-tions, as in other environments, but also by feed-ing rates.129,130 Methanosaeta perform better at highfeeding-rate digestors, such as an upward-flow anaer-obic sludge blanket (UASB), presumably due to theirefficient adhesion and granulation.131,132 In contrast,Methanosarcina are more sensitive to turbulence andshear, and they frequently dominate in fixed- andstirred-tank digestors.133

The H2 partial pressures in anaerobic digestorsrange from 2 Pa to 1200 Pa.129 Low H2 levels indi-cate efficient hydrogenotrophic methanogenesis andare usually associated with stable performance. HighH2 levels (>10 Pa) lead to inhibition of the anaero-bic fermentation, and accumulation of electron sinksas lactate, ethanol, propionate, and butyrate. There-fore, efficient interspecies hydrogen transfer is quiteimportant for good performance of anaerobic di-gestors. Formation of granular sludge facilitates in-terspecies hydrogen transfer by reducing the distancebetween H2-producing bacteria and H2-consumingmethanogens.134 A wide variety of hydrogenotrophicmethanogens belonging to Methanomicrobiales andMethanobacteriales are detected in and cultured fromanaerobic digestors.43,135

Temperature and pH are two main parametersthat influence methanogenesis in anaerobic digestors.Slightly thermophilic temperatures (50–60◦C) are de-sired in certain anaerobic digestors to increase reactionrates and decrease retention times. The most commonhydrogenotrophic methanogens in thermophilic di-gestors include M. thermoautotrophicus and Methanoculleus

thermophilicum.43,136 Aceticlastic methanogens gener-ally cannot grow at temperatures higher than 65◦C,thus limiting the temperature range of digestors.The most common aceticlastic methanogens in ther-mophilic digestors include Methanosarcina thermophila

and thermophilic Methanosaeta.43,137 The pH is anotherimportant parameter. Increases of loading rates leadto increases in the concentration of fatty acids anddecreases in pH. Most methanogens grow optimallyunder neutral to slightly alkaline conditions (pH 6.8–8.5). Thus, acid tolerant methanogens are desired toimprove the stability of anaerobic digestion. Methanobre-

vibacter acididurans, a methanogen isolated from a souranaerobic digestor, grows in the pH range of 5.0–7.5.138 Additions of M . acididurans to acidic digestorsshow better methanogenesis and decreases in accumu-lation of fatty acids.139


Conclusions

Methanogens are very diverse in terms of phylogenyand ecology. Their ability to use methanogenesisas an anaerobic respiration allows them to occupya physiologically unique niche that is unavailableto the Bacteria. They play an important role in theanaerobic food chain, driving anaerobic fermentationthrough removal of excess H2 and formate. Theirphysiological diversity makes them truly cosmopolitanin anaerobic environments. The relative abundanceof different types of methanogens is regulated by theavailability of substrates and other parameters, for in-stance, temperature, pH, and salinity. The diversity ofmethanogens is underestimated as indicated by recentculture-independent studies. Further physiological andecological investigations are required to fully reveal thediversity and importance of methanogens.

Conflict of Interest

The authors declare no conflicts of interest.

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Metabolic, Phylogenetic, and Ecological Diversity of the Methanogenic Archaea